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DEVELOPMENT OF IMMUNOLOGICAL REAGENTS FOR DETECTING
Salmonella enterica SEROVAR TYPHIMURIUM
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
I would like to thank my mentor, Paul Gulig, for his support and guidance in my
education. I greatly appreciate his contribution to developing my scientific thinking. I
extend my thanks to the other members of my committee, Donna Duckworth and
Shouguang Jin, for their helpful ideas and support. Thanks also go to my labmates
especially Angela, Julio, and Qiu, for their friendship and help.
A very special thanks to my parents, Kotaiah and Prabhavathi, Suji, Shreesha,
Chinni, Ravi, and Deepthi for all their love and trust in me. Thanks also to Nori, Jayanth,
Saurav, and Archit.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iii
LIST OF TABLES ............. ...................... ......... .... ..... .......... vi
L IST O F FIG U R E S .... ...... ...................... ........................ .. ....... .............. vii
A B S T R A C T .......................................... .................................................. v iii
1 IN TR OD U CTION ............................................... .. ......................... ..
Traditional Methods of Bacteriological Detection and Analysis ...............................
T he B iosensor Sy stem ..................... ........................................ ................ 3
Antigenic Structure of S. Typhimurium .............. .......... .......... .................... 4
M onoclonal A ntibody Production ........................................... ......................... 6
Recombinant Phage Display Antibodies and scFv.............................. .............7
M 13 Phage B iology ................ ..... .. .. ......... ...... .... ..........8...... .8
Griffin. 1 and Tomlinson I + J Human Synthetic VH + VL Phagemid Libraries ....9
2 M ATERIALS AND M ETHOD S ........................................... ........................ 14
Bacterial Strains, Phage Strains, and Growth Conditions .......................................14
ELISA ......................................... .... ............................... 16
Biopanning of the Phage Display Library ............... ...... ......... .. ............. 18
Panning on Im m unotubes ....................................................... ............... 18
P anning in Suspension ........... ........................................ ...... ...... .............. 19
Titering the Phage.......................... ........... ... .. ........... .............. 20
Amplification of the Selected Phage ................................ ................ 21
Panning w without A m plification ................................ .......... ............... .... 22
Production of Soluble Antibody Fragments (scFv Antibodies) ........................23
D N A M anipulations...... ............................................................ ........ .... .... ....... 24
Plasm id E xtractions ........................................................... .. .. .... ........ 24
Enzym e M anipulations ......................... ............................... ............... 24
A garose G el Electrophoresis ........................................ .......................... 24
Polymerase Chain Reaction (PCR) ........................................ ............... 24
Construction of scFv-Avitag Plasmid Vectors ............... ...............25
Protein and LPS M anipulations ........................................................ ............... 27
Extraction of Flagella ...............................................................................27
Determination of Protein Concentration ...........................................................27
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)....27
Coomassie Blue Staining for Proteins................................. ............. ........... 28
Immunoblotting .. ............. ... ........ ..................... ............ ...... 28
Concentration of scFv Antibodies from Culture Supernatant.............................29
Extraction of Soluble Proteins from Periplasmic Space..................................29
3 R E S U L T S ............................................................................. 3 0
R rationale for Study ........... .... .... ...... ... .... ....... .... ... .. .... ... .. ..... ................. .. 30
Specific Aim 1: Standardization of a Protocol for Whole Bacterial Cell ELISAs.....30
R action D evelopm ent Tim e..................................... ............................ ........ 31
A ntigen Concentration for Coating ........................................ .....................32
Comparing the Blocking Efficiencies of Casein and BSA......................... 34
Specific Aim 2: Characterization of Commercially Available Monoclonal Antibodies
to S. Typhim urium ............... .............. .. ................ ... ........ .. .. ... ........... ..35
Specific Aim 3: Isolation of Recombinant Phage Display Antibodies to
S. Typhimurium and Genetically Fusing scFv Antibodies with Ligands or
R reporter M olecules ................ ..................................................... .. ... ..............39
Preliminary Experiments for Optimizing Production and Analysis of Phage
A antibodies .......................................... .... ..... .. .....................40
Isolation of Phage Antibodies Recognizing S. Typhimurium...........................42
Determining the Activity of scFv Antibodies vs. Phage Antibodies...................50
Genetically Fusing scFv Antibodies to Biotin................... ........... ............... 52
4 D ISC U SSIO N ........... .............................................. .......................... 55
Specific Aim 1: Standardization of a Protocol for Whole Bacterial Cell ELISAs.....56
Specific Aim 2: Characterization of Commercially Available Monoclonal Antibodies
to S. Typhim urium ............... .............. .. ................ ... ........ .. .. ... ........... ..59
Specific Aim 3: Isolation of Recombinant Phage Display Antibodies to
S. Typhimurium and Genetically Fusing scFv Antibodies with Ligands or
R reporter M olecules ........................ .... ... ........ .. .... ............ ................ 63
Overcoming the Problem of Insert-less Phage in the Phage Pool .......................64
Reactivity of Phage Antibodies .................................................... ............... 65
Reactivity of scFv Antibodies vs. Phage Antibodies .......................................68
Future D directions ................................................ ........ .. ............ 71
L IST O F R E F E R E N C E S ....................................................................... .... .................. 73
BIO GRAPH ICAL SK ETCH .................................................. ............................... 80
LIST OF TABLES
2 -1: Strain s of b bacteria ........ .................................................................. ....... ....... .. 15
2-2: List of m onoclonal antibodies ........................................................... ..................... 18
3-1: Comparing the blocking efficiencies of casein and BSA........................................35
3-2: Comparing the Activities of Various Monoclonal Antibodies...............................37
3-3: Potency of USB10 and Biosp53 Anti-LPS Monoclonal Antibodies..........................38
3-4: Activity of anti-flagellar m onoclonal antibodies........................... .....................38
3-5: Specificity ofUSB10 and Biosp53 anti-LPS monoclonal antibodies........................39
3-6: Comparing the Activity of Phage Produced Using Hyperphage or helper phage
M 13K 07 by ELISA ........... .... .... .. .................. .. .......... .. ............ 42
3-7: Activity of phage obtained after panning on flagellar extracts .............................48
3-8: Activities of unmodified and biotinylated scFv antibodies................... ..............54
LIST OF FIGURES
3-1: Effect of varying antigen concentration on signal ............................ ..................33
3-2: Activity of phage obtained after each round of panning on S. Typhimurium cells in
3-3: Western blot to determine the specificity of SF1 and SF2 anti-flagella phage
antib bodies .....................................................................................49
3-4: Confirmation of synthesis of biotinylated scFv antibodies. ............. ... .............. 53
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
DEVELOPMENT OF IMMUNOLOGICAL REAGENTS FOR DETECTING
Salmonella enterica SEROVAR TYPHIMURIUM
Chair: Paul A. Gulig
Major Department: Molecular Genetics and Microbiology
Salmonella enterica serovar Typhimurium (S. Typhimurium) is bacterium
responsible for over a million cases of gastroenteritis per year in the United States.
Extremist groups have also used this bacterium as an agent of biological terrorism.
Salmonella infections cause huge economic losses to the food industry, especially the
poultry and diary industries. Effective measures to counter the dangers and economic
losses caused by this bacterium should include rapid, sensitive, and specific detection of
the bacterium in the samples suspected of contamination.
A biosensor system consisting of an optic fiber probe with antibodies attached to
the plastic surface of the probe connected to a photodiode and a computer monitor was
developed at the University of South Florida. When the optic fiber probe is placed in the
samples, the antibodies on the surface of the probe capture the relevant microbe.
Detection antibodies labeled with a fluorescent dye are then added forming a sandwich
by the two antibodies with the microbe in between. When light of a certain wavelength is
passed through the optic fiber probe, the fluorescent dye is excited and emits a light of
different wavelength. This emitted light is captured by the photodiode and quantified.
This entire process can be completed in as little time as 20 min.
We developed an ELISA protocol for using whole bacterial cells as antigens and
screened several commercially available murine monoclonal antibodies recognizing
surface epitopes of S. Typhimurium using the standardized procedure. ELISA was
chosen as the method of choice, because it closely resembles the conditions of the
biosensor system and has a much higher throughput than the biosensor system.
Recombinant antibody phage display libraries offer a powerful, economical, and rapid
method of screening libraries of large complexities of recombinant antibody molecules
on the order of 109 variants. We used the Griffin. 1 and Tomlinson I Human Synthetic
scFv phagemid libraries to isolate antibodies to surface epitopes of S. Typhimurium.
Panning was done under varying conditions using whole bacterial cells, purified LPS, or
flagella as antigens. Two antibodies recognizing the flagella of S. Typhimurium, named
SF1 and SF2, were isolated.
We used the SF1, SF2, and the provided anti-BSA positive control phagemids to
obtain soluble antibody molecules (scFv) instead of fusion proteins displayed on the
phage surface, and genetically modified the antibodies to increase their usefulness. These
studies will aid in the continuing development of immunological tools for use with the
real time fiber optic biosensor system.
Bioterrorism is a problem of ever-increasing magnitude confronting civil societies
all over the world. The cost of development and ease of deployment of agents of
bioterrorism make these weapons appealing to extremists (1-3). Mailing of envelopes
containing spores of anthrax after September 11, 2001 serves as a good example to
illustrate the fact. Fringe extremist groups with limited means to access sophisticated
weapons may use agents of bioterrorism to further their interests and cause panic in
society. A famous example was the contamination of salad bars with Salmonella enterica
serovar Typhimurium (S. Typhimurium) by the Ranjneeshee religious cult in Dulles,
Oregon in 1984 to prevent local people from voting in a county election (4,5). The
economic and psychological impacts of these attacks can be far more devastating than the
direct threat to human health caused by these weapons (6). As the saying goes,
prevention of these attacks is the best form of protection that can be offered.
Nevertheless, when an attack is suspected, law enforcement officials should be able to
verify and confirm actual use of biological agents in a timely manner to take corrective
actions. With the increasing incidence of bioterrorism, it is imperative that sensitive,
specific, rapid, and reliable systems be available to detect agents of bioterrorism in
complex matrices such as food, water, and on surfaces. Such detection systems also
facilitate better analysis of clinical samples in a hospital setting leading to a better and
quick diagnosis and therapy (7). Such detection systems also increase the efficiency of
Contamination of food in food processing industries is a problem of huge economic
and practical concern. Monitoring food samples at various levels of processing would
ensure better control over food quality (8-11). Tests used in such situations, in which
multiple samples from multiple sources need to be analyzed quickly, should be easy and
rapid to perform, should be sensitive and specific, and should give results in a short time
The characteristics of the ideal tests or systems used for detecting contamination
with microbes and toxins should be sensitive, specific, rapid, portable, less time
consuming, involve minimal handling and processing of the samples, and obviate the
need for costly and bulky equipment. The need for skilled technicians should be minimal
Traditional Methods of Bacteriological Detection and Analysis
Some of the traditional methods of determining microbial contamination involve
enumeration of coliforms by the Most Probable Number (MPN) method and biochemical
tests. Estimating contamination by the MPN method is not very specific and not
sensitive (13). Biochemical methods identify the microbes in a very specific manner;
however, they are time consuming and not very sensitive. Generally, the detection of
microbes involves a pre-enrichment step for 6 to 8 hours, followed by growth in an
enrichment medium and then in a selective medium. Several biochemical tests are
needed for characterizing the microbes (14-16). Some of the modern techniques used in
industry and laboratories for rapid and sensitive detection of microbes involve
amplification of specific nucleic acid sequences by Polymerase Chain Reaction (PCR)
(17,18); immunomagnetic separation (19); enzyme tests with synthetic chromogenic or
fluorogenic substrates (20); real-time PCR (21); and flow cytometry (7,9,22). Many
commercially available kits including API Rapid 20E (bioMerieux, Los Angeles, CA),
GN Microplate (Biolog, San Jose, CA), Directigen (BD Diagnostic Systems, Franklin
Lakes, NJ) are available for detection of microbes (23). Many of the above-mentioned
techniques identify microbes sensitively and specifically; but require costly and sensitive
equipment, skilled personnel, and sterile conditions to perform the analysis. Most of
these systems are not portable and cannot be used in field conditions.
Antibodies show exquisite specificity toward their target. Murine monoclonal
antibodies have been used for many years in research for detecting the antigens and
separating the particles from a complex mixture (24). Some of the above-mentioned
detection systems employ antibodies in conjunction with some other technique to identify
the pathogen. Examples for such techniques using immunological reagents include
immunofluorescence (25), fluorescence automated cell sorting (25),
immunoprecipitation, immunohistochemistry, and immunomagnetic separation (19).
The Biosensor System
Transportable fiberoptic biosensor systems are being developed that will enable
real-time or near-real-time detection of microbes and toxins (26-28). These systems
combine the advantage of sensitivity and specificity afforded by antibodies and a robust
architecture enabling their use in field conditions. The evanescent wave fiberoptic
biosensor works on the principle of a sandwich immunoassay. Monoclonal or polyclonal
antibodies are used as capture antibody and reporter antibody. The capture antibodies are
attached to the surface of the optical fiber probe. Reporter antibodies are tagged with a
fluorescent dye. The evanescent field-sensing region is formed by the final 7.5 cm of the
fiber, where the cladding is removed to expose the silica core. When the samples (such
as contaminated food and water) that need to be analyzed are brought in contact with the
probe, the capture antibodies bind to the antigen. When the reporter antibodies are added
to the solution, they bind to the relevant epitopes of the specific antigen to be detected,
forming a sandwich. A 635 nm laser diode provides the excitation light, which is
launched into the proximal end of the probe. Fluorescent molecules within
approximately 1000 nm of the fiber are excited by the evanescent field; and a portion of
their emission energy recouples into the fiber. A photodiode is used to quantitate the
collected emission light at wavelengths of 650 nm and above, and the data can be
displayed on a computer monitor (26). Similar systems using chemiluminescence are
also in use (27).
The advantages of this method over the traditional methods of detection are
"Dirty" samples can be processed without a need for pre-enrichment or
The biosensor is reported to be detecting as low as 3 CFU/mL of E. coli 0157:H7
in seeded ground beef (26).
The architecture of the apparatus is robust, enabling the use in field conditions.
Results are obtained in as little time as 20 min, thereby greatly aiding the
law-enforcement officials and health personnel in times of emergencies.
Antigenic Structure of S. Typhimurium
Salmonella enterica serovar Typhimurium is a gram-negative bacterium that is
responsible for over a million cases of gastroenteritis in the United States every year (29).
Salmonella spp. are responsible for three classes of diseases: enteric fevers, septicemia
and gastroenteritis. Typhoid fevers are caused by S. Typhi and S. Paratyphi, while
S. Cholerasuis causes septicemia. However, the most common disease caused by
Salmonella is gastroenteritis, the most common causative agent of which is
S. Typhimurium (30,31). Salmonella gastroenteritis is generally self-limiting.
Salmonella infections, with the exception of enteric fevers, are typically food-borne
illnesses that are transmitted to humans by animal products such as poultry, pork, meat,
and eggs. S. Typhimurium can also be used as an agent ofbioterrorism by contaminating
food and water supplies with the bacterial culture (3). Salmonella infections cause major
economic losses in food industry (32). A biosensor system for detecting S. Typhimurium
in various matrices would be of great practical use. This thesis describes the efforts done
to characterize existing monoclonal antibodies and to isolate recombinant phage display
antibodies for use in such a system. Antibodies recognizing the cell surface epitopes
were chosen for this study to enable the detection of whole cells, without the need for
preparing cell extracts.
The outer membrane of S. Typhimurium is composed of lipopolysaccharide (LPS),
phospholipids, outer membrane proteins such as porins, and matrix proteins such as
OmpA. Lipopolysaccharide is found only in the outer membrane of gram-negative
bacteria. Lipopolysaccharide is composed of three components viz., lipid A, core
oligosaccharide, and O antigen. Lipid A is the major lipid constituent of the outer
membrane and is highly conserved among different gram-negative organisms. Lipid A is
responsible for the endotoxic activity of gram-negative bacteria. The core
oligosaccharide connects the lipid A moiety to the peripheral O-antigen and is conserved
among different bacteria. The outer core region consists of a branched pentasaccharide
chain made up of sugars such as glucose, galactose, and 2-acetamido-2-deoxy-D-glucose.
The inner core region is made up of L-glycero-D-mannoheptose and
3-deoxy-D-manno-octulosonic acid. Variation in O antigen results from the variation in
sugar components and from the variation in the nature of covalent bonds between the
sugars and from the order of the sugar molecules in the oligosaccharide. The repeating
units are composed of trisaccharides, pentasaccharides, or branched chain
oligosaccharides. The differences in the O side chains are exploited to differentiate
between various serogroups by immunological reagents. Thus, O-antigen makes a good
target for characterizing gram-negative bacteria (33).
Flagella are another good target for immunological detection of whole cells.
Flagella are made up of an external filament, a hook region, and a basal body (34). The
hook region, which lies outside the outer membrane, connects the external filament to the
basal body, which spans both the outer and inner membranes. The external filament is a
polymer of a 60-kD flagellin molecule, which provides many sites for binding of
anti-flagellar antibodies (34). Flagellin is encoded by two different genes in
S. Typhimurium, which are highly homologous, but the two forms of flagellin are
antigenically distinct. For every 103 to 105 generations, there is a reversal in the
expression of the genes, but at any given time, only one of the genes is expressed. This is
due to a switching in the expression of the promoters for these genes and this
phenomenon is called "phase variation" of flagella (35-37)
Murine monoclonal antibodies recognizing various O antigens such as 04 and 012,
antibodies of undetermined specificity to the bacterial cell surface, and antibodies
recognizing flagella were chosen for this study based on the above-mentioned criteria.
Monoclonal Antibody Production
Kohler and Milstein first described the idea of monoclonal antibodies (38).
Monoclonal antibodies have since been used in many varied applications (24). The
production of monoclonal antibodies involves immunizing mice with the antigen and
boosting the immune response by periodical booster doses of the antigen. Serum is
collected periodically, and the response to the antigen is determined by ELISA or
Western blot. When the mice have a satisfactory titer of the antibody, an assay is done to
determine the predominant isotype of the antibodies (i.e., IgG or IgM). The mice having
good titers and class switching (IgM to IgG) are selected. The spleen of the selected
mouse is harvested and separated into single cells. These single cells are combined with
mouse cells of the myeloma cell line SP2/0. Polyethylene glycol (PEG) is added to
promote the fusion of the cells, so these cells are called hybridomas. The cells are then
resuspended in selective medium that kills off nonfused cells and are grown in a 96-well
plate. After several days of growth at 37 C and intermittent feeding with tumor-
conditioned medium, the supernatant is tested for activity by ELISA, and cells in the
positive wells are transferred to a 24-well plate and grown further for a week. A
secondary screen is done with the supernatant from these wells. Finally, since it is
possible that the initial cultures contain different hybridomas, positive cultures are
subjected to limiting dilution and reexamined for activity. Antibody-positive cell lines
are now called clones and can now be grown in larger cultures for mass production of the
antibody from the culture supernatant followed by affinity purification.
Recombinant Phage Display Antibodies and scFv
Phage display is a technique of displaying antibodies or peptide fragments as fusion
proteins to one of the coat proteins of the phage. M13 is the commonly used phage for
this purpose (39). Single chain variable fragment (scFv) antibody molecules are
composed of the variable portions of the heavy and light chains of immunoglobulin
linked by a spacer region. The variable portion of the antibody molecule is responsible
for the specificity of the antibody. Antibodies can be displayed on the phage particle in
either scFv format or in Fab format. The phage antibodies and the scFv fragments can be
used in all immunological assays, similarly to a monoclonal antibody (40,41).
M13 Phage Biology
M13 is a nonlytic filamentous bacteriophage of the Ff class of phages, which infect
E. coli displaying the F conjugative pilus. The phage contains a circular, single-stranded
DNA (ssDNA) of approximately 6,400 nucleotides as genomic DNA and is surrounded
by a flexible protein coat. pVIII, a 50-amino acid protein present in approximately 2,700
copies, forms the major part of the protein coat. pIII, a 406-amino acid protein, is a
minor coat protein present at one end of the phage particle and is present in
approximately 5 copies along with another minor coat protein, pVI (39).
pIII is composed of three domains two amino-terminal domains, N1 and N2, and
a carboxy-terminal domain, CT, separated by a glycine-rich region. The first domain,
N1, contains the amino-terminal 68 amino acids and is required during infection for the
translocation of the phage DNA into the cytoplasm and insertion of coat proteins into the
membrane. The second domain, N2, is composed of residues 87 to 217 and is
responsible for binding to the F pilus. Both domains contain cysteine molecules that are
involved in intramolecular disulfide bonds within each domain. N1 and N2 are exposed
on the surface of the phage particles; removal of these domains by protease treatment
produces noninfectious phage. The carboxy-terminal 150 residues make up the third
domain, CT, which is essential for forming a stable phage particle (39).
Infection is initiated by the binding of the tip of the F pilus to the N2 domain of the
phage pIII protein. After the phage binds to the pilus, the pilus retracts bringing the pIII
end of the phage particle to the periplasm. N1 interacts with the TolA protein, and pVIII
and minor capsid proteins disassemble into the cytoplasmic membrane as the phage DNA
is translocated into the cytoplasm.
After the phage ssDNA (+ strand) enters the cytoplasm, the complementary
(- strand) is synthesized, and a covalently closed, supercoiled, double-stranded DNA
called the replicative form, RF, DNA, is formed. New DNA molecules are synthesized
by rolling circle mode of replication. mRNA is synthesized using the (-) strand as the
template. The bacteria then synthesize phage proteins. The phage particles are
assembled when the concentration of pV reaches a critical concentration. M13 is a
non-lytic phage. Therefore, the phage particles bud out of the E. coli cell membrane
without killing the cell (39).
Phagemid vectors are plasmid vectors that contain both E. coli ColE1 ori and M13
ori sequences. Therefore, phagemids can be propagated as plasmids in bacteria. These
vectors do not encode all of the genes necessary for the formation of phage particles by
themselves. However, when E. coli containing phagemids are superinfected with a
helper phage to supply all of the necessary proteins for replication and packaging, the
phagemid DNA can replicate and be packaged into viable infectious phage particles
Griffin.1 and Tomlinson I + J Human Synthetic VH + VL Phagemid Libraries
Griffin. 1 and Tomlinson I + J Human synthetic VH + VL phagemid libraries are
scFv phagemid libraries displaying human antibody sequences as fusion protein to the
minor coat protein, pIII. These libraries were constructed by amplifying the genes coding
for the variable portions of immunoglobulin molecules from the peripheral blood
lymphocytes of unimmunized donors (41,43).
The nucleotide sequence at the 5' end of the exons coding for V-genes is conserved
in human beings. Using universal degenerate primers, the antisense strands of the heavy
and light chain V-genes were amplified. Heavy and light chain V-genes were linked by
PCR by using overlap extension technique. The V-genes were amplified by PCR, and the
repertoires were combined with linker DNA, which has regions of sequence homology
with the 3' end of the VH gene and 5' end of the VL gene. Then PCR was done with
primers hybridizing to outer flanking sequences of the VH and VL genes. The final
product contains the VH and VL genes linked by the linker region, (Gly4Ser)3, with
restriction sites for facilitating cloning into phagemid vectors (43). The vector used for
constructing the Griffin. 1 library was pHEN2, and the Tomlinson libraries were
constructed in pIT2 vector. Antibody molecules of this form heavy and light chain
regions of the variable portion of the immunoglobulin molecules linked by a peptide
linker are called scFv (single chain variable fragment) antibodies.
For displaying the scFv sequence on the surface of the phage as fusion protein, the
scFv gene was cloned in frame with the gIII gene coding for the pIII protein in the
phagemid vectors. When E. coli harboring these phagemids are superinfected with a
helper phage, phage particles are produced which display the scFv-pIII fusion protein.
Some other important features (42) engineered into these libraries are
The phagemid encodes the bla gene, which can be used as a selectable marker.
When E. coli cells harboring the phagemid are grown on selective media, E. coli
form colonies rather than plaques because the phagemid cannot form complete
phage particles without superinfection by helper phage.
A bacterial leader peptide sequence, pelB, is present at the 5' end of the scFv
gene. PelB directs the scFv protein into the secretary pathway, for exporting the
protein into the periplasm.
A c-myc epitope and a hexahistidine tag are present at the 3' end of the scFv gene
to allow for affinity purification and detection using appropriate antibodies and
nickel matrices, respectively.
Protein A and protein L binding sites are present in the antibodies produced from
Tomlinson libraries. These sites offer additional options for detection and affinity
purification. Also, all of the phage antibodies produced from the Tomlinson
library are pre-selected for their ability to bind to protein A and protein L to
ensure that most of the phage have the scFv-gIII gene, instead of wild type gill.
A TAG amber stop codon is present at the junction of the scFv gene and gill. The
presence of amber stop codon allows the production of scFv molecules as soluble
antibody molecules instead of scFv-pIII fusion proteins. When the phagemid is
present in a suppressor strain of E. coi, the amber stop codon is translated as
glutamine and instead of termination of the peptide chain synthesis, and scFv-pIII
fusion protein is synthesized. When the phagemid is in a non-suppressor strain of
E. coi, the synthesis of peptide chain is terminated at amber stop codon, releasing
a soluble scFv molecule.
SThe scFv-gIII is expressed from the wild type lac promoter control of Lad, so
that the synthesis of the protein, which could be toxic to the host E. coi, can be
suppressed by glucose. Alternatively, when scFv antibody molecules are intended
to be produced, synthesis can be induced by IPTG.
Phage display offers a powerful, economic, and rapid method of screening libraries
of huge complexity in the order of 109 library size (41). Short peptides or antibody
molecules can be displayed on the surface of the phage. The phage displaying the
sequence of interest can be isolated by incubating the library with the antigen to allow for
specific binding, and the unwanted phage are washed away. Abundant amounts of the
phage of interest can be produced by propagating them in E. coi. Antibodies can be
obtained as soluble molecules or as fusion proteins. The advantages of this technology
can be considered under two aspects ease of production and ease of manipulation.
The advantages of recombinant phage display antibodies over murine monoclonal
antibodies in terms of their production are:
The Tomlinson I + J libraries and the Griffin. 1 library have a complexity of
approximately 1.2 x 109, which approximates the complexity of the human
Conventional hybridoma technology involves immunizing animals with the
antigen and harvesting the spleen cells for creating a hybridoma. There is no need
for animal immunization for creating a phage display library and isolating the
desired antibodies from the library.
Isolating a monoclonal antibody takes from 3 to 6 months, while phage display
can be as short as a couple of days!
Isolating antibodies by screening phage display libraries is immensely economical
when compared to the production of murine monoclonals.
Ethical and legal constraints limit injecting toxic substances into mice for
antibody production. Antibodies cannot be produced for antigens that are lethal
to the host animal. However, antibodies to theoretically any antigen can be
isolated from phage display libraries because the screening is done in vitro rather
than in vivo.
The recombinant antibodies isolated from phage display libraries can be
genetically manipulated relatively easily compared with murine monoclonal
Antibodies obtained by screening human antibody libraries can be used for
therapeutic or diagnostic purposes in humans without the fear of eliciting host
immune response to the antibodies. Although murine monoclonal antibodies can
be humanized by grafting the variable portion of the antibodies to constant region
of human immunoglobulins, the antibodies lose their affinity to the antigen
(24,44-46). Human monoclonal antibodies can be produced by creating
heterohybridomas between human lymphocytes and mouse myeloma cell lines,
but success is limited (47,48).
The gene encoding the antibody is immediately available for manipulation, and
effects are immediately observable on the activity of the phage and/or antibody.
Therefore, effects of genetic manipulation of the antibody sequence and structure
can be studied easily.
scFv antibodies can be genetically modified to be conjugated to various ligands
and reporter molecules, thereby greatly increasing the spectrum of their potential
Affinities of the antibodies can be increased by techniques such as error-prone
PCR, shuffling heavy or light chains, or propagating the phagemids in mutator
strains of E. coli (49-52). These methods mimic the affinity maturation by
somatic hypermutatation in B cells.
This thesis describes the efforts to isolate recombinant phage display antibodies to
S. Typhimurium and the attempts at genetically modifying the antibodies for their
potential use in the biosensor system.
The specific aims for this study are:
1. Standardization of a protocol for whole bacterial cell ELISAs: The biosensor is
intended to detect whole bacterial cells. However, it is not a very high throughput
system for screening antibodies. ELISA closely resembles the conditions of the
biosensor system and enables screening large number of antibodies. Therefore, a
protocol for whole-cell ELISAs needs to be developed and standardized for
screening commercially available murine monoclonal antibodies, recombinant
phage display antibodies, or scFv antibodies using whole bacterial cells as
2. Characterization of commercially available monoclonal antibodies to
S. Typhimurium: Lim et al. (26) are developing a biosensor at the University of
South Florida for the detection of S. Typhimurium. We screened commercially
available antibodies in a whole-cell ELISA for use in that biosensor system.
3. Isolation of recombinant phage display antibodies to S. Typhimurium: As
mentioned above, isolating phage display antibodies is much easier and more
efficient than isolating a murine monoclonal antibody. We proposed to isolate
phage display antibodies recognizing surface antigens of S. Typhimurium from
the Griffin. 1 and the Tomlinson libraries. We explored the possibility of
genetically modifying recombinant antibodies for optimal use.
MATERIALS AND METHODS
Bacterial Strains, Phage Strains, and Growth Conditions
The bacterial strains used and their genotypes are listed in Table 2-1. Griffin. 1 and
Tomlinson I + J Human Synthetic VH + VL phagemid libraries constructed by the
Medical Research Council, Cambridge, U.K. were obtained from the Interdisciplinary
Center for Biotechnology Research Hybridoma Core, University of Florida. Helper
phage M13K07 was obtained from New England BioLabs (Beverly, MA). Hyperphage
was obtained from Progen Biotechnik (Heidelberg, Germany).
E. coli TG1 cells were grown in 2xTY broth (16 g tryptone, 10 g yeast extract, 5 g NaC1,
and 3 mL of 1 M NaOH in 1 L water) or on 2xTY agar plates containing 1.5% (w/v)
agar. E. coli TG1 cells infected with phage from the Griffin. 1 or Tomlinson libraries or
with the phage obtained after elution and neutralization after a round of panning were
grown on 2xTY agar plates with 100 [g/mL ampicillin and 1% (w/v) glucose (2xTY AG
plates). When the bacteria were superinfected with helper phage or Hyperphage to
produce phage particles, they were grown in 2xTY broth with 100 tg/mL ampicillin and
50 [g/mL kanamycin. All of the other bacteria were grown in modified Luria-Bertani
broth (LB-N: 10 g tryptone, 5 g yeast extract, 8.5 g NaC1, and 3 mL of 1 M NaOH in 1 L
water) or on LB-N plates containing 1.5% (w/v) agar. All of the bacteria were grown as
an overnight standing cultures in either LB-N or 2xTY at 37 C. The bacteria from the
starter culture were diluted 1:10 into fresh medium and grown with aeration at 37 C until
the optical density at 600 nm (OD600) reached approximately 0.4. The bacteria thus
obtained were in exponential phase of growth. All the experiments were done using
exponentially growing bacteria to maintain consistency.
Table 2-1: Strains of bacteria
Strain Genotype / Antigenic formula Source / Reference
S. Typhimurium X3000 1,4,,12:i:1,2 (53)
S. Typhimurium (fliC) fliC::Tn 10 (54)
S. Typhimurium (flhD) flhD (55)
E. coli LE392 F- hsdR514 (r-,m+) supE44 (56)
E. coli x1918 HfrH lacZ-x90 argEam-210 metB- Roy Curtis III
E. coli TG1 K12 A(lac-proAB) supE thi (42)
hsdD5/F' traD36proA B lacI
E. coli AVB100 K12 MC1061 araD139 Avidity
A(ara-leu)7696 A(lac)174 galU
galK hsdR2(rK- mK+) mcrB1
S. Cholerasuis 3246 6,7:c:1,5 Anita C. Wright
S. Enteritids 14213 1,9,12:g,m:- Anita C. Wright
S. Rubislaw 10717 11:r:e,n,x Anita C. Wright
S. Worthington 1,13,23:z:l,w Anita C. Wright
S. Gaminara H0662 16:d:1,7 Anita C. Wright
S. Urbana 9261 30:b:e,n,x Anita C. Wright
S. Adelaide 35:f,g:- Anita C. Wright
When biotinylated scFv antibodies were produced in E. coli AVB 100, d-biotin
(Sigma, St. Louis, MO) was added to the culture medium to a final concentration of
50 [iM, and birA was induced by adding L-arabinose (Sigma) to a final concentration of
0.4% (w/v). The gene encoding the scFv-Avitag was induced by adding IPTG to a final
concentration of 1 mM.
Enzyme Linked ImmunoSorbent Assay was used for characterizing the activities of
antibodies. The procedure consisted of coating the antigen at desired concentration in
either carbonate buffer (13 mM Na2CO3, 87 mM NaHCO3, pH 9.2) or phosphate buffered
saline (PBS) (137 mM NaC1, 2.7 mM KC1, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3).
For bacterial cell ELISAs, exponentially growing bacteria were centrifuged at 6,000 x g
for 10 min at 4 C. The cells were washed with PBS by suspending the pellet in 10 mL
PBS and centrifuging at 6,000 x g for 10 min at 4 oC. The pellet was suspended in an
appropriate amount of carbonate buffer to give a final concentration of 107 CFU/mL, and
8% (w/v) aqueous grade 1 glutaraldehyde (Sigma) was added to a final concentration of
0.05% (w/v) (57). For LPS assays, the antigen was suspended in PBS or carbonate buffer
at 5 [g/mL. For protein ELISAs the antigen was suspended in either coating buffer or
PBS at 10 [g/mL. One hundred microliters of the antigen suspension was added per well
of a 96 well non-tissue culture treated polystyrene microtiter plate (Becton-Dickinson,
CA) for coating, and the plate was incubated overnight at 4 oC.
The wells were washed three times with 200 [L of PBS containing 0.05% (w/v)
Tween 20 (Sigma) (PBS-T (0.05)) in ELx 800 Strip Washer (BioTek, VT). Two hundred
microliters of casein blocking buffer (1% (w/v) casein in PBS with 0.05% (w/v)
Tween 20) (Sigma) was added to each well for blocking the nonspecific binding sites.
The wells were washed as described above after 2 h incubation at 4 oC.
One hundred microliters of the primary antibody polyclonall or monoclonal
antibodies, phage particles, or scFv antibodies) diluted in casein blocking buffer at
desired concentrations was added per well. The murine monoclonal antibodies used with
their specificities are listed in Table 2-2. In ELISAs using PEG-precipitated phage as the
primary antibody the concentration of the phage was 109 phage/mL. When using phage
from culture supernatant, the overnight culture supernatant was diluted 1:2 in casein
blocking buffer. After an incubation period of 2 h at 4 oC, the wells were washed as
described above, and secondary antibody was added.
The secondary antibodies were conjugated to either horseradish peroxidase or
alkaline phosphatase. The antibody was diluted in casein blocking buffer and used
generally at a dilution of 1:2000 at 100 [iL per well. The plate was incubated at 4 oC for
2 h, and the wells were washed as described above.
Substrate for development reaction for HRP-conjugated secondary enzymes was
prepared by dissolving one capsule of phosphate citrate buffer with sodium perborate
(Sigma) in 100 mL of water (0.05 M phosphate-citrate buffer pH 5.0, 0.03% (w/v)
sodium perborate). A 10 mg tablet of 3, 3', 5, 5'-tetramethylbenzidine substrate (Sigma)
was added to 10 mL of the buffer to give a final concentration of 1 mg/mL. For alkaline
phosphatase-conjugated secondary enzymes, the substrate was prepared by dissolving a
20 mg tablet of p-nitrophenylphosphate (pNPP) substrate (Sigma) in 20 mL of
bicarbonate buffer (15 mM NaHCO3, 15 mM Na2CO3, 2 mM MgC12). Two hundred
microliters of the substrate solution was added to each well, and the plate was read in an
ELx 800 UV plate reader (BioTek, VT) at 405 nm for HRP conjugates or 630 nm for
alkaline phosphatase conjugates for 40 min. The data were analyzed with Kc Junior
(BioTek, VT) and Microsoft Excel software programs.
Table 2-2: List of monoclonal antibodies
1 US Biologicals S0060-10
2 US Biologicals S0060-15
3 Biospacific A60580228P
4 Biospacific A60530228P
5 RDI 10D9H
6 Virostat 6341
7 Virostat 6321
8 Biodesign 8C11C
9 Virostat 6301
10 Accurate YVS6301
Groups A, B, D Salmonella
Groups A, B, D Salmonella
Group B Salmonella
Biopanning of the Phage Display Library
Isolation of the recombinant antibodies to the antigens on whole bacterial cells,
purified LPS, or flagellar extracts was done by panning the phage display libraries on the
antigen either coated on immunotubes or suspended in blocking buffer.
Panning on Immunotubes
The antigen was suspended in the appropriate buffer (whole bacterial cells in
coating buffer at 109 CFU/mL, flagellar extract or LPS at various concentrations in PBS
or coating buffer), and immunotubes (Nunc) were filled with the antigen suspension and
incubated at 4 oC overnight. The antigen suspension was drained, and the tube was
washed three times by filling with PBS and aspirating the wash solution without
agitation. The nonspecific binding sites on the immunotube were blocked by filling the
tube with casein blocking buffer and incubating for 2 h at 4 oC. The blocking buffer was
aspirated, and the tube was washed three times with PBS. Approximately 1012 phage
were suspended in 4 mL of casein blocking buffer and used for panning. In the first
round of panning, phage from the Griffin. 1 or Tomlinson libraries were used. In the
subsequent rounds of panning, phage from the previous round of elution and / or
amplification were used. The immunotube was filled with the phage suspension and
incubated at 4 oC in the case of whole cells or at room temperature in the case of LPS or
flagellar extracts for at least 2 h with continuous rotation. The phage suspension was
discarded, and the tube was washed with PBS-T (0.1%) followed by PBS. The number
of washes varied depending on the round of panning 10 times in the first round and 20
times in the subsequent rounds of panning.
The phage bound to the antigen were eluted with either 0.1 M glycine or trypsin
depending on the source of phage. Elution was done with 0.1 M glycine, pH 2.8, if the
phage were from the Griffin. 1 library. One milliliter of glycine was added to the tube and
incubated at room temperature for 10 min with continuous rotation. The eluate was
transferred to another tube, and the solution was brought to neutral pH by adding 50 itL
of 1 M Tris, pH 8.0. Trypsin XIII from bovine pancreas (Sigma) in PBS at a
concentration of 1 mg/mL was used for elution if the phage were from Tomlinson I
library. For elution with trypsin, 500 [iL of 1 mg/mL trypsin solution was added to the
tube and incubated for 30 min at room temperature with continuous rotation. The phage
were titered and used to infect E. coli TG1 for amplification or used in the next round of
Panning in Suspension
Panning on the antigen in suspension was done only for whole bacterial cells.
Exponentially growing S. Typhimurium cells were suspended in 1 mL of casein blocking
buffer in a microcentrifuge tube to a final concentration of 109 CFU/mL. Approximately
1012 phage from the Griffin. 1 or Tomlinson libraries for the first round of panning or
from amplified phage of the previous round were suspended in 200 [L of casein blocking
buffer and incubated with the bacteria at 4 oC for at least 2 h with continuous rotation.
The mixture was centrifuged at 5,000 x g for 3 min. The supernatant was discarded, and
the bacterial pellet containing cells with the phage bound to the surface antigens were
suspended in 1 mL of PBS with 0.1% (w/v) Tween 20 by pipetting. This wash procedure
was repeated for at least 10 times in the first round of panning and 15 to 20 times in later
rounds. The wash procedure was stopped before all the washes were done if the pellet
size seemed to be decreasing. After the final wash the bound phage were eluted by
suspending the pellet in 1 mL of 0.5 M glycine, pH 2.8 and incubating for 10 min with
continuous rotation. The bacteria were centrifuged, the supernatant was transferred to
another tube, and 50 [L of 1 M Tris, pH 8.0 was added to neutralize the solution. When
phage from Tomlinson libraries were used elution was done by suspending the pellet in
0.5 mL of 1 mg/mL trypsin and incubating for 30 min at room temperature. The phage
thus obtained were used to infect exponentially growing E. coli TG1 for titering and
Titering the Phage
The phagemid lacks all the genes necessary for the production of proteins required
for phage assembly and packaging, so the phagemid acts as a plasmid in E. coli.
E. coli TG1 infected with the phage, therefore, produce colonies when plated on selective
media. The concentration of the phage was determined by counting the colonies
produced from infecting E. coli. Serial dilutions of phage suspensions were made in
PBS, and 10 iL of 10-4, 10-6, 10-7, and 10-8 dilutions were used to infect 1 mL of
exponentially growing E. coli TG1 for 30 min at 37 C. One hundred microliters of each
infected culture was plated on 2xTY AG plates and incubated at 37 C overnight. The
colonies were counted, and the titer was determined.
Amplification of the Selected Phage
The phage obtained after each round of panning and elution were amplified in
E. coli. This increased the number of the phage bearing the specific antibody sequence
relative to the nonspecific phage or the phage having the wild type pIII. To 0.5 mL of the
eluted phage, 1.5 mL of exponentially growing E. coli TG1 cells at OD600 approximately
0.4 were added and incubated at 37 C for 30 min. The cells were centrifuged at
6,000 x g for 10 min at 4 oC. The bacterial pellet was suspended in 0.4 mL of 2xTY, and
0.1 mL each was plated on four 2xTY AG plates and incubated at 37 C overnight.
The bacterial mass was gently scraped off the plates with a glass spreader after
adding 0.5 mL of 2xTY to loosen the cells. Fifty microliters of cells was added to 50 mL
of 2xTY AG, and the culture was grown at 37 C with shaking until the OD600 was
approximately 0.4. Glycerol was added to the remaining cells to a final concentration of
15% (v/v) and stored at -80 oC after freezing in a dry ice-ethanol bath. Helper phage
M13K07 or Hyperphage were added to 10 mL of the 2xTY AG culture with OD600
approximately 0.4 at a multiplicity of infection (MOI) of 20. The Hyperphage were at a
concentration of 1012 phage/mL according to the supplier. However, when titered by
plating infected E. coli TG1 on LBN-Kan plates, the concentration was only
109 phage/mL. It was impractical to use the measured concentration if a MOI of 20 was
to be achieved, so Hyperphage were used at the concentration stated by the supplier.
Using Hyperphage at the concentration stated by the manufacturer, however, did not
affect the yield of phage particles. The culture was incubated for 30 min at 37 C and
then centrifuged at 6,000 x g for 10 min at 4 oC. The bacterial pellet was suspended in
50 mL of 2xTY with 100 [tg/mL ampicillin and 50 [tg/mL kanamycin and grown with
shaking at 30 oC overnight. The culture was centrifuged at 6,000 x g for 10 min at 4 oC,
and the bacterial pellet was discarded. To precipitate the phage, 10 mL of 20% (w/v)
polyethylene glycol (PEG)/2.5 M NaCl was added to 40 mL of the culture supernatant
and incubated at 4 oC for at least 4 h. The suspension was centrifuged at 6,000 x g for
10 min at 4 C, and the supernatant was discarded. A brief centrifugation was done to
remove any remaining PEG/NaC1, and the pellet was suspended in 1 mL of PBS. The
suspended phage were centrifuged at 5,000 x g for 3 min to remove any remaining
bacterial debris (42). The phage were titered, as described above.
Panning without Amplification
This method differed from panning on immunotubes in that the amplification of
eluted phage in between the rounds of panning was eliminated. The eluted phage from
the previous round were suspended in 4 mL of casein blocking buffer and used for
panning on the antigen-coated on immunotubes. The scFv-pIII fusion protein has a
c-myc site, which is susceptible to trypsin digestion, in between the amino terminal N1
and N2 domains and the carboxy terminal CT domain of the pIII protein. Elution with
trypsin cleaves this c-myc site leaving the antibody sequence and N1 and N2 domains of
pIII attached to the antigen, while the phage particle is released into the fluid phase.
When panning was done without amplification between rounds, elution in the first two
rounds was done with 0.1 M glycine, pH 2.8 to maintain the antibody sequence on the
phage particle to be able to bind the antigen in the next round. If the phage were from
Tomlinson library, elution in the third round was done with 1 mg/mL trypsin. If elution
had been done with trypsin in the first and second rounds also, the antibody sequence
would have been cleaved off the fusion protein, and phage could not have bound to the
antigen coated on the immunotubes.
Production of Soluble Antibody Fragments (scFv antibodies)
The gene coding for the scFv is in frame with gIII, the gene coding for the coat
protein pIII, separated by a TAG amber stop codon. When the phagemid is in an amber
suppressor strain ofE. coli (supE) such as TG1, the TAG codon is translated and
glutamine is incorporated into the peptide chain instead of termination. This results in
the production of a fusion protein, scFv-pIII. When the phagemid is in a nonsuppressor
strain such as E. coli x1918, the translation of the peptide is terminated at the TAG codon
releasing the soluble scFv antibody molecule. The phagemid has thepelB leader
sequence at the 5' end of the scFv-gIII gene, which directs the peptides into secretary
pathway. Therefore, when soluble fusion protein is produced, it is directed to the
periplasmic compartment and/or released into the culture supernatant.
scFv antibodies were produced from the phage clones testing positive for antibody
activity by ELISA by infecting E. coli x1918 with the selected phage. Phage were added
to 1 mL of cells at a MOI of 20 and incubated at 37 C for 30 min. The cells were added
to 9 mL of 2xTY with 0.1% (w/v) glucose and 100 [g/mL ampicillin and were grown
with shaking at 37 C. When the culture was in exponential phase of growth, IPTG was
added to a final concentration of 1 mM to induce the expression of scFv-pIII synthesis,
and the culture was grown overnight with aeration at 37 C (42).
When biotinylated scFv antibodies were produced from E. coli AVB 100 the culture
was brought to 50 [M d-biotin (Sigma) 1 hr prior to the induction of scFv antibody
production with 1 mM IPTG. When the culture is at an OD600 of approximately 0.8, birA
was induced with 0.4% (wt/vol) L-arabinose the scFv antibody production was induced
with 1 mM IPTG.
The scFv antibodies released into the culture supernatant were harvested by
centrifuging the culture at 6,000 x g for 10 min at 4 oC and discarding the bacterial pellet.
The culture supernatant containing the scFv was diluted 1:2 in casein blocking buffer and
used in ELISA or Western blot.
Plasmid extractions were done using QIAprep Spin Miniprep kit (Qiagen) for
cultures up to 10 mL and Plasmid Midi kit (Qiagen) for culture volumes of 50 to 100 mL,
according to the manufacturer instructions.
Restriction enzyme digestions were done with enzymes purchased from Invitrogen,
Rockville, MD; New England BioLabs, Beverly, MA; and Promega, Madison, WI, and
were used according to manufacturer instructions.
Agarose Gel Electrophoresis
DNA was resolved on 0.7 to 1% (w/v) agarose gels using Tris-borate-EDTA buffer
(89 mM Tris, 89 mM boric acid, 2 mM EDTA) with 10 tg/mL ethidium bromide. Gel
electrophoresis was done at 100 V, and the DNA bands were visualized on a UV
Polymerase Chain Reaction (PCR)
PCR was done to verify the presence of scFv insert DNA sequences in phagemids
and to ascertain the number of insert-positive phage in the original Tomlinson I library.
The primers used were Lmb3-extend (hybridizing upstream ofpelB leader sequence) and
FDseql-extend (hybridizing in the 5' region of gIII in the antisense orientation).
The PCR conditions were: 9 min at 95 oC; 30 cycles of (45 sec at 95 oC, 45 sec at
60 C, 90 sec at 72 C), and 10 min at 72 C. The PCR products were resolved on
0.7% (w/v) agarose gels, and the DNA bands were visualized on a UV transilluminator.
Construction of scFv-Avitag Plasmid Vectors
To improve the usefulness of the scFv antibodies, they were biotinylated by cloning
the scFv gene into pAC Avitag vectors and expressing them in E. coli AVB 100 (Avidity).
Avitag is a 15-peptide sequence specifically biotinylated by the BirA enzyme ofE. coli.
Plasmid vectors pAC4, pAC5, and pAC6 contain the sequence coding for Avitag peptide
distal to a multiple cloning site. The E. coli AVB 100 strain has birA stably integrated
into the chromosome under the control of AraC.
The genes coding for anti-flagella scFv and anti-BSA scFv were cloned into the
pAC5 Avitag vector. Anti-BSA antibodies were biotinylated and tested as a model for
the scFv antibodies.
Plasmid DNA was extracted from E. coli TG1 cells containing plasmids encoding
the anti-flagella and anti-BSA scFv antibodies. Restriction digestion was done with
HindIII and NotI to obtain the fragment containing the ribosome binding site, the pelB
leader sequence, and the entire scFv gene (Figure 2-1). The 5' overhangs were filled in
with Klenow fragment of DNA polymerase I.
The digestion mixture was resolved on a 0.7% (w/v) agarose gel, and the fragment
corresponding to HindIII/NotI band was excised. The DNA was extracted from the
agarose gel using GenElute Minus EtBr Spin columns (Sigma). Plasmid vector pAC5
was digested with Smal to linearize the plasmid, and the HindIIllNotI or Ncol/NotI
fragment was blunt end ligated into the vector.
With anti-BSA scFv, another plasmid was constructed without the pelB leader
sequence by digesting the plasmid with NcoI and NotI to obtain only the scFv gene.
After digestion with NotI, the 5' overhang was filled in with Klenow fragment of DNA
polymerase I and then digested with NcoI, which cuts at the 5' end of the scFv gene. The
digestion mixture was resolved on a 0.7% (w/v) agarose gel, and the fragment
corresponding to Ncol/NotI band was excised. pAC5 Avitag vector was digested with
Ncol and SmaI, the gel-purified fragment was ligated into the vector.
HindIII Sfil/NcoI XhoI Sal NotI Amber
promote RR peB VH VL c-myc tag gill
Figure 2-1: Vector map of pIT2 phagemid vector pIT2. The vector map of pIT2
phagemid vector used in constructing the Tomlinson libraries. RBS-Ribosome binding
site. pelB-leader peptide sequence. VH and VL-genes coding for heavy and light chains,
respectively. Linker- (Gly4Ser)3 linker sequence. Amber stop codon is at the junction of
c-myc tag and gIII gene. Not shown in the figure are E. coli ColEl ori, M13 ori, and bla.
Escherichia coli AVB 100 cells were electroporated with the ligation mixture and
plated on LBN-Amp plates for selecting the transformed clones. Plasmid DNA was
extracted from randomly selected clones and checked for the presence of the insert.
Protein and LPS Manipulations
Extraction of Flagella
Flagella were extracted from S. Typhimurium and used for coating immunotubes
(34). Briefly, 10 mL of standing overnight culture was added to 1 L of LBN and grown
with shaking for 4 h. The cells were pelleted by centrifuging at 6,000 x g for 10 min at
4 C. The supernatant was discarded, and the pellet was suspended in 100 mL of 0.5 M
Tris-HC1, pH 7.5. The suspension was homogenized in an Osterizer blender at maximum
speed for 60 sec to mechanically shear off the flagella. The cells were pelleted by
centrifuging at 6,000 x g for 10 min at 4 oC and were discarded. The supernatant was
centrifuged at 100,000 x g for 90 min at 4 oC to pellet the flagella. The pellet was
suspended in 5 mL of 0.5 M Tris-HC1, pH 7.5, and the protein concentration was
determined by the Bradford method.
Determination of Protein Concentration
Protein concentration was determined by the Bradford method using the Dc Protein
Assay reagent (Bio-Rad, Hercules, CA) according to manufacturer instructions.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
ReadyGel Tris-glycine 10% (w/v) PAGE gels (Bio-Rad) were used with the
MiniProtean Electrophoresis system (Bio-Rad) for SDS-PAGE of protein samples. The
samples were diluted 1:2 in Laemmli sample buffer (Bio-Rad), and dilutions were made
as necessary. When whole bacterial cells were used as antigens, 10s CFU were
suspended in Laemmli sample buffer and boiled for 10 min. Electrode buffer was 25 mM
Tris, 0.17 M glycine, and 0.1% (w/v) SDS. The samples were electrophorosed for 1 hour
at 100 V. The gel was stained with Coomassie Blue for visualizing proteins.
Alternatively, some gels were used for immunoblotting.
Coomassie Blue Staining for Proteins
Proteins in SDS-PAGE were fixed in 50% (v/v) methanol, 10% (v/v) glacial acetic
acid in water for 30 min. The gel was stained with 0.1% (w/v) Coomassie Blue R,
50% (v/v) methanol, 7% (v/v) glacial acetic acid) and agitated gently for 30 min. The gel
was destined with 5% (v/v) methanol, 7% (v/v) glacial acetic acid in water. When the
protein bands appeared the gel was dried, and a photograph was taken (58).
The antigens in the SDS-PAGE gels were transferred onto a nitrocellulose
membrane for reaction with monoclonal antibodies, phage, or scFv antibodies according
to the procedure of Towbin et al (59). A Mini TransBlotting cell (Bio-Rad) was used for
the wet blotting technique. The transfer buffer used was 25 mM Tris, 192 mM glycine,
and 20% (v/v) methanol. A voltage of 100 V was applied for 1 hour at room
temperature. The nonspecific binding sites on the membrane were blocked with casein
blocking buffer. Primary antibody diluted in casein blocking buffer was added and
incubated for 2 h with continuous rotation. The membrane was washed with 100 mL Tris
buffered saline with 0.1% (w/v) Tween 20 with gentle agitation. The membrane was
incubated with secondary antibody diluted in blocking buffer for 2 h with continuous
rotation followed by another three washes as described above. The development of the
reaction was done by placing the membrane in 100 mL of substrate solution and
observing the appearance of bands. The substrate used for secondary antibodies
conjugated with horseradish peroxidase was 4-chloro-1-naphthol (4CN) (Fisher) prepared
according to manufacturer instructions. For alkaline phosphatase the substrate used was
nitro blue tetrazolium-5-bromo-4-chloro-3-indoylphosphate (Sigma) prepared according
to manufacturer instructions. The development reaction was stopped by removing the
membrane from the substrate and placing in water. The membrane was scanned using an
UMax photo scanner (60).
Concentration of scFv Antibodies from Culture Supernatant
scFv antibodies produced from the Tomlinson libraries have protein A and
protein L binding sites. scFv antibodies secreted into the culture medium were
concentrated by passing the culture supernatant over a protein A-Sepharose column (61).
The binding buffer was 0.02 M sodium phosphate, pH 7.0. For elution 0.1 M citric acid,
pH 3 was used, and the eluate was neutralized with 1 M Tris-HC1, pH 9.0. The column
was first washed with three column volumes of binding buffer, and the sample was
passed through the column. The column was washed with ten column volumes of
binding buffer, and the bound protein was eluted with three column volumes of elution
buffer. The eluate was neutralized with 60 [L of 1 M Tris-HC1, pH 9.0.
Extraction of Soluble Proteins from Periplasmic Space
scFv protein present in the periplasmic compartment ofE. coli was extracted by the
osmotic shock method (62). Briefly, exponentially growing E. coli x1918 cells
containing the phagemids were harvested by centrifugation at 6,000 x g for 10 min at
4 C. The cells were washed with 40 volumes of cold 10 mM Tris-HC1, 30 mM NaC1,
pH 7.1. The pellet was suspended in 40 mL of 33 mM Tris-HC1, pH 7.1. The cells were
diluted in 40 mL of 40% (w/v) sucrose in 33 mM Tris-HC1, pH 7.1, and 0.1 M disodium
EDTA, pH 7.1 was added to a final concentration of 0.1 mM EDTA. The suspension was
shaken in a rotary shaker for 10 min at 24 C. The cells were centrifuged at 13,000 x g
for 10 min at 4 C. The bacterial pellet was then dispersed in 5 mL of ice-cold water.
After incubation for 10 min in an ice bath, the cells were centrifuged at 6,000 x g for
10 min. The supernatant containing the scFv antibodies was saved.
Rationale for Study
In a world facing increasing threats of bioterrorism, rapid, sensitive, and specific
detection of microbes and toxins would enable the law enforcement agencies to react to
the situation effectively and appropriately. Such a detection system would also greatly
aid in solving problems of public health importance. A biosensor system consisting of an
optical fiber probe with an attached capture antibody and a fluorescently labeled antibody
for detection was developed as a step in that direction (26). This thesis describes the
efforts made to characterize existing monoclonal antibodies and to isolate new
recombinant phage display antibodies to S. Typhimurium to be ultimately used in the
biosensor system as capture antibodies and detection antibodies for detecting
The specific aims for this study were:
1. Standardization of a protocol for whole bacterial cell ELISAs
2. Characterization of commercially available monoclonal antibodies to
3. Isolation of recombinant phage display antibodies to S. Typhimurium and
genetically fusing scFv antibodies with ligands or reporter molecules
Specific Aim 1: Standardization of a Protocol for Whole Bacterial Cell ELISAs
A protocol for ELISA using whole bacterial cells as antigens was developed and
standardized for characterizing the monoclonal antibodies. The conditions that were
taken into consideration for standardizing the protocol included antigen concentration,
coating buffer, blocking buffer, and reaction development time. Each condition was
tested by coating the antigen in triplicate. The mean absorbance values were calculated,
and the signal to noise ratios were calculated by dividing the mean value of the
positive-antigen wells with the mean of the negative-antigen wells. The optimal
conditions from these experiments were adapted for characterizing monoclonal
antibodies, recombinant phage display antibodies, and scFv antibodies.
Reaction Development Time
Rather than chemically stopping the development reaction at some arbitrary time,
we chose to let the development reaction proceed and take multiple reads so that we
could use data from the optimal stage of development. The ELx 800 UV microplate
reader used for reading the absorbance values in ELISAs takes approximately 1 min for
reading all of the 96 wells, so if the reaction time is short enough, the time required to
read the plate could significantly add to the reaction time for wells read last. To study the
effect of duration of development of the reaction on the signal obtained, bacteria were
coated at same concentration in the first three wells of the first row and the last three
wells of the last row, and the ELISA was done. Reading the absorbance values 5 to
10 min after starting the reaction resulted in an error because the values obtained in the
wells in the last row were higher than the wells in the first row as the reaction was
proceeding in the last well while the plate was being read. However, if the reaction was
allowed to continue for 40 min there was no difference in the absorbances for the first
and last wells. Therefore, the absorbances values in all the subsequent ELISAs were read
after 40 min.
Antigen Concentration for Coating
The concentration and amount of the antigen coated in the wells determine the
signal obtained and the background activity. If the concentration of the antigen is too
high, antibodies might not bind to the antigen because of steric hindrance and also might
give false positive values because of nonspecific binding of the antibodies to the antigen
(63). The concentration of the antigen needs to be adjusted to maximize the signal and
minimize the background activity.
Five-fold serial dilutions of the S. Typhimurium cells from 1.0 x 109 CFU/mL to
6.4 x 104 CFU/mL were used for coating wells. Wells coated with either E. coli LE392
or coating buffer alone were used as negative antigen controls. Rabbit
anti-S. Typhimurium serum at a dilution of 1:1000 was used as the primary antibody, and
donkey anti-rabbit IgG-HRP conjugate at a dilution of 1:2000 was used as the secondary
antibody. The development of color was monitored for 40 min, and the absorbance was
read at 630 nm. The peak absorbance value was around 1.72 and stayed the same for
antigen concentrations ranging from 1.0 x 109 CFU/mL to 8.0 x 106 CFU/mL
(Figure 3-1). Further decreasing the antigen concentration resulted in a significant
decrease in the peak signal obtained. The development of the reaction was gradual and
linear with respect to time at the antigen concentration of 8.0 x 106 CFU/mL, as opposed
to higher antigen concentrations for which the peak signal was reached more rapidly. In
all the subsequent experiments for characterizing the monoclonal antibodies, phage
antibodies, or scFv antibodies bacteria were coated at a concentration of
1.0 x 107 CFU/mL.
To enhance the binding of bacterial cells to the microtiter plate, glutaraldehyde was
added to the antigen suspension to a final concentration of 0.05% (w/v) (57). In an
2.5 -- 1.0 x 109 CFU/mL
2.0 x 10 CFU/mL
'- 4.0 x 10 CFU/mL
1.5 -0 8.0 x 106 CFU/mL
C _-- 1.6 x 106 CFU/mL
S- 3.2 x 105 CFU/mL
-+- 6.4 x 104 CFU/mL
o -- Blank
5 10 15 20 25 30 35 40
Figure 3-1: Effect of varying antigen concentration on signal. S. Typhimurium cells were
serially diluted five-fold from 1.0 x 109 CFU/mL to 6.4 x 104 CFU/mL and were coated
in triplicate onto a microtiter plate. ELISA was performed with a 1:1000 dilution of
rabbit anti-S. Typhimurium serum and a 1:2000 dilution of HRP-conjugated donkey
anti-rabbit IgG antibody as primary and secondary antibodies, respectively. Data are
shown as mean and standard deviation.
ELISA done to study the effect of glutaraldehyde on increasing the signal using
S. Typhimurium whole cells as antigen, goat anti-S. Typhimurium serum as primary
antibody, and anti-goat IgG conjugated with alkaline phosphatase as secondary enzyme,
the absorbance value obtained in wells containing glutaraldehyde was 2.67 while the
signal in the wells not containing glutaraldehyde was 2.72, and the difference was not
statistically significant. The addition of glutaraldehyde did not significantly enhance the
signal obtained. However, we continued adding glutaraldehyde to the antigen
The effect of coating buffer on the signal obtained was tested by suspending
S. Typhimurium in either carbonate buffer or PBS. S. Typhimurium cells were
suspended in either of the buffers at a concentration of 107 CFU/mL, and ELISA was
done using USB10 as primary antibody. The signal obtained in wells coated with PBS as
the coating buffer was 1.11, while in the wells with carbonate buffer as the coating buffer
the signal was 1.14, showing no statistically significant difference. Carbonate buffer was
used for suspending the bacteria in all the subsequent experiments.
Comparing the Blocking Efficiencies of Casein and BSA
A common problem in immunological analysis is the nonspecific binding of
antibodies to the matrix on which the antigen is bound, such as the wells of a microtiter
plate or a nitrocellulose membrane. To overcome this problem all of the sites not coated
by the antigen are made inaccessible to antibodies by blocking with agents such as BSA,
casein, Tween 20, dextran sulfate, and skim milk (64).
To determine the blocking efficiencies of casein and BSA, 1% (w/v) casein in PBS,
1% (w/v) BSA in PBS, or a mixture of 0.5% (w/v) casein and 0.5% (w/v) BSA in PBS
was used as blocking buffer in a whole-cell ELISA. All of the blocking buffers had
0.1% (w/v) Tween 20. The wells of a microtiter plate were coated with S. Typhimurium
cells at a concentration of 107 CFU/mL. Goat anti-S. Typhimurium serum at 1:1000
dilution was used as primary antibody, and secondary antibody was donkey anti-goat IgG
conjugated with alkaline phosphatase used at 1:2000 dilution. Both primary and
secondary antibodies were diluted in the blocking buffer. Wells coated only with coating
buffer were used as negative antigen controls. There were no significant differences in
the signals obtained in each of the cases, the values ranged from 3.35 0.10 to
3.55 0.07; however, the background activity in the negative-antigen wells was half as
much in wells blocked with casein when compared wells blocked with BSA (Table 3-1).
Casein mixed with BSA gave background activity similar to that for casein alone.
Therefore, for all subsequent experiments 1% (w/v) casein with 0.1% (w/v) Tween 20
was used as the blocking buffer.
Table 3-1: Comparing the blocking efficiencies of casein and BSA
Blocking buffer Salmonella Blank Signal/Noise
1% (w/v) BSA 3.42 + 0.06 0.34 0.05 10
0.5% (w/v) BSA + 0.5% (w/v) casein 3.55 0.07 0.16 0.04 23
1% (w/v) casein 3.35 0.10 0.13 0.05 26
The wells of a microtiter plated were coated in triplicate either with 107 CFU/mL of
S. Typhimurium (Salmonella) or with coating buffer (Blank), and ELISA was
performed with goat anti-S. Typhimurium serum (1:1000 dilution) and alkaline
phosphatase conjugated donkey anti-goat IgG (1:2000 dilution) as primary and
secondary antibodies, respectively. The antibodies were diluted in the same buffer
that was used as blocking buffer. The data are presented as mean with standard
deviation. Signal/Noise value was calculated by dividing the signal obtained in
antigen-positive wells with the signal obtained in antigen-negative wells. The
absorbance values were not significantly different, but the blank values were
significantly different with p < 0.008.
In some of the initial experiments to determine the appropriate concentration of
bacteria for coating the wells, the secondary antibodies used were conjugated to alkaline
phosphatase. For all of the subsequent experiments to characterize the monoclonal
antibodies, phage antibodies, and scFv antibodies, the secondary antibodies were
conjugated to horseradish peroxidase.
Specific Aim 2: Characterization of Commercially Available Monoclonal Antibodies
to S. Typhimurium
Murine monoclonal antibodies recognizing various surface epitopes of
S. Typhimurium including LPS and flagella and monoclonal antibodies of undetermined
specificity were tested by ELISA to determine their potential usefulness in the biosensor
system as capture antibodies and detection antibodies (Table 2-2). Antibodies
recognizing surface epitopes were chosen to facilitate detection of whole bacterial cells
using the biosensor system and to obviate the need for preparing cell extracts to identify
internal antigens. The monoclonal antibodies were characterized by an ELISA because,
in addition to closely resembling the conditions of the biosensor system, ELISA is a
simple, rapid, and economical alternative to other methods such as Western blotting (64).
The antibodies were screened by using the standardized conditions determined above.
An absorbance value of greater than 1.0 unit was considered satisfactory.
The monoclonal antibodies recognizing LPS or other undetermined surface
epitopes were tested by ELISA using whole cells and purified LPS of S. Typhimurium as
antigens. LPS was suspended in either PBS or coating buffer at a concentration of
5 [tg/mL, and 100 [iL per well was used for coating the wells of the microtiter plate. A
concentration of 5 tg/mL of LPS gave a signal comparable to the signal obtained when
107 CFU/mL of whole cells were coated. Of all the monoclonal antibodies screened,
US BiologicalslO (USB10) monoclonal antibody to LPS of S. Typhimurium performed
far superiorly in a whole-cell ELISA, giving absorbance values higher than 1.5
(Table 3-2). USB10 monoclonal antibody gave a similar signal with purified LPS of
S. Typhimurium in a LPS ELISA compared with a whole-cell ELISA. USB10
monoclonal antibody was tested by an ELISA at varying concentrations and performed
well at concentrations as low as 0.01 [tg/mL giving a signal of 0.44 units (Table 3-3).
Biospacific53 (Biosp53) monoclonal antibody to LPS performed best among the
remaining antibodies and gave a signal higher than 1.3 absorbance units at concentrations
of 0.1 tg/mL. Of the remaining antibodies, RDI and Virostat6341 worked moderately
well at a concentration of 10 tg/mL giving a signal of 0.7 absorbance units. All of the
remaining antibodies to the LPS of S. Typhimurium or other surface antigens gave
signals less than 0.6 absorbance units and were considered unsuitable for further study.
The anti-flagellar antibodies were tested by ELISA using either whole bacteria or
flagellar extracts of S. Typhimurium. Flagella were suspended in PBS at a concentration
of 10 [tg/mL, and 100 tiL was used for coating the wells of a microtiter plate. Of the
anti-flagellar monoclonal antibodies, Virostat6321 did not give a signal higher than 0.2
units either with whole cells or with flagellar extracts. AccurateYVS6301 reacted well
with flagellar extract giving a signal of 1.0 units, but gave signal less than 0.2 absorbance
units with whole cells (Table 3-4). Since it was possible that an inadequate amount of the
Table 3-2: Comparing the Activities of Various Monoclonal Antibodies
Monoclonal Antigen Signal /
Antibody Specificity Salmonella Blank Noise
US BiologicalslO 04 1.19 + 0.05 0.08 15
US Biologicalsl5 04 0.17 + 0.02 0.08 2
Biospacific53 LPS 1.07 0.02 0.11 10
Biospacific58 04 0.16 + 0.04 0.08 2
RDI 012 0.47 + 0.09 0.09 5
Virostat6301 Flagellum 0.25 0.02 0.08 3
Virostat6321 Unknown 0.18 0.07 0.13 1
Virostat6341 012 0.70 + 0.06 0.09 8
Biodesign 04 0.18 0.08 0.15 1
AccurateYVS6301 Flagellum 0.12 0.01 0.09 1
The wells of a microtiter plated were coated in triplicate either with 107 CFU/mL of
S. Typhimurium (Salmonella) or with coating buffer (Blank), and ELISA was
performed with each of the monoclonal antibodies as the primary antibodies.
US BiologicalslO and Biospacific53 were at 1 [tg/mL, and all of the remaining
antibodies were at 10 tg/mL. Goat anti-mouse IgG antibody conjugated with HRP was
used as secondary antibody at a dilution of 1:2000. The data are the mean and standard
deviation of the absorbance values at 630 nm and signal / noise values.
antigen was responsible for the weak signal, bacteria were coated at 109 CFU/mL, one
hundred-fold higher than used in a typical assay, but no higher signal was obtained.
To determine the specificity ofUSB10 and Biosp53 monoclonal antibodies,
whole-cell ELISA was done using Salmonella belonging to various serogroups as
Table 3-3: Potency of USB10 and Biosp53 Anti-LPS Monoclonal Antibodies
Concentration of antibody
1.37 + 0.08
1.11 + 0.08
0.44 + 0.08
1.35 + 0.05
1.09 + 0.02
0.35 + 0.03
S. Typhimurium cells at a concentration of 107 CFU/mL were used as
antigen, coated in triplicate. USB10 and Biosp53 were diluted in casein
blocking buffer at the indicated concentrations and used as primary antibody
for the ELISA. The data presented are mean and standard deviation for
absorbance values at 630 nm 40 min after the addition of the substrate.
Table 3-4: Activity of anti-flagellar monoclonal antibodies
1.05 + 0.03
0.20 + 0.01
0.18 + 0.01
ELISA was done using whole S. Typhimurium cells (Salmonella) at a concentration of
107 CFU/mL or flagellar extract (Flagella) at a concentration of 10 [g/mL as antigen.
Accurate YVS6301 and Virostat6321 anti-flagellar monoclonal antibodies were used as
primary antibody at a concentration of 10 [g/mL. The data are presented as the mean
values of the absorbances with one standard deviation. The differences are statistically
significant with p < 0.02.
antigens. The signal obtained with non-group B salmonellae was approximately 0.2
units, while for group B salmonellae it was 0.7 to 1.2 absorbance units (Table 3-5). With
S. Worthington, which belongs to group G, the signal was approximately 0.4 units.
USB10 and Biosp53 were determined to be suitable antibodies for further study in
the biosensor system, as they were giving a satisfactory signal at concentrations as low as
0.1 [g/mL by a whole-cell ELISA and were specific in recognizing S. Typhimurium.
Table 3-5: Specificity of USB10 and Biosp53 anti-LPS monoclonal antibodies
Strain Serogroup USB10 Biosp53
S. Typhimurium x3000 B 1.1 + 0.01 1.2 + 0.04
S. Typhimurium 13311 B 0.6 + 0.01 0.7 0.05
S. Cholerasuis 3246 C 0.1 0.01 0.1 0.01
S. Enteritidis 14213 D 0.1 0.01 0.1 0.01
S. Rubislaw 10717 F 0.1 0.01 0.1 0.02
S. Worthington G 0.4 0.03 0.4 0.04
S. Gaminara H0662 I 0.2 0.02 0.1 0.01
S. Urbana 9261 N 0.1 0.02 0.1 0.01
S. Adelaide 0 0.2 + 0.01 0.1 + 0.01
Salmonellae belonging to various serogroups as indicated were used as antigen in a
whole-cell ELISA at a concentration of 107 CFU/mL. USB10 and Biosp53 were used as
primary antibodies at a concentration of 1 [g/mL for the ELISA. The data are presented
as mean values of the absorbances at 630 nm after 40 min after the addition of the
Specific Aim 3: Isolation of Recombinant Phage Display Antibodies to
S. Typhimurium and Genetically Fusing scFv Antibodies with Ligands or Reporter
Recombinant phage display antibodies to antigens were isolated from a naive
combinatorial library by panning the library on the antigen either in suspension or
immobilized on an immunotube. For isolating phage bearing antibody to surface
epitopes of S. Typhimurium, panning was done with phage from the Griffin. 1 or the
Tomlinson I Human Synthetic VH + VL libraries. The phage from the Tomlinson I or
Griffin. 1 libraries or phage obtained from a previous round of amplification were used for
panning on whole cells, LPS, or flagellar extracts. The phage antibodies bound to the
antigen were eluted either with 0.1 M glycine-HC1, pH 2.8 if the phage were from
Griffin. 1 library or with trypsin if the phage were from Tomlinson I library. The phage
antibodies were eluted and amplified in a suppressor strain ofE. coli to increase the
proportion of phage displaying specific antibody sequence over the nonspecific phage.
Phage were produced by superinfection with either helper phage or Hyperphage. The
cycle of panning and amplification was done three or more times to enrich for the desired
phage. The culture supernatant was used for immunological analysis or was used for
concentrating phage by PEG precipitation. The amplified phage thus obtained were
tested by an ELISA either with the phage produced from all the pooled clones ofE. coli
polyclonall phage ELISA) or with the phage produced from each individual clone
(monoclonal phage ELISA).
Preliminary Experiments for Optimizing Production and Analysis of Phage
The Griffin. 1 and Tomlinson libraries are in a phagemid format. The plasmid has
both plasmid ColEl ori and phage M13 ori sequences. The phagemid is deficient in all
of the genes necessary for phage replication and packaging, except the scFv-gIII coding
for the scFv-pIII fusion protein. For this phagemid to be propagated and viable phage
particles to be produced, E. coli are superinfected with helper phage M13K07, which
provides all of the necessary proteins required for phage assembly. However, most of the
phage particles produced have wild type pIII rather than the recombinant protein, thereby
greatly decreasing the reactivity of the phage population (65,66).
Hyperphage is derived from M13K07 by deleting most of the gIII from its genome
(65). Superinfection of E. coli with Hyperphage forces the display of the scFv
recombinant protein pill on the phage particles, as that is the only available source of
pill. Although this greatly decreases the phage output, most of the phage produced have
the recombinant protein leading to a greatly increased activity.
To test the efficiency of Hyperphage over helper phage M13K07 in improving the
potency of the phage antibodies, phage were produced from anti-human thyroglobulin
clones, which were included with the Griffin. 1 library as a positive control phage, using
either Hyperphage or helper phage. The amount of phage obtained with Hyperphage was
about two orders of magnitude lower than that obtained with helper phage. The phage
were tested by ELISA after normalizing for the titer. The phage derived from
superinfection with Hyperphage showed higher activity than those produced with helper
phage (Table 3-6). When phage were used at a concentration of 109 phage/mL, phage
produced with helper phage gave a signal of 1.5 units, while phage produced with
Hyperphage gave a signal of 1.7 units. However, when phage were used at a
concentration of 107 phage/mL the signal obtained with phage produced from
Hyperphage was 0.9, twice that of the phage produced with helper phage.
The activity of the phage was primarily measured by ELISA, and the phage giving a
signal of greater than one absorbance unit in ELISA were tested in Western blots.
Anti-human thyroglobulin phage obtained with the Griffin. 1 library and anti-BSA phage
obtained with the Tomlinson libraries were used as positive controls, using thyroglobulin
or BSA as antigens, respectively. To determine the concentration of phage particles to be
used in ELISA, an experiment was done using human thyroglobulin as antigen and
anti-thyroglobulin phage as primary antibody. Human thyroglobulin (Sigma) at a
concentration of 10 [g/mL in PBS was used for coating. Anti-thyroglobulin phage were
produced by superinfecting anti-thyroglobulin phagemid-bearing E. coli TG1 with helper
phage M13K07. Phage were suspended in casein blocking buffer at serial ten-fold
concentrations from 1011 phage/mL to 106 phage/mL. A concentration of 109 phage/mL
gave a good signal of approximately 1.5 absorbance units. In all the subsequent
experiments in which PEG precipitated phage were used as primary antibody, a
concentration of 109 phage/mL was used.
Table 3-6: Comparing the Activity of Phage Produced Using Hyperphage or helper
phage M13K07 by ELISA
Signal obtained with phage produced
Concentration of phage used as primary
antibody (phage/mL) Hyperphage M13K07
1.0 x 109 1.7 0.02 1.5 0.03
1.0 x 108 1.3 + 0.04 0.9 + 0.04
1.0 x 107 0.9 + 0.03 0.4 + 0.05
Anti-thyroglobulin phage were produced in E. coli TG1 by superinfection with either
Hyperphage or M13K07. Human thyroglobulin at a concentration of 10 [g/mL in PBS
was used as antigen. The phage were normalized for the titer, and serial ten-fold
dilutions from 1.0 x 109 phage/mL to 1.0 x 107 phage/mL were used as primary
antibody. HRP-conjugated anti-M13 antibody at a dilution of 1:2000 was used as
secondary antibody. The data presented are mean and standard deviation. The
differences are statistically significant with p < 0.0001.
Isolation of Phage Antibodies Recognizing S. Typhimurium
The first phage library we had available was the Griffin. 1, the original phage
display library. The phage obtained from the Griffin. 1 library were used for panning for
three rounds, either with or without amplification of phage in between each round, on
whole bacterial cells or LPS. For whole cells, panning was done with the antigen either
suspended in casein blocking buffer or coated on immunotubes in coating buffer. LPS
was coated on immunotubes in either coating buffer or PBS. The phage obtained after
each round of panning and amplification were tested by a polyclonal phage ELISA using
whole cells or LPS as antigen. Wells coated with coating buffer alone were used as
negative antigen controls. The proportion of phage showing nonspecific binding or
phage binding to plastic increased with each round. There was no increase in specific
activity with each round. This occurred consistently, irrespective of the antigen (whole
cells or LPS) or the type of panning method (panning in suspension or panning on
immunotubes). We tried panning on both of the antigens on immunotubes without
amplifying the eluted phage in between each panning round. However, when the eluted
phage at the end of third round were tested in a monoclonal phage ELISA, there was no
useful signal either with whole cells or LPS. We also tried coating the bacteria in the
presence and absence of glutaraldehyde in the antigen suspension. We used 3% (w/v)
BSA instead of casein to suspend the phage particles in panning to rule out the potential
detrimental effects of Tween on the binding of LPS. However, we did not obtain any
useful phage antibodies. In all, 10 attempts were made using the phage from Griffin. 1
We then obtained the next generation of phage the Tomlinson I and J libraries.
These phage have several advantages over the Griffin. 1 library detailed in the
Introduction and Materials and Methods. Phage from the Tomlinson I library were
panned on whole S. Typhimurium cells, LPS, or flagellar extracts. When the library was
panned on whole cells either in suspension or on immunotubes, with amplification in
between rounds, the number of eluted phage increased with each round, suggesting
enrichment of specific phage. The phage obtained after each round were tested by a
polyclonal phage ELISA using whole cells and LPS as antigens, and wells coated with
Vibrio cholerae cells or with coating buffer were used as negative-antigen controls. The
background activity in the wells coated only with coating buffer did not increase with
each round. The reactivity against purified LPS was very low and was only slightly
higher than the wells not coated with any antigen (Figure 3-2). There was a gradual
increase in reactivity against S. Typhimurium with each round, but there was higher
activity against V cholerae cells. Assuming cross reactivity of phage antibodies with the
surface antigens of V. cholerae, we incubated the cross-reactive phage with excess of
V. cholerae cells to absorb the cross-reactive phage antibodies. Potentially cross-reactive
phage should have been removed by pelleting V. cholerae cells by centrifugation.
C 1.00 Salmonella
1 2 3
Rounds of panning
Figure 3-2: Activity of phage obtained after each round of panning on S. Typhimurium
cells in suspension. Phage from the Tomlinson I library were used for panning on whole
S. Typhimurium cells in suspension. Three rounds of panning were done with
amplification of eluted phage in between rounds. Phage from rounds 1, 2, and 3 were
tested for their activity against S. Typhimurium (Salmonella) cells, V. cholerae (Vibrio)
cells, purified LPS of S. Typhimurium (LPS), and no-antigen (Blank) control wells in a
polyclonal phage ELISA. The data presented are the means and standard deviations of
the absorbance values at 630 nm 40 min after addition of the substrate.
Unexpectedly, there was no increased activity against S. Typhimurium or a decrease in
binding to V cholerae when the phage remaining in the supernatant were tested by an
ELISA. To isolate clones specific to S. Typhimurium from the phage pool showing
reactivity towards both S. Typhimurium and V cholerae, phage were absorbed on
S. Typhimurium, and the specific phage antibodies were eluted with trypsin. The eluted
phage were used to infect E. coli TG1 and plated on 2xTY AG plates. Each colony was
screened individually by whole-cell ELISA to see if the same colony had activity against
V cholerae and S. Typhimurium or if there were two populations of phage with separate
activities. However, the activity against V cholerae remained higher than for
S. Typhimurium for all of the clones screened. None of the phage obtained by panning
on S. Typhimurium reacted with purified LPS, although LPS is the predominant antigen
on the cell surface.
As an alternative to panning on whole cells, purified LPS of S. Typhimurium
suspended in either PBS or carbonate buffer was used as an antigen for coating on
immunotubes. Successive rounds of panning were done either with or without
amplification in between the panning rounds. When panning was done in succession
without amplification, LPS was coated at progressively decreasing concentrations for
each round, starting from 10 [g/mL to 0.1 [g/mL, to isolate phage with higher affinities
Phage obtained after three rounds of panning with amplification in between rounds
of panning were tested in polyclonal phage ELISA. The phage pool did not show any
specific activity against either purified LPS or whole bacterial cells. The phage obtained
after three rounds of panning without amplification were screened in a monoclonal phage
ELISA. Of the 120 clones examined, some clones showed moderately high activity
against purified LPS giving a signal of 1.1 units, but with whole cells the highest signal
obtained was in the range of 0.4 units. Some clones showed equally high reactivity
giving signal in the range of 0.5 units with both S. Typhimurium and V. cholerae, which
was used as a negative-antigen control. The phage obtained from the clones showing
reactivity with both S. Typhimurium and V. cholerae were used as primary antibody in a
Western blot using S. Typhimurium and V. cholerae whole cells as antigens. The phage
did not react with proteins or LPS of either S. Typhimurium or V. cholerae. Therefore,
we were unable to determine the specificity and nature of these dual-reactive phage.
Study of these clones was not pursued further.
As an alternative to LPS, flagellar extract was used as the antigen for panning.
Isolating phage antibodies to proteins is relatively easier than for carbohydrate antigens,
because the affinity of the anti-carbohydrate antibodies is generally a 1000-fold lower
than the affinities of the antibodies that can be isolated by panning (68,69). The flagella
of S. Typhimurium were extracted (62) and analyzed by SDS-PAGE. A single protein
band of approximately 58 kD was observed, which corresponded to the molecular weight
of flagellin (34,70). The flagellar extract was tested by ELISA with Accurate YVS6301
anti-flagellar and USB10 anti-LPS antibodies. Both of the antibodies gave a signal of
approximately 1.2, indicating the presence of LPS also. Flagellar extract was coated on
immunotubes at a concentration of 10 [g/mL in PBS. Three pannings were done with
phage from the Tomlinson I library without amplification in between rounds of panning.
As explained in the Materials and Methods, when trypsin was used for elution, the eluted
phage could not be used for panning without amplification, as the scFv antibody
sequence is lost. Therefore, when panning without amplification between rounds of
panning was done with phage from the Tomlinson library, elution was done with 0.1 M
glycine, pH 2.8 in the first two rounds, and in the third round 1 tg/mL trypsin was used
for elution. Fifty-nine colonies were obtained after three rounds of panning. Phage were
produced from each colony separately and were screened by monoclonal phage ELISA
using flagellar extracts and whole bacterial cells as antigens. Thirteen clones showed
very good reactivity giving a signal higher than 1.6 units with flagellar extract and poor
activity against whole cells (Table 3-7). As detailed below, it turned out that 11 of the 13
were multiple isolates of the same clone and the remaining two were isolates of another
To determine how many of the thirteen clones were clonal representing multiple
copies of the same phage, restriction digestion of the plasmids extracted from E. coli
infected with each of these phages was done. The four base pair recognizing restriction
enzymes BstN1, HaeIII, AluI, or Sau3A were used to cut the plasmids to produce a
restriction pattern for each separate clone. The restriction patterns were not conclusive
because of the small size of the insert compared to the whole plasmid and the relatively
low probability of a novel restriction site being present in the variable portion of the
scFv-encoding sequences. All of the thirteen clones and even the anti-BSA clone gave an
apparently similar pattern. These plasmids were sequenced with primers Fdseql,
hybridizing upstream of the scFv gene, and Lmb3, binding to DNA in the 5' region of
gIll. Analysis of the DNA sequences with GCG software package showed that there
were only two different clones among the thirteen. The distinct phage clones were
named SF1 and SF2. As expected, the anti-BSA phage had a unique DNA sequence.
Table 3-7: Activity of phage obtained after panning on flagellar extracts
Clone number Salmonella Flagella
2 0.38 + 0.02 1.98 + 0.09
4 0.45 + 0.03 1.98 + 0.07
6 0.39 + 0.04 1.98 + 0.03
9 0.56 + 0.03 1.96 + 0.03
11 0.30 + 0.08 1.94 + 0.05
12 0.30 + 0.04 1.95 + 0.03
17 0.35 0.02 1.97 0.06
29 0.4 0.03 1.7 0.04
44 0.27 + 0.01 1.83 + 0.08
45 0.39 + 0.02 1.79 + 0.10
48 0.29 + 0.03 1.83 + 0.05
54 0.28 + 0.04 2.04 + 0.09
55 0.31+ 0.10 2.01+ 0.11
59 colonies obtained after three rounds of panning on flagellar extracts of S. Typhimurium
were screened by a monoclonal phage ELISA using either 10 tg/mL flagellar extract
(Flagella) or whole S. Typhimurium cells (Salmonella) at 107 CFU/mL as antigen. The
data presented are the mean absorbance and standard deviation values of the positive
To examine the specificity of SF1 and SF2 anti-flagellar antibodies,
S. Typhimurium cells and flagellar extracts were resolved by SDS-PAGE, and the
antigens were transferred onto a nitrocellulose membrane. The blot was probed with SF1
or SF2 phage antibodies at concentration of 108 phage/mL or with Accurate YVS6301
anti-flagellar monoclonal antibody at a concentration of 10 tg/mL. HRP conjugated
anti-M13 or anti-mouse IgG antibodies at a dilution of 1:2000 were used as secondary
antibody, and 4-CN was used as the substrate. The monoclonal antibody failed to
recognize the flagellar protein, while both of the phage antibodies recognized the band in
both whole cell sample and flagellar extract (Figure 3-3).
A B C D MM AB C D
Figure 3-3: Western blot to determine the specificity of SF1 and SF2 anti-flagella phage
antibodies. Whole cells of two non-flagellated strains of Salmonella, S. Typhimurium
(fliC) (A) and S. Typhimurium (flhD) (B), S. Typhimurium (wild-type) (C), and flagellar
extract of S. Typhimurium (D) were resolved on SDS-PAGE and a Western blotting was
done using SF1 (1) or SF2 (2) anti-flagella phage as primary antibody. Molecular weight
marker (M). The position of the flagellar protein is shown by asterisk (*).
The specificities of SF1 and SF2 anti-flagellar phage antibodies were tested using two
non-flagellated strains of S. Typhimurium. Approximately 108 cells each of
S. Typhimurium X3000 (wild-type), S. Typhimurium (fliC::Tnl0),
S. Typhimurium (flhD), and 5 pg/mL of purified flagellar protein were resolved by
SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. The blot
was probed with SF1 or SF2 phage antibodies at concentration of 108 phage/mL or with
Accurate YVS6301 anti-flagellar monoclonal antibody at a concentration of 10 [g/mL as
primary antibody. HRP conjugated anti-M13 or anti-mouse IgG antibodies at a dilution
of 1:2000 were used as secondary antibody, and 4-CN was used as the substrate. The
monoclonal antibody failed to recognize the flagellar protein, while both of the phage
antibodies recognized the band in both whole cell sample and flagellar extract
Determining the Activity of scFv Antibodies vs. Phage Antibodies
scFv antibodies can be obtained as soluble antibody molecules instead of scFv-pIII
fusion proteins expressed in the context of intact phage. The genes for scFv and gIII are
separated by a TAG amber stop codon. When the phagemid is present in a
non-suppressor strain such as E. coli x1918, synthesis of the peptide chain is terminated
at the amber stop codon, and a soluble antibody molecule is released. The scFv gene has
apelB leader sequence at the 5' end of the gene enabling the secretion of scFv antibody
molecules into periplasm and culture supernatant. scFv antibodies can be harvested
directly from the culture supernatant or from periplasmic by osmotic shock (62). scFv
antibody molecules produced from phage obtained from the Tomlinson library have
protein A and protein L sites in addition to the hexahistidine tag and c-myc epitope
present in scFv antibodies obtained from the Griffin. 1 library. It should therefore be
possible to determine the activity of scFv antibodies by ELISA or Western blotting using
protein A conjugates or anti-c-myc conjugates.
scFv antibodies were produced by infecting E. coli x1918 with phage that gave a
good signal by monoclonal phage ELISA. To understand the properties of scFv
antibodies in general, anti-BSA scFv antibody, which was obtained as a positive control
with the Tomlinson library, was used. One hundred microliters ofBSA (10 tg /mL) in
PBS was used as antigen. Anti-BSA scFv antibody produced in E. coli x1918 and
harvested from the culture supernatant was diluted 1:2 in casein blocking buffer and used
as primary antibody. Protein A-HRP conjugate was used as secondary antibody at a
dilution of 1:2000. The signal obtained with the anti-BSA scFv antibody was 1.2 units,
comparable to the signal obtained with anti-BSA phage antibody. Anti-BSA scFv
antibodies recognized the band corresponding to BSA when used as primary antibody in
a Western blot with BSA as antigen (data not shown).
scFv antibodies were produced from SF1 and SF2 phage clones. The culture
supernatant containing the scFv antibodies were diluted 1:2 in casein blocking buffer and
used for immunological analysis. The activities of the scFv antibodies were tested by an
ELISA and a Western blot with whole bacterial cells and flagellar extracts as antigens.
Protein A-HRP conjugate was used at a dilution of 1:2000 as secondary antibody. There
was no signal with any of the anti-flagellar scFv antibodies against flagellar extract or
against whole cells in an ELISA. scFv antibodies also failed to recognize the flagellar
protein in a Western blot.
To determine if scFv antibodies were being produced and secreted into the culture
medium, ELISA was done using the culture supernatants having SF1, SF2, and anti-BSA
scFv antibodies as antigens for coating the wells. Protein A-HRP conjugate was used at a
dilution of 1:2000 as primary antibody. There was no signal with any of the scFv
antibodies. The same samples of culture supernatant were resolved by SDS-PAGE, and
the antigens were blotted onto a nitrocellulose membrane and probed with protein A-HRP
conjugate. There were no bands corresponding to any of the scFv antibodies. Fifteen
milliliters of culture supernatant containing anti-BSA scFv antibody was concentrated by
affinity chromatography with a protein A-Sepharose column and eluting with 0.1 M citric
acid, pH 3 (61). The eluted fraction was resolved by SDS-PAGE, and the gel was stained
with Coomassie Blue. Alternatively, the antigens in the gel were transferred onto a
nitrocellulose membrane, and the membrane was probed with protein A-HRP. No bands
were observed on either gel. The scFv antibodies might be present in concentrations
below the limits of detection by a Coomassie Blue staining or Western blotting.
Genetically Fusing scFv Antibodies to Biotin
Biotinylation of antibody molecules and detection with streptavidin conjugates is a
common strategy to amplify the signal obtained in immunological analysis (71-73).
Proteins such as scFv or monoclonal antibodies are most often chemically derivitaized
with biotin; however, chemical biotinylation has disadvantages of variability and possibly
inactivating the antibody. Therefore, we chose to take advantage of the ability of
genetically modifying scFv antibodies to enable enzymatically mediated and site-directed
Avitag is a 15-peptide sequence that is specifically recognized and biotinylated by
BirA enzyme of E. coli (Avidity). birA is stably integrated into the chromosome of
E. coli AVB 100. Avitag vectors pAC4, pAC5, and pAC6 have the sequence coding for
Avitag peptide at the 3' end of a multiple cloning site. The genes for SF1, SF2, and
anti-BSA scFv were subcloned as HindIIl/NotI fragment into the pAC5 vector, and
electroporated into E. coli AVB 100 cells. Production of scFv was induced with 1 mM
IPTG. birA was induced with 0.4% (w/v) L-arabinose after adding d-biotin to the culture
medium to a final concentration of 50 [M. To confirm the synthesis of the biotinylated
antibodies, approximately 108 cells of E. coli AVB 100 bearing the pAC5 plasmid with
the anti-BSA scFv, SF1, or SF2 genes were boiled and resolved by SDS-PAGE. The
antigens were transferred to a nitrocellulose membrane and probed with streptavidin-HRP
conjugate. All of the clones showed a band of 28 kD corresponding to biotinylated scFv
MABC DMEFGH I
Figure 3-4: Confirmation of synthesis of biotinylated scFv antibodies. Escherichia coli
AVB 100 cells containing the SF1, SF2, or anti-BSA scFv genes cloned into pAC5 Avitag
vectors or the culture supernatants were resolved on SDS-PAGE and the antigens were
transferred onto a nitrocellulose membrane. Streptavidin-HRP conjugate was used for
probing. Molecular weight marker (M), Biotinylated anti-BSA scFv positive control
(A,D), SF1-Avitag in whole cells (B) or culture supernatant (C), SF2-Avitag in whole
cells (F) or in culture supernatant (E), anti-BSA scFv-Avitag in whole cells (H) or culture
scFv antibodies were harvested from either culture supernatant or from periplasm.
scFv antibodies in the periplasmic space were obtained by cold osmotic shock method
(62). The culture supernatant and periplasmic extract were diluted 1:2 in casein blocking
buffer and used as primary antibody in an ELISA using BSA or flagella as antigens as
appropriate. Protein A-HRP conjugate was used as secondary antibody for unmodified
scFv antibodies, while streptavidin-HRP conjugate was used as secondary antibody for
biotinylated antibodies. With anti-BSA scFv antibodies in the culture supernatant, the
signal obtained with biotinylated molecules was lower than with unmodified antibodies,
the absorbance values being 1.42 and 1.08, respectively (Table 3-8). The periplasmic
extracts showed a slightly higher activity than the culture supernatant (Table 3-8). There
was no activity with the SF1 and SF2 scFv antibodies either in the unmodified or
biotinylated forms in either the culture supernatant or periplasmic extract. Biotinylation
of SF 1, SF2 and anti-B SA scFv antibodies did not have any benefit over the unmodified
antibodies. However, as seen with anti-BSA scFv, the genetic fusion of scFv to a
biotinylation cassette was successful.
Table 3-8: Activities of unmodified and biotinylated scFv antibodies
Anti-BSA scFv SF1 scFv SF2 scFv
Unmodified scFv 1.42 + 0.03 0.16 + 0.02 0.10 + 0.02
Biotinylated scFv 1.08 + 0.06 0.18 + 0.02 0.07 0.02
Unmodified scFv 1.67 + 0.05 0.11 0.04 0.09 + 0.01
Biotinylated scFv 1.31 + 0.03 0.12 + 0.01 0.09 + 0.01
The activities of unmodified and biotinylated anti-BSA, SF1, and SF2 scFv antibodies
harvested from either culture supernatant or periplasm were tested by ELISA using
10 tg/mL BSA or 10 tg/mL flagellar extract as antigen, as appropriate. Protein A-HRP
conjugate or streptavidin-HRP conjugate at a dilution of 1:2000 was used as secondary
antibody for unmodified and biotinylated scFv antibodies, respectively. The data
presented are the mean and standard deviation of the absorbance values after 40 min
after the addition of the substrate. However, the amounts of protein in each sample were
S. Typhimurium accounts for over a million cases of gastroenteritis in the United
States every year caused by eating contaminated food products (29). Salmonella
infections also cause huge losses in the poultry and diary industries (74).
S. Typhimurium can also be used as an agent ofbioterrorism (3,5). The first step in
managing the above-mentioned problems would be accurate and timely detection of
contamination. A biosensor system with capture antibodies attached to an optic fiber and
detection antibodies labeled with fluorescent dyes has been shown to be able to detect
bacteria sensitively and rapidly (26). Such systems would be of great practical and
economic utility. Efforts are underway at the University of South Florida to develop such
a system for detecting S. Typhimurium. As a part of that project, we screened several
murine monoclonal antibodies recognizing surface epitopes of S. Typhimurium by
ELISA for their potential use in the biosensor system.
Recombinant phage display antibodies recognizing an antigen can be isolated from
a phage display library by panning the library on the desired antigen and isolating the
phage that bound specifically to the antigen. In this procedure antibodies can be isolated
very rapidly, when compared with producing a monoclonal antibody using hybridoma
technology. Griffin. 1 and Tomlinson I human synthetic scFv phagemid libraries were
used for panning on whole S. Typhimurium cells or cell extracts to isolate antibodies
recognizing surface epitopes of S. Typhimurium.
This thesis describes the efforts done to develop a protocol for ELISAs using
bacterial cells as antigens, to characterize several commercially available monoclonal
antibodies for their use in the biosensor system, and to isolate recombinant phage display
antibodies recognizing S. Typhimurium and test their potential usefulness in the
Specific Aim 1: Standardization of a Protocol for Whole Bacterial Cell ELISAs
ELISA is a very simple, economical, and rapid method for immunological analysis
of antibodies (64). There are many different variations of the basic protocol, each
suitable for detecting antigens or antibodies in a complex mixture or for characterizing
antibodies. The basic procedure has been adapted by us to suit the requirements for using
whole bacterial cells as antigens. We examined the following parameters in optimizing
our procedure: antigen concentration, coating conditions such as buffer for suspending
the antigen, blocking buffer, and reaction development time. In standardizing these
conditions, we used goat anti-S. Typhimurium serum as the primary antibody and donkey
anti-goat IgG-alkaline phosphatase conjugate as the secondary antibody. All of the
incubations were done at 4 C. To enhance the binding of the antibodies to antigens,
incubations were done for 2 h rather than 30 min to 1 h as used in ELISAs using proteins
as antigens (64). Glutaraldehyde stabilizes the cell membrane by cross-linking the amine
groups of the surface proteins (57). Therefore, glutaraldehyde was added to the antigen
suspension to a final concentration of 0.05% (w/v) to stabilize the cell membranes. (It
does not have a role in increasing adhesion as we were thinking all these days). Each
condition was tested in triplicate, and the mean absorbance value was calculated. The
signal to noise ratio was calculated by dividing the mean value of the antigen-positive
wells with mean value of antigen-negative wells.
We chose to use enzyme-linked secondary antibodies, as opposed to fluorescently
labeled antibodies or fluorogenic substrates, mainly for economy. The ELx 800UV
microtiter plate reader was used to read the absorbance values from product development
in ELISAs. Rather than stopping the enzyme reactions at an arbitrary time, as is
commonly done, we allowed color development to continue during reading so that we
could use data at the optimum, rather than arbitrary, time of reaction. However, the
ELx 800UV plate reader takes approximately 1 min for reading all of the 96 wells of a
microtiter plate. Absorbance values in wells coated with the same concentrations of
bacteria and treated in identical conditions differed by a significant amount depending on
the location of wells on the plate when the plate was read after only a short time, e.g., 5 to
10 min after the addition of substrate (i.e., the plate reading time contributed significantly
to the development time of the different wells). However, there was no difference in
absorbance values if the plate was read after approximately 30 min, since the one minute
required to read the plate constituted an insignificant part of the total development time.
Therefore, the development reaction was allowed to proceed for 40 min to prevent
discrepancies introduced by the slowness of the plate reader. The components of the
ELISA were therefore adjusted for optimum development over such an extended period.
Experiments were done to optimize the concentration of bacteria that needed to be
coated for obtaining a good signal without increasing the background activity. When the
concentration of bacteria is high, the coating of the cells might not be even, leading to
formation of clumps and thereby inhibiting optimal binding of antibody due to steric
hindrances (63). If the concentration of the antigen is low, the signal obtained might be
too low for interpreting the results properly. Initial experiments were done with bacteria
at an arbitrarily chosen concentration of 1.0 x 109 CFU/mL. When the wells were coated
with bacteria at serial five-fold dilutions and the change in the intensity of signal was
observed, a concentration of 8.0 x 106 CFU/mL gave signal of 1.8 units, which was
similar to the signal obtained with higher concentrations (Figure 3-1). In addition, at a
concentration of 8.0 x 106 CFU/mL the rate of development of reaction was linear with
respect to time and reached peak intensity towards the end of the monitoring period of
40 min. The signal to noise ratios were similar for all the concentrations of bacteria
above 8.0 x 106 CFU/mL, because wells coated with only coating buffer were used as
antigen-negative control wells. Further lowering of the concentration of bacteria affected
the peak signal obtained (Figure 3-1). Therefore, a concentration of 1.0 x 107 CFU/mL
was determined to be appropriate for the whole-cell ELISAs.
Nonspecific binding of the antibodies to the matrix on which the antigen is
deposited, such as the wells of a microtiter plate or a nitrocellulose membrane, is a
common problem in immunological analysis (63). Several different types of agents are
used for preventing this nonspecific binding, the common ones being casein, BSA, and
dextran sulfate (75,76). These blocking agents bind to the sites where the antigen is not
bound and prevent nonspecific binding of primary and secondary antibodies to the
matrix. The primary and secondary antibodies are diluted in blocking buffer for the same
purpose. Some blocking buffers have detergents such as Tween which might affect the
binding of molecules such as LPS to the matrix. Two common agents used for blocking,
casein and BSA, suspended in PBS were tested for their blocking efficiencies in
whole-cell ELISAs. Both of the blocking buffers had 0.1% (w/v) Tween 20. One
percent (w/v) BSA, 1% (w/v) casein, or a mixture of 0.5% (w/v) casein and 0.5% (w/v)
BSA was used as blocking buffer in an ELISA with S. Typhimurium cells as antigen.
Casein performed better than either BSA alone or the mixture of BSA and casein. The
signal obtained in each case was in the range of 3.35 to 3.55 absorbance units. The
background activity was 0.13 units with casein, while with BSA the value was 0.34 units.
The signal to noise ratio was therefore doubled with casein, and casein was used as the
blocking buffer for subsequent experiments.
S. Typhimurium was suspended in either the carbonate buffer or PBS to study the
effect of coating buffer on the signal obtained. The antigen was at a concentration of
107 CFU/mL, and the ELISA was done using USB10 as primary antibody. There was no
significant difference in the signal obtained, with the values being 1.11 and 1.14 for
carbonate buffer and PBS, respectively.
Based on the above results, we determined that for characterizing monoclonal
antibodies S. Typhimurium would be used at a concentration of 107 CFU/mL suspended
in carbonate buffer with glutaraldehyde at final concentration of 0.05% (w/v). PBS-T
(0.1% (w/v)) containing 1% (w/v) casein would be used as the blocking buffer. The
reaction would be allowed to develop for 40 min.
Specific Aim 2: Characterization of Commercially Available Monoclonal Antibodies
to S. Typhimurium
Several monoclonal antibodies to various antigens of S. Typhimurium are available
commercially. We analyzed monoclonal antibodies recognizing surface antigens for their
suitability in the biosensor system. Since the final conditions of the biosensor probe
closely resemble the conditions of an indirect sandwich ELISA, rather than other
immunological methods such as immunoblotting, all the antibodies were characterized by
indirect ELISA. We considered that an indirect ELISA would be more appropriate than
an indirect sandwich ELISA, as we were primarily interested in the reactivities of the
antibodies rather than their ability to stick to the microtiter plate and serve as capture
LPS was chosen as the principal antigen for study because the purpose of the
biosensor is to detect intact cells rather than cell extracts. LPS is abundant on the cell
surface enabling the binding of many antibody molecules on the surface rather than other
antigens such as outer membrane proteins, which might be present only in few copies
(33). Antibodies recognizing LPS have the added advantage of being able to be used
both as capture antibody and detection antibody, as LPS is present in many copies on the
bacterium. The selected antibodies recognized different epitopes such as 04 and 012
Another antigen of interest was the flagellar protein. Flagella are made up of
repeating units of monomeric flagellin, a 60-kD protein. Antibodies recognizing flagella
offer all of the advantages of those recognizing LPS. The disadvantages include their
inability to recognize non-flagellated strains. S. Typhimurium also exhibits phase
variation of their flagella (34,70). The genes coding for the two forms of flagella are
highly homologous, but are not identical, making the two forms of flagella antigenically
distinct (34). The antibodies recognizing one form of flagella might not recognize the
other, giving false-negative results.
Using the standardized protocol, antibodies were tested at various concentrations,
and their activities were noted. In the initial experiments E. coli LE392 was used as a
negative-antigen control. With the monoclonal antibodies the signal obtained with E. coli
coated wells was equal to wells coated with coating buffer only. For subsequent
experiments wells coated with coating buffer alone were used as negative-antigen
controls. The antibodies were ranked on both the peak signal obtained and the signal to
noise ratio. A signal greater than one absorbance unit was considered satisfactory.
Antibodies recognizing LPS and other undetermined cell surface antigens were
characterized by ELISA using whole bacterial cells and purified LPS as antigens. USB10
anti-S. Typhimurium LPS monoclonal antibody recognizing the 04 epitope performed
superiorly to all of the antibodies screened (Table 3-2). This antibody gave a signal
higher than 1.3 in whole-cell ELISAs. It gave a similar signal when purified LPS at a
concentration of 5 [g/mL was used as antigen. This antibody was tested at various
concentrations, and it gave a signal of 0.3 absorbance units at concentrations as low as
0.1 [g/mL (Table 3-3). Biosp53 anti-S. Typhimurium LPS antibody also performed
similarly at concentrations as low as 0.1 [g/mL. RDI and Virostat 6341 monoclonal
antibodies specific to groups A, B, and D Salmonella performed moderately well at
concentrations of 10 [g/mL giving a signal of approximately 0.6 absorbance units. The
other antibodies recognizing LPS or uncharacterized whole cell epitopes did not give any
satisfactory signal in whole-cell ELISAs.
It is interesting to note that there is a hundred-fold difference in the potencies of the
antibodies allegedly recognizing the same epitopes. For example, USB10 recognizing
04 epitope gave a good signal at an antibody concentration of 0.1 [g/mL, while USB 15
recognizing the same epitope was not satisfactory even at concentrations as high as
10 [g/mL. The difference in the activity could be due to difference in affinity of the
antibodies produced from different clones, even though they recognize the same epitope.
Antibodies recognizing flagella were characterized by ELISA and Western blotting
using whole bacterial cells and flagellar extracts as antigens. Of the anti-flagellar
monoclonal antibodies Accurate YVS 6301 performed well at concentrations of
10 [g/mL giving a signal of 1.03 absorbance units when flagellar extract at a
concentration of 10 [g/mL in PBS was used as the antigen (Table 3-4). However, the
signal obtained in a whole-cell ELISA was less than 0.2 absorbance units. The other
anti-flagellar monoclonal antibody, Virostat 6301, did not give any signal higher than 0.2
units either with flagellar extracts or whole cells. The lack of activity with Virostat 6301
antibody may be explained by the fact that it recognizes a different phase of flagellum
than present on the bacterium used in the experiment. Neither of the antibodies
recognized the flagellar protein band on a Western blot. This could be explained by the
possibility that the antibodies were recognizing conformational epitopes, which could
have been destroyed by denaturation in the SDS-PAGE. As described in the Introduction
section, anti-flagellar antibodies have most of the advantages of anti-LPS antibodies in
recognizing whole cells, though detecting non-flagellated strains would be difficult.
Whole-cell ELISAs were done using S. Enteritidis of different serogroups to
determine the specificity of USB10 and Biosp53 monoclonal antibodies. Both of them
showed neglible activity with non-group B Salmonella, except that they gave a moderate
signal of 0.4 units with S. Worthington, belonging to group G (Table 3-5). However, the
authenticity of the strain cannot be verified, and the strain may in fact be a group B
salmonella. Additionally, there could be some partial cross-reactivity between groups B
USB10 and Biosp53 were determined to be useful for further study in the biosensor
system because of their potency and specificity.
Specific Aim 3: Isolation of Recombinant Phage Display Antibodies to
S. Typhimurium and Genetically Fusing scFv Antibodies with Ligands or Reporter
Phage display of antibody fragments offers a powerful, rapid, and economical
approach to screen libraries of large complexities ranging from 107 to 109 variants
(66,67). Griffin. 1 and Tomlinson I + J Human Synthetic VH + VL scFv libraries are scFv
phagemid libraries made from synthetic V-gene segments by cloning the heavy and light
chain variable regions of human immunoglobulin genes into phagemid vectors (43). The
genes coding for the antibody fragments are cloned in frame with gene gill coding for the
coat protein pill. Genes coding for a hexahistidine tag and a c-myc tag are cloned at the
carboxyl terminus of the antibody gene for the purposes of purification of scFv from
culture supernatant. Sequences coding for protein A and protein L binding sites also are
present in the constructs of Tomlinson library. A TAG amber stop codon is engineered at
the junction of the scFv gene and gIII (42).
Isolating antibodies from phage display libraries by panning the phage libraries on
antigen of choice is an efficient, rapid, and economical method of obtaining antibodies of
desired specificity. We used Griffin. 1 and Tomlinson I libraries for panning on whole
bacteria, purified LPS, or flagellar extracts for isolating antibodies. Phage obtained from
Griffin. 1 library were used for panning on whole S. Typhimurium cells and purified LPS.
Phage obtained from Tomlinson I library were used for panning on flagellar extracts of
S. Typhimurium, in addition to whole cells and purified LPS.
Overcoming the Problem of Insert-less Phage in the Phage Pool
Generally, three rounds of panning were done with each antigen. The eluted phage
after each round were used for infecting E. coli TG1 and amplification of the phage
bearing antibody sequences of desired specificity, or the phage were used in the next
round of panning. Phage particles were produced by superinfecting E. coli TG1 with
helper phage. This amplified phage pool was used for panning in the subsequent round.
However, in each amplification step a significant fraction of the output phage do not
display the scFv-pIII fusion protein. This might be because of the relative growth
advantage of the phagemids not bearing the scFv gene (67). To overcome the problem of
insert-less phage dominating the culture, panning was also done without the amplification
step in between the rounds of panning. The eluted phage were suspended in blocking
buffer and used for panning immediately. The number of phage particles input in each
subsequent round of panning would be exponentially decreasing if the eluted phage are
not amplified, thereby greatly decreasing the non-specific phage obtained after the third
round. The number of phage particles obtained at the end of the third round were
typically around 100, enabling screening of each clone by a monoclonal phage ELISA.
When a phage pool gave a good signal by polyclonal phage ELISA, the E. coli TG1
bearing the phagemids were serially diluted and plated to obtain single colonies, phage
produced from which were screened by a monoclonal phage ELISA. By eliminating the
amplification step in between, the need for isolating single clones from the pool was
obviated. This method has added advantages of being less time-consuming and being
cost-effective than the traditional method of panning. However, a disadvantage is that
specific phage might be lost and not recovered because they are not amplified.
Reactivity of Phage Antibodies
Phage from Griffin. 1 library were used for panning on either whole cells or purified
LPS. The phage obtained after three rounds of panning both in suspension and on
immunotubes did not show any significant specific activity against either LPS or whole
cells. The phage pool showed nonspecific binding or binding to the plastic surface of the
microtiter plate. This result was the same with either of the antigens and was irrespective
of the method of antigen coating. Panning was done without amplifying the eluted phage
in between rounds of panning, and the output phage of the third round were screened in a
monoclonal phage ELISA. Most of the phage clones showed no activity, and the
remaining phage showed nonspecific activity. Many clones gave a very good signal
irrespective of the presence or absence of any antigen. These apparently "plastic
binding" phage were considered useful, because these phage could be used in the
biosensor system for attaching the capture antibodies to the probe by constructing a
hybrid scFv antibody with the plastic-specific scFv coupled to the desired antigen-
specific scFv. Since, the genes coding for the antibodies are readily available, they can
be genetically manipulated to create an antibody showing specificity towards the desired
antigen and towards plastic. These antibodies can be used to coat the plastic surface of
the optic fiber, such that the antibody recognizing the microbes is exposed into the
solution. This would be better than attaching the antibodies to the probe by chemical
means, as the process might destroy the structure and hence the function of the antibody.
However, such experiments were not performed in these studies.
When panning was done with phage from Tomlinson I library on whole bacteria
with amplification in between rounds, there was a gradual increase in the activity of the
phage pools against whole bacteria (Figure 3-2). There was no increase in the
background activity with each round, as was observed with phage from Griffin. 1 library.
This could be the result of better elimination of nonspecific phage in the eluted fraction
by trypsin treatment. However, the activity against antigen-negative control V cholerae
cells was higher than with Salmonella. We considered the possibility of the two
gram-negative bacteria sharing some common epitopes, and so we tried to remove the
cross-reactive phage from the pool. The phage pool was incubated with excess of
V. cholerae cells, and the cells with the dual-reactive phage bound to them were removed
by centrifugation. The supernatant was tested for activity by ELISA. However, there
was no change in the specificity of the phage pool. As another approach to isolate the
phage antibodies specific to S. Typhimurium from the pool containing cross-reactive
phage, the phage pool was incubated with S. Typhimurium cells and the phage bound to
the antigens were eluted with trypsin. Instead of amplifying the eluted phage, they were
used to infect E. coli TG1, and the infected cells were plated on 2xTY AG plates. Phage
were produced from each of the clones, and the phage were tested by a monoclonal phage
ELISA. However, there was no increased specificity with any of the clones.
S. Typhimurium and V cholerae cells were run on a SDS-PAGE, and the antigens were
transferred onto a nitrocellulose membrane. Phage from a few representative clones
showing dual reactivity were used as primary antibody. None of the phage recognized
any antigen of either bacterium. The study of these clones was not pursued further.
The difficulty in isolating a phage specific to LPS or whole cell epitopes might be
explained by the fact that the affinities of anti-carbohydrate antibodies are in low
micromolar range (68). The panning procedure involves many washing steps, and it is
possible that the phage bearing antibody sequences against carbohydrates were being lost
in the washing process. With the phage display technique the lowest affinity of the
antibodies isolated is generally in the nanomolar range (68). This could explain the fact
that there are very few instances in literature of isolation anti-carbohydrate antibodies,
while antibodies to proteins seem to be relatively easy to isolate. Therefore, flagella were
chosen as an alternative antigen for panning.
Flagella were extracted from S. Typhimurium and used as antigen for coating
immunotubes. Three successive rounds of panning were done without amplification
steps between rounds of panning. Fifty-nine colonies were obtained when the eluate of
the third round was used to infect E. coli and plated on 2xTY AG plates. When these
colonies were screened by monoclonal phage ELISA using flagellar extracts and
S. Typhimurium cells as antigens, thirteen clones showed good reactivity with flagella
giving a signal of 1 absorbance unit but showed poor reactivity with whole cells
(Table 3-8). To investigate the reason for lower reactivity of the phage with whole cells
when compared to flagellar extracts, bacteria were coated at hundred-fold higher
concentration, and the ELISA was repeated, but there was no increased signal. This
result is similar to the one obtained with anti-LPS monoclonals, in which bacteria coated
at 107 or 109 CFU/mL gave a similar absorbance value.
To determine if each of the thirteen clones was different or if they represented
repeated isolations of the same clone, restriction digestion of the plasmid DNA of each
clone was done. Plasmid DNA was extracted from E. coli infected with each of these
phage, and restriction digestion was done with either BstNI, Sau3A, AluI, or Hhal, all
being four-base pair recognizing enzymes, to generate a restriction pattern. The
restriction patterns were inconclusive. The size of the vector pIT2 is 4.2 kb, while the
size of the scFv insert is approximately 900 bp. The relatively smaller size of the insert
when compared with the vector made analyzing the restriction pattern difficult.
Additionally, the variable portion of the scFv-encoding sequences is even a smaller target
for potentially distinctive restriction enzymes. All of the thirteen clones and the
anti-BSA clone gave apparently an identical restriction pattern. Therefore, the plasmid
DNAs from the thirteen anti-flagellar clones were sequenced, and the sequences were
aligned using GCG software package. Eleven of the thirteen clones turned out to be
siblings. Approximately 1012 phage particles were used in the first round of panning, and
the size of the library is approximately 1.2 x 109 variants. Therefore, statistically a
thousand copies of each antibody were present in the initial suspension. It was not
unlikely that multiple copies of the same antibody were isolated, even though the eluted
phage were not amplified in between rounds of panning. The diversity in the sequence
was observed in the VH region. The two anti-flagellar clones were named SF1 and SF2.
The specificities of SF1 and SF2 were tested in a Western blot using wild type
S. Typhimurium and two non-flagellated mutants of S. Typhimurium cells and flagellar
extract of S. Typhimurium as antigens. Both of the anti-flagellar phage antibodies
recognized the flagella in the whole cell sample and purified flagellar extract. No protein
band corresponding to flagella was seen in the lanes with either of the two non-flagellated
strains (Figure 3-3).
Reactivity of scFv Antibodies vs. Phage Antibodies
Antibody molecules can also be obtained as soluble scFv molecules instead of
fusion proteins displayed on the pIII protein of the phage particle by expressing the
phagemids in a non-suppressor strain of E. coli.
The anti-BSA scFv showed a moderate activity giving a signal of 0.6 units when
tested by an ELISA using 10 tg/mL BSA as antigen and anti-c-myc-HRP conjugate as
secondary antibody. However, there was no activity with either of the anti-flagellar
antibodies with either whole cells or flagellar extracts. To determine if the protein was
being made, the culture supernatant was used in an ELISA as the antigen and
protein A-HRP conjugate was used for detection. There was no signal either with
anti-BSA scFv or with the anti-flagellar scFvs. The same supernatants were resolved on
a SDS-PAGE, and the antigens were transferred onto a nitrocellulose membrane. When
probed with protein A-HRP, no bands appeared corresponding to any of the scFvs. The
scFv antibodies were concentrated by passing the culture supernatant on a
protein A-sepharose column and eluting with 0.1 M citric acid, pH 3 (61). The eluted
fractions were tested for their activity by ELISA and Western blot. None of the fractions
showed any activity by either an ELISA or a Western blot.
The instability of the scFv molecule might explain the activity of these antibodies
as scFv-pIII fusion proteins and not as soluble molecules. The factors on which the
stability of the scFv molecules depend include intradomain disulfide bonds and the
stability of VH-VL interface (77). Alternatively, there could be a problem in the secretion
of different scFvs. The amount of scFv antibodies secreted into the culture supernatant
might be very low for use as detection reagents.
Two strategies were used to obtain increased yields of scFv antibodies, using
anti-BSA antibody as a model. Periplasmic extracts were analyzed for the presence of
scFv protein and its activity to rule out the possibility that most of the protein was present
in the periplasm and was not being released into the supernatant. As an alternative
approach, sucrose was added to a final concentration of 0.4 M while the culture was in
mid-exponential phase to increase the yield of scFv antibodies in the supernatant as
shown by Kipriyanov et al (78). The culture supernatant and the periplasmic extract were
tested for the presence of scFv antibodies by a Western blot using protein A-HRP
conjugate as detection reagent. No band was observed corresponding to the antibody in
either periplasmic extract and culture supernatant. The activity of the antibodies in the
periplasmic extract and culture supernatant was measured by a Western blot and an
ELISA using BSA as antigen. There was no useful signal in either of the assays.
Biotinylation of antibodies is a common technique used to amplify the signal in
immunological analysis by taking advantage of the high affinity of biotin for the
multivalent binding sites of streptavidin and the wide variety of streptavidin conjugates
available for detection (72,73). SF1, SF2, and anti-BSA scFv antibody molecules were
biotinylated in vivo by cloning the respective genes into pAC5 Avitag vector and
expressed in E. coli AVB 100. scFvs were harvested from periplasm and culture
supernatant and were tested for their activity by ELISA. There was no activity with the
biotinylated or unmodified SF1 and SF2 harvested from either the periplasm or culture
supernatant (Table 3-8). The biotinylated anti-BSA scFv gave a lower signal than
unmodified scFv, the absorbance values being 1.08 and 1.42, respectively with the
antibodies harvested from culture supernatant, 1.31 and 1.67, respectively with the
antibodies from periplasmic extract. However, the amount of protein in each sample was
not quantified, making the comparison of activities inaccurate. These experiments
showed that scFv antibodies can be biotinylated easily without the loss of activity.
Studies are being done in our lab to isolate phage antibodies to whole cell epitopes
of S. Typhimurium by panning on whole cells or purified LPS. Two strategies are being
used to isolate antibodies to LPS. One of the strategies being followed is to conjugate the
O antigen portion of the LPS molecule to a carrier molecule such as BSA for use as
antigen in panning. The rationale behind this approach is that purified LPS being a
hydrophobic molecule might not be sticking in enough amounts to immunotubes for
serving as the antigen for panning. By conjugating the O antigen to a carrier protein such
as BSA, it is possible that the antigen could be coated on immunotubes in adequate
amounts. Another strategy is to obtain a new version of Tomlinson library that is
obtained by superinfecting the original bacterial library with Hyperphage instead of
KM13 helper phage. When phage were obtained from the original library for the first
time, KM13 helper phage was used for superinfection. The antibodies thus obtained
might be displaying wild type pIII and scFv-pIII molecules, instead of only scFv-pIII
molecules (65). When such phage were used for panning, the phage specifically
recognizing LPS might be washed away in the first round of panning because of the low
affinity of anti-carbohydrate antibodies. However, in theory if the phage were obtained
with Hyperphage, all copies of pIII would be scFv-pIII. Avidity effects could play a role
in increasing the binding of the antibodies to the antigen and hence an improved chance
of isolating anti-carbohydrate antibodies, as shown by MacKenzie et al (68).
Incubating biotinylated scFv antibodies with streptavidin could yield a tetrameric
form of the antibody, as each streptavidin can bind to four biotin molecules (71).
Multivalency of the antibody could be useful with anti-carbohydrate antibodies with
intrinsic low affinities (65). When streptavidin conjugates are used for forming such a
tetrameric complex, the same complex can be used both as primary and secondary
A biosensor system that can detect microbial contamination in food and water and
on surfaces is of practical and economic importance. This thesis describes the efforts
done towards developing such a system for detecting S. Typhimurium. Commercially
available murine monoclonal antibodies were screened for their potential usefulness in
the biosensor system. Recombinant phage display antibodies that recognize flagellar
protein of S. Typhimurium were isolated. Although the phage display antibodies we
isolated were not very useful in recognizing whole cells, the general methods developed
for panning and isolating antibodies could be used to isolate antibodies with greater
affinities and higher potencies. Alternatively, the isolated anti-flagellar antibodies could
be modified genetically to increase their affinity.
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Gopal Sapparapu was born in 1974 in Guntur, India. He did his schooling at Sainik
School Korukonda. In 1994 he joined the MBBS program at Guntur Medical College,
Guntur and graduated in June, 2000. He joined the MS program in Molecular Genetics
and Microbiology at the University of Florida in Fall 2001.