• TABLE OF CONTENTS
HIDE
 Front Cover
 Summary
 Table of Contents
 I. Introduction
 II. Materials and methods
 III. Some factors affecting results...
 IV. Uses in plant virology for...
 V. Concluding remarks
 VI. References on techniques and...
 Literature cited
 Back Cover
 Historic note






Group Title: Bulletin - University of Florida. Agricultural Experiment Station ; 788
Title: Immunodiffusion tests with sodium dodecyl sulfate (SDS)-treated plant viruses and plant viral inclusions
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00027447/00001
 Material Information
Title: Immunodiffusion tests with sodium dodecyl sulfate (SDS)-treated plant viruses and plant viral inclusions
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: i, 39 p. : ill. ; 23 cm.
Language: English
Creator: Purcifull, Dan E
Batchelor, Dallas L
Publisher: Agricultural Experiment Stations, Institute of Food and Agricultural Sciences, University of Florida
Place of Publication: Gainesville Fla
Publication Date: 1977
 Subjects
Subject: Immunodiffusion   ( lcsh )
Plant viruses   ( lcsh )
Immune serums   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 31-39.
Statement of Responsibility: Dan E. Purcifull and Dallas L. Batchelor.
General Note: Cover title.
 Record Information
Bibliographic ID: UF00027447
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 001597007
oclc - 03417535
notis - AHM1137

Table of Contents
    Front Cover
        Front Cover
    Summary
        Page i
    Table of Contents
        Page ii
        Page iii
        Page iv
    I. Introduction
        Page 1
        Page 2
    II. Materials and methods
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
    III. Some factors affecting results of immunodiffusion tests with SDS-treated plant viral antigens
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
    IV. Uses in plant virology for immunodiffusion tests with SDS-treated antigens
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
    V. Concluding remarks
        Page 29
    VI. References on techniques and on the effects of SDS on viruses and proteins
        Page 30
    Literature cited
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
    Back Cover
        Page 40
    Historic note
        Page 41
Full Text

Bulletin 788 (technical)
MARCH 1977


Agricultural Experiment Stations
Institute of Food and Agricultural Sciences
University of Florida, Gainesville


Immunodiffusion Tests
with Sodium Dodecyl
Sulfate (SDS) -Treated
Plant Viruses and
Plant Viral Inclusions


Dan E. Purcifull and
Dallas L. Batchelor













IMMUNODIFFUSION TESTS WITH SODIUM
DODECYL SULFATE(SDS)-TREATED PLANT VIRUSES
AND PLANT VIRAL INCLUSIONS

Dan E. Purcifull and Dallas L. Batchelor'

Department of Plant Pathology, Plant Virus Laboratory, University
of Florida, Gainesville, Florida 32611.

SUMMARY
Procedures are described for conducting double diffusion
tests in agar gels containing sodium dodecyl sulfate (SDS) and
sodium azide. Antisera were prepared by injecting rabbits intra-
muscularly with purified immunogen emulsified with Freund
adjuvant. In some cases it was necessary to treat the immunogen
itself with SDS in order to produce antisera which reacted satis-
factorily with SDS-treated antigens. Unfractionated, undiluted
antisera were used routinely. When diluted antiserum was re-
quired, normal serum was used as the diluent. Antisera diluted
with normal sera had titers twofold to eightfold higher than cor-
responding antisera diluted with Tris-buffered saline. Methods
are given for avoiding some of the various types of nonspecific
precipitation that may occur in immunodiffusion media in the
presence of SDS. Based on the literature and on the results pre-
sented herein, double diffusion tests in the SDS gels have been
used successfully for studies with a wide variety of antigens.
Included are 19 rod-shaped viruses (15 potyviruses, 3 potex
viruses, and tobacco mosaic virus), 4 isometric viruses (citrus
variegation, cowpea mosaic, southern bean mosaic, and squash
mosaic viruses), one plant rhabdovirus, pinwheel inclusion body
proteins induced by 5 potyviruses, and tobacco etch virus-induced
nuclear inclusions. The techniques were suitable either for
studies with purified viral antigens or for detection of virus-
specific antigens in crude plant extracts.

1The current address of the junior author is: Department of Plant
Pathology, Montana State University, Bozeman, Montana 59715.







CONTENTS


Page
Summary .......................................... i

I. Introduction ........................................ 1

II. Materials and Methods ................................. 3
A. Source and Purity of SDS .......................... 3
B. Preparation of Immunogen and Immunization ......... 4
1. Use of untreated viruses or virus-induced
inclusions as immunogens ....................... 4
2. Use of SDS-treated immunogen ................... 4
3. Use of denaturing agents other than SDS
for immunogen preparation ....................... 5
4. Emulsification of immunogen and immunization .... 5
C. Processing of Sera ................................. 6
D. Immunodiffusion Media ............................ 6
E. Antigen Preparation and Preservation ............... 7
F. Gel patterns, Addition of Reactants,
and Recording of Results ........................... 9
G. Titering of Antisera ................................ 9
H. Summary of Methods ............................... 10

III. Some Factors Affecting Results of Immunodiffusion
Tests with SDS-treated Plant Viral Antigens ............ 11
A. Antiserum ........................................ 11
B. Treatment of Test Antigen .......................... 12
C. Composition of Media .............................. 12
1. Agar ........................................ 12
2. Salts .......................................... 13
3.SDS ......................................... 14
D. Effects of SDS on Immunoprecipitin Formation
and on Serum Titers ............................... 15
1. Ephemerality of precipitin lines ................. 15
2. Effects of SDS on serum titers .................... 15







Page
E. Immunospecific Reactions of Normal Plant Proteins .. 17
F. Nonspecific Precipitates ............................ 17
1. Serum-SDS interactions ......................... 18
2. Serum-plant sap interactions .................... 18
3. Plant sap-plant sap interactions .................. 21
4. Chemical additives to media or to reactants ........ 21

IV. Uses in Plant Virology for Immunodiffusion Tests
with SDS-treated Antigens .......................... 22
A. Diagnosis and Detection of Plant Viruses ............ 22
B. Relationships of Plant Viruses ...................... 22
C. Analyses of Proteinaceous Inclusion Bodies Induced
by Potato Y Group (PVY) Viruses ................... 26
D. Characterization of Viral Proteins .................. 28

V. Concluding Remarks ................................ 29

VI. References on Techniques and on the Effects
of SDS on Viruses and Proteins ....................... 30
Acknowledgements .................................... 30
Literature Cited ..................................... 31













This public document was promulgated at an annual cost
of $1,888.73 or a cost of $.63 per copy to present information
on immunodiffusion tests with plant viruses and plant
viral inclusions.






I. INTRODUCTION
During the past 15 years, immunodiffusion tests have been
used extensively in plant virology for studies involving the na-
ture of plant virus proteins, virus diagnosis, and virus relation-
ships. In the first immunodiffusion tests with some of the rod-
shaped viruses, the technique failed because the viruses involved
were too long to diffuse readily into agar gels (129, 133)1. To
alleviate this problem, several rod-shaped viruses were chem-
ically dissociated (degraded) into diffusible, immunoreactive
components (27, 83). Sodium dodecyl sulfate (5, 21, 22, 35, 82
and Table 1) and a variety of other dissociating agents (3, 5, 22,
50, 81, 97, 99, 102) have been used for this purpose in recent
years. Ultrasonics have also been used to degrade viruses into
diffusible antigens (122).
Sodium dodecyl sulfate (SDS) is an anionic detergent which
has the formula: CH3(CH2)I1OSO3Na (sodium lauryl sulfate is
a synonym). SDS binds to proteins (e.g., 32, 116, 117, 131), and
it is an effective agent for degrading viruses and many proteins.
The effects of SDS on plant viruses were first demonstrated
nearly 40 years ago (6, 107). Bawden and Pirie (6) reported
that treatment of PVX' with SDS resulted in a loss of infec-
tivity, serological activity, flow birefringence, and sediment-
ability (under conditions that sedimented untreated virus).
According to Sreenivasaya and Pirie (107), SDS had similar
effects on TMV. They reported that SDS treatment of TMV re-
sulted in dissociation of the nucleic acid from the protein. They
also determined that the rate of action of SDS on TMV depended
on temperature, concentration of SDS, and pH. They attempted
unsuccessfully to prepare an antiserum to the disrupted virus.
Virologists and biochemists now widely use SDS as a de-
naturant in conjunction with analyses of proteins on polyacry-
lamide gels (124, 131, 132), for preparation of viral nucleic
acids (20, 43, 87, 104), and for studying protein-RNA interac-
tions in plant viruses (9, 10). Gooding and Bing (22) recently
extended the use of SDS as a dissociating agent for plant virus
serological work by incorporating SDS in agar immunodiffusion
media. This technique (or some modification of it) has been used
successfully for immunodiffusion tests with various rod-shaped,
isometric and bacilliform viruses (e.g., 5, 21, 79, and Table 1)
and for virus-induced inclusions (5, 35, 45, 63, 82 and Table 1).

1Numbers in parentheses refer to Literature Cited.
2For meaning of abbreviations of virus names, see Table 1.







Table 1. Reports of immunodiffusion studies With SDS-treated plant viruses
or virus-induced inclusions.

Virus or inclusion studied Abbreviation Citation


Barley stripe mosaic virus
Bean pod mottle virus
Bean yellow mosaic virus

Bidens mottle virus
Bidens mottle pinwheel inclusions
Blackeye cowpea mosaic virus
Cactus virus X
Carnation vein mottle virus
Citrus variegation virus


Clover yellow mosaic virus
Clover yellow vein virus
Cowpea mosaic virus
Cucumber mosaic virus
Cymbidium mosaic virus
Dasheen mosaic virus
Fulva iris mosaic virus

Lettuce mosaic virus
Narcissus mosaic virus
Papaya mosaic virus
Peanut mottle virus
Pepper mottle virus
Pepper mottle pinwheel inclusions
Pepper veinal mottle virus
Potato virus X

Potato virus Y



Potato virus Y pinwheel inclusions
Potato yellow dwarf virus
Sonchus yellow net virus

Southern bean mosaic virus

Soybean mosaic virus
Squash mosaic virus


BSMV
BPMV
BYMV

BMoV
BMoV-I
BICMV
CVX
CVMV
CVV


CYMV
CYVV
CPMV
CMV
CyMV
DMV
FIMV

LMV
NaMV
PMV
PMoV
PeMV
PeMV-I
PVMV
PVX

PVY



PVY-I
PYDV
SYNV


2
121
4,25,40,76,77,78,126,
127
5,79,80,82,84,141
33,82
53
46,47
36
Purcifull, Garnsey
and Gonsalves, un-
published
5,47,79
4,36
Lima, unpublished
137
47
1
Barnett and Alper,
unpublished
5,79,84,141
47
5,47,79
49,69,113,120,121
5,66,79,82,85,139,140
5,33,82,85
85
5,21,24,46,47,79,97,
100,103
5,21,22,23,24,35,54,55,
56,59,60,66,74,79,82,
85,101,106,113,138,139,
140
33,35,82
44
Jackson and Christie.


unpublished
SBMV 5; Purcifull and
Christie, unpublished
SoyMV 120,121
SMV Purcifull and Christie,
unpublished


continued








Virus or inclusion studied Abbreviation Citation

Tobacco etch virus TEV 5,13,21,22,24,35,45,59,
60,66,74,79,82,85,101,
113,138,139,140
Tobacco etch pinwheel inclusions TEV-I 5,33,35,45,82
Tobacco etch nuclear inclusions TEV-NI 5,45
Tobacco mosaic virus TMV 28,29,79
Tobacco vein mottle virus TVMV 5,74,106,112
Turnip mosaic virus TuMV 5,34,62,63,79,82
Turnip mosaic pinwheel inclusions TuMV-I 33,62,63,82
Turnip yellow mosaic virus TYMV Koenig, unpublished
Watermelon mosaic virus WMV Purcifull and Hiebert,
unpublished
White clover mosaic virus WCMV 47


The primary purposes of this bulletin are to present and dis-
cuss specific methods for immunodiffusional anaylses of SDS-
treated plant viruses and virus-related proteins, and to briefly
discuss various applications of the techniques in plant virology.
For information on the fundamentals of plant virus serology and
immunodiffusion, the reader is referred to various reviews (3,
7, 16, 61, 67, 68, 99, 129, 133). References on SDS-protein inter-
actions and on techniques for use with SDS are given in Section
VI.

II. MATERIALS AND METHODS
Many of the methods described below are ones which have
been used in our laboratory for the past several years, but spe-
cific methods reported by others are also presented. Experimen-
tation may be required to find reliable procedures for any par-
ticular virus-host system.

A. Source and Purity of SDS
Commercial preparations of SDS (Sigma Chemical Co., St.
Louis, Mo., U.S.A.) were used without further purification, un-
less noted otherwise.3 The purity of SDS may be important, how-
ever, and commercial preparations sometimes contain undesir-
able impurities. Methods for purification of SDS by recrystalli-
zation from hot ethanol have been described (15, 87).
3No endorsement of any product over other products of similar
composition or capability is implied.







B. Preparation of Immunogen and Immunization
Three basic approaches have been used to prepare immu-
nogens in order to obtain antisera which react with SDS-treated
viral antigens. The choice of these or other techniques will de-
pend on the nature of the antigen itself and on the objectives of
subsequent serological tests.

1. Use of untreated viruses or virus-induced inclusions as
immunogens
Depending on the virus or protein, this simple approach may
give good results. It has proved particularly useful for numerous
viruses in the potyvirus group (e.g., 22, 63, 85) and for inclusion
body proteins (35, 45, 63, 82). Antisera prepared against un-
treated virus also have been used for detecting SDS-treated TMV
proteins (79) and for several isometric viruses (e.g., CVV,
SBMV, SMV).

2. Use of SDS-treated immunogen
An antiserum to SDS-degraded SBMV was produced using
the following procedure. Two mg of lyophilized, purified virus
was resuspended in 1 ml of 1%7 SDS and 20 ,l of 2-mercapto-
ethanol (2-ME). The solution was boiled for 2-4 min, then emul-
sified directly with Freund's complete adjuvant, and injected
intramuscularly into a rabbit. Serum collected 6 weeks later re-
acted strongly with SDS-treated, boiled extracts from SBMV-
infected plants, but not with extracts from healthy plants. (See
Fig. 1 in Section III. A.)
Batchelor (5) prepared specific antisera reactive to SDS-
treated antigens of three viruses in the potato X group, three in
the potato Y group, SBMV, and the inclusion bodies induced by
two viruses in the potato Y group. His method involved dissocia-
tion of the purified virus or inclusions in solutions containing
SDS and 2-ME, separation of the immunogen from 2-ME and
most of the free SDS, and reconcentration of the immunogen.
SDS (Sigma Chemical Co.) was recrystallized once from hot
ethanol prior to use. To 1 ml of a viral or viral inclusion prepara-
tion, SDS and 2-ME were each added to give final concentrations
of 3% (w/v and v/v, respectively). The virus preparations were
used at concentrations of 2-4 mg/ml, and the inclusion prepara-
tions were adjusted to 3-5 absorbance units/ml at 280 nm. The
solutions were then placed in a boiling water bath for 1-2 min.






After heat treatment, the inclusion preparations were centri-
fuged at 4,500 rpm for 15 min in a Servall Model SP table cen-
trifuge to remove insoluble material. One ml was layered onto
a K15/30 Sephadex column packed with G-50-150 Sephadex gel.
The samples were eluted by gravity flow using 0.02 M sodium
borate, pH 8.0. A flow rate of 1-2 ml/min was employed, and the
effluent was monitored by an ISCO absorbance instrument and
optical unit Model UA-4. The optical unit was fitted with a 1 cm
flow cell, and a Model 610 ISCO external strip chart recorder
was used to record ultraviolet absorbancy during chromatogra-
phic events. Column calibration was performed using intact
papaya mosaic virus (1 mg/ml) or TMV (1 mg/ml) as the ex-
clusion volume (Vo) marker and 2-ME (1%) as the total vol-
ume (V,) marker. The peak appearing at the column Vo was
collected in a 12 ml syringe fitted with a 400 mesh nylon net,
which was sealed at the tip with Parafilm. To concentrate the
preparation, Sephadex G-25-150 was added to the liquid in the
syringe and allowed to swell for 10-15 min. The Parafilm seal
covering the syringe tip was removed and the syringe was cen-
trifuged at 1500 rpm for 15 min in the Servall Model SP table
centrifuge. The liquid containing the denatured protein was
collected and this procedure was repeated until the desired vol-
ume was obtained (Technical Data Sheet-"Solute Concen-
trating with Sephadex," Pharmacia Fine Chemicals, Piscataway,
New Jersey). Based on UV spectrophotometry, at least 90% of
the sample applied to the column was recovered for use as im-
munogen.

3. Use of denaturing agents other than SDS for immunogen
preparation
Antisera prepared to pyridine-degraded potato virus X
(PVX) also reacted with SDS-degraded PVX protein (97).
Similarly, antiserum to guanidine-HCL-denatured papaya mosaic
virus (PMV) also reacted with SDS-treated PMV (5), and anti-
serum to pyrrolidine-treated tobacco etch virus (TEV) reacted
with SDS-treated TEV (101).

4. Emulsification of immunogen and immunization
Emulsification of immunogen with Freund's adjuvant was
done directly in the syringe to be used for injection. The tip of
a plastic disposable syringe was stoppered and the syringe was
partially immersed in an ice bath. The adjuvant was placed in the






syringe first and then an equal volume of antigen was added
gradually while stirring vigorously. A convenient stirring ap-
paratus consisted of a Phillips screwdriver (with handle re-
moved) inserted into the chuck of a mechanical stirrer. After
stirring at about 5,000-7,000 rpm for a few minutes, a thick
emulsion suitable for injection was formed.
The following general immunization procedure (5) has given
satisfactory results for preparation of antiserum either to un-
treated or to denatured viral antigens. The immunogen (con-
sisting of 2-4 mg in a volume of about 1 ml for denatured viruses
or 1-3 OD2so units in a volume of about 1 ml for inclusions) was
emulsified 1:1 with Freund's complete adjuvant (Difco) and 1
ml of the mixture was injected into the thigh muscle of each hind
leg of a New Zealand white rabbit. About 1 month later each
animal was administered a similar amount of antigen emulsified
1:1 with Freund's incomplete adjuvant. Serum collection was
commenced 1-2 weeks after the second injection and carried out
over a period of several months.

C. Processing of Sera
Although we have successfully used standard procedures for
processing serum (3, 99), the following method (5) results in
good serum yields and minimizes processing time. Whole blood
taken by nicking the marginal ear vein was collected in glass,
round-bottom centrifuge tubes. The tubes were kept in a water
bath at 370C for 1 hour, and then centrifuged for 10 min at
2000 rpm in a Model SP Servall table centrifuge. The serum was
drained from the clots directly into conical-bottom tubes and cen-
trifuged for 15 min at 4000-4500 rpm in the Servall SP centri-
fuge. The clear supernatant fluid from the second centrifugation
was collected and stored either by freezing or by freeze-drying
until use.

D. Immunodiffusion Media
The medium originally reported by Gooding and Bing (22)
has proven useful for analysis of numerous SDS-treated plant
viruses and virus-induced inclusion proteins (e.g., 5, 79, 82).
The medium consists of 0.8% Noble agar (Difco Labs., Detroit,
Mich., U.S.A.), 0.5% SDS (Sigma Chemical Co., St. Louis, Mo.,
U.S.A.), and 1% sodium azide (Matheson, Coleman, and Bell,
Norwood, Ohio, U.S.A., or Sigma), all w/v in water. The pur-
pose of the SDS is to act as a degrading agent and to minimize






the occurrence of confusing nonspecific precipitates when SDS-
treated antigens are used. The sodium azide contributes to ionic
strength and inhibits microbial growth. CAUTION: Sodium
azide is poisonous and also may react with certain metals in
plumbing systems over a period of time to form potentially ex-
plosive compounds. If sodium azide is discarded into metal
plumbing, the system should be thoroughly flushed immediately
with running water. Information about decontaminating plumb-
ing systems is available from the National Institute for Occu-
pational Safety and Health, Rockville, Maryland 20852 U.S.A.
To prepare enough medium for about 40 plates (plastic dis-
posable Petri plates, 100 x 15 mm), 4 g Noble agar was added to
about 300 ml of distilled or deionized water and autoclaved for
5 minutes at 2500F. After autoclaving, 5 g sodium azide was
added and dissolved by stirring. Then 2.5 g SDS was dissolved in
about 150 ml of hot water. This was added to the agar-azide
mixture, and the medium was brought up to a final volume of
500 ml with hot water. The Petri plates were placed on a level
surface and 12 ml of agar medium were added per plate. After
the agar solidified, plates were put in plastic bags or plastic
boxes and stored at room temperature or at 2-40C for use over a
period of several weeks. Since SDS precipitates at 2-40C, the
medium becomes opaque; however, it will clear again on warm-
ing to room temperature.
Various other media, some with SDS (21, 39, 48, 121, 126,
127) and some without (126, 137) also have been used satisfac-
torily for immunodiffusion of SDS-treated antigens.

E. Antigen preparation and preservation

For routine serodiagnostic work, sap expressed from infected
leaves with a mortar and pestle or hand sap press has provided
a reliable source of antigen for certain viruses (21). To detect
virus-induced inclusions in leaf extracts (82), leaves are ground
with a mortar and pestle in 1 ml of water per g of tissue. After
adding 1 ml of 3% SDS (w/v, in water) per g of tissue, the sap
is expressed through cheesecloth, and added directly to reactant
wells. This method also has worked satisfactorily for detecting
coat protein antigens of numerous potyviruses and several potex-
viruses (79).
Purified viruses or inclusion preparations either can be used
directly or can be treated first with SDS, depending on what is
found necessary to dissociate a particular antigen. Proteins ex-








tracted from polyacrylamide gels also give specific reactions in
SDS-immunodiffusion tests (5, 33, 82).
Lyophilization of crude extracts from infected plants has
recently been proposed as a means of maintaining a variety of
viral antigens for routine SDS-immunodiffusion tests (79; Table
2). Reactions of serological identity (no spur formation) have


Table 2. Viruses which reacted positively in SDS-immunodiffusion tests when
resuspended, freeze-dried sap from infected plants was used as
antigen.

Host used for
Virus crude extract Preparationa,b Citation


Bidens mottle


Clover yellow
mosaic
Lettuce mosaic


Papaya mosaic
Pepper mottle


Pepper veinal
mottle
Potato Y



Potato X
Southern bean
mosaic
Tobacco etch



Tobacco mosaic

Turnip mosaic

Watermelon
mosaic


Cichorium endivia L.
Lactuca sativa L.
Nicotiana hybrid
Pisum sativum L.

Cichorium endivia L.
Lactuca sativa L.
Pisum sativum L.
Carica papaya L.
Capsicum annuum L.
N. hybrid
N. tabacum L.
C. annuum L.
N. hybrid
C. annuum L.
N. tabacum L.
Lycopersicon esculentum
Mill.
N. tabacum L.
Phaseolus vulgaris L.

C. annuum L.
Datura stramonium L.
N. tabacum L.
L. esculentum Mill.
N. tabacum L.
L. esculentum Mill.
N. hybrid
Brassica perviridis Bailey
Cucurbita pepo L.


SDS
SDS
SDS
SDS, W

SDS
SDS
SDS
W
W
W
SDS, W
W
W
SDS, W
SDS, W
W


84
79,84
79
5,79

84
79,84
5,79
5,79
79
79
5
85
5
79,85
5,79
Unpublished


SDS, W 5,79
W 5


W
SDS, W
SDS, W
W
W
W
SDS
SDS, W
SDS


79,85
79
79
Unpublished
79
Unpublished
79
5,63
Purcifull &
Hiebert Un-
published


aSDS=Crude plant extracts prepared in 1.5%
pended in water prior to testing.


SDS, freeze-dried, and resus-


bW=Crude plant extracts prepared in water, freeze-dried, and resuspended in
water or in 1.5% SDS prior to testing.






been obtained thus far in tests comparing viral antigens in
lyophilized extracts to those in freshly prepared extracts (79).
The lyophilized antigens, however, usually gave weaker immuno-
precipitin lines than freshly prepared antigens (79). For each
virus, it would seem advisable to check for antigenic differences
between fresh and freeze-dried viral antigens before using the
latter as reference antigens in virus identification. Other tech-
niques also have been reported to be useful for preserving anti-
genic activity, such as the addition of 1%/ sodium azide to crude
plant juice (21), dried infected leaf tissue ground and resus-
pended in water (21), and antigens frozen in crude juice or in
SDS-treated juice (63).

F. Gel Patterns, Addition of Reactants, and
Recording of Results
Reactant wells are punched in the agar with cork borers and
the agar plugs are aspirated with a Pasteur pipet or glass tubing
connected to a vacuum line. An adjustable gel cutting device
(Grafar Corp., Detroit, Mich.) was used for cutting the patterns.
A useful gel pattern consists of up to 6 peripheral antigen wells
(7 mm in diameter), surrounding a central serum well (also
7 mm in diameter). Each peripheral well is 4-5 mm from the
central well at the closest point. Due to the presence of SDS,
somewhat more care than usual is required in loading reactant
wells and in handling plates to avoid spillage.
Reactions usually appear within 12 hours and are complete
within 24-48 hours when the SDS-immunodiffusion plates are
incubated at room temperature (about 240C). Reactions should
be recorded at this time because the immunoprecipitin lines will
usually disappear within 5-10 days after addition of reactants.
The results can be viewed and recorded photographically using
dark-field illumination (16). Alternatively, the plates can be
washed in sodium phosphate-buffered saline and stained with
nigrosin or coomassie brilliant blue (16; Christie and Purcifull,
unpublished).
Methods for avoiding some of the nonspecific reactions that
can be encountered in SDS-immunodiffusion tests are given in
a later section (III. F).

G. Titering of Antisera
We recommend that plant virus antisera be diluted in normal
serum rather than in buffered saline when titers are to be deter-







mined in media containing SDS. Alternatively, a diluent con-
sisting of 5% bovine serum albumin (Fraction V powder, Sigma
Chemical Co.) and 0.85% NaCl (both w/v), in 0.05 M Tris ad-
justed to a final pH of 7.2, has given results similar to normal
serum in limited tests (see Section III. D. 2. for more informa-
tion on titering).

H. Summary of Methods
The following summary briefly describes general procedures
which have given good results with many viruses. Details of
these methods and various modifications of them are given either
in the preceding portions of Section II or in Section III.
(1) The immunodiffusion medium consisted of 0.8% Noble
agar, 0.5% SDS, and 1.0% sodium azide (all w/v) in
deionized water. Twelve ml of medium were poured into
each 100 mm x 15 mm plastic Petri dish. After the agar
solidified, the plates were placed in a humid container and
stored at 40C.
(2) Antisera were prepared by injecting rabbits intramuscu-
larly with preparations emulsified with Freund's adjuvant.
In some cases it was necessary to immunize with SDS-
treated antigen (e. g., with some potexviruses), but in
other cases (e. g., potyviruses) antisera obtained by im-
munizing either with untreated or SDS-treated virus gave
satisfactory results. Experimentation may be required to
determine the best procedure for a particular virus.
Antisera were stored frozen or lyophilized. The anti-
sera were usually used undiluted, but if dilutions were
required, normal serum or a solution of 5% bovine serum
albumin in 0.85% NaC1 0.05 M Tris (final pH of 7.2)
were used as diluents.
(3) Antigens were prepared from virus-infected and healthy
leaves by grinding tissue in HO (1 ml per g of tissue).
One ml of 3% SDS was then added per g of tissue, and
the mixture was expressed through cheesecloth. The ex-
tracts were used within 1-2 hrs after preparation. Lyo-
philization was a convenient means of preserving antigens
in leaf extracts. Purified antigens were either untreated
or were treated with 0.1%-1.5% SDS prior to use.
(4) Normal serum and extracts from healthy plants were in-
cluded as controls.






(5) For routine tests a gel pattern consisting of 6 peripheral
antigen wells surrounding a central serum well (each well
7 mm in diam) was used. The edge of the center well was
4-5 mm from the edges of the peripheral wells.
(6) After addition of reactants, the plates were incubated at
24C and results were recorded after 24-48 hrs.


III. SOME FACTORS AFFECTING RESULTS OF IMMUNO-
DIFFUSION TESTS WITH SDS-TREATED PLANT VIRAL
ANTIGENS

The following illustrations and discussion document the im-
portance of various factors which may influence results of im-
munodiffusion tests with SDS-treated antigens. Methods are
presented for preventing or solving problems which might be
encountered.

A. Antiserum
Antigenic disparity between SDS-treated viral proteins and
the untreated virus has been indicated for PVX (97, 102), CMV
(137), and SBMV (5 and Fig. 1). Therefore, it is essential to
determine that a virus antiserum contains antibodies to the SDS-


Fig. 1. Evidence
for antigenic disparity
between untreated
southern bean mosaic
virus (SBMV) and
SDS-treated SBMV. (
The peripheral wells
were filled with SDS-
treated extracts from
SBMV-infected bean 4
(1 and 3) or healthy 4
bean (2 and 4). Ex- SDS
tracts added to wells
3 and 4 were boiled in SDS prior to use. The center wells were filled
with antiserum to untreated SBMV (A) or with antiserum to SDS-treated
SBMV (B). When SBMV-infected bean sap was boiled in SDS, a virus
degradation product was formed which reacted strongly with anti-
serum to SDS-treated virus (reaction between wells B and 3) but not
with antiserum to untreated virus. The antiserum to intact virus, how-
ever, reacted with unboiled, SDS-treated SBMV (reaction between wells
A and 1) apparently because the virus is not as extensively degraded
without boiling in SDS.






denatured antigens, by including appropriate positive (immuno-
reactive) controls in immunodiffusion tests (e.g., known PVX
antigen should be included when testing an unknown virus
against PVX antiserum).
When using antisera provided by other investigators, it is
very important to know the means they used for preparation of
immunogen. It is also important to know if the serum has been
diluted and the nature of the diluent (see section III. D. 2).

B. Treatment of Test Antigen
Procedures necessary to achieve satisfactory results may vary
because of several factors, including the nature of the antigen
involved, its purity, and prior treatment. With some viruses, all
that is necessary is to place crude sap from infected plants into
reactant wells (21; Fig. 2); presumably enough SDS diffuses
from the medium into the well to effect virus degradation. With
other antigens, such as potato Y group "pinwheel" inclusions,
reactions may not be obtained unless SDS is added to the leaf
extracts (82; Fig. 2B).
Batchelor (5) found that SBMV did not react against anti-
serum to SDS-denatured SBMV, unless crude sap containing the
virus was first boiled in SDS (Fig. 1) or lyophilized prior to
treatment with SDS. Sehgal and Das (96) have recently re-
ported that lyophilized SBMV is more susceptible to disruption
by SDS. It should be noted that different viruses (9, 10) or even
strains of the same virus (123) may differ markedly in their sus-
ceptibility to SDS. Likewise, SDS concentration, pH, and tem-
perature may also influence the effects of SDS on virus disrup-
tion (9, 10, 107, 123).

C. Composition of Media
The results of immunodiffusion tests can be markedly af-
fected by various factors, particularly the concentrations of SDS
and sodium salts.

1. Agar

lonagar No. 2 (121), Difco Noble agar (5, 35, 79) and
agarose (21) have been used successfully, but we have not made
critical comparisons of these agars. Gooding (21), however, sug-
gested that some types of agar may contain impurities that
would make their use undesirable.






























Fig. 2. Effects of adding SDS to the agar and to leaf extracts on
immunoprecipitin patterns. The central wells contain antiserum to SDS-
treated TEV (A, B, C) and antiserum to TEV pinwheel inclusions (D, E,
F). Peripheral wells contain: extracts from TEV-infected tobacco pre-
pared in 1.5% SDS (1) or in water (4); extracts from healthy tobacco
prepared in 1.5% SDS (2) or in water (5); 1.5% SDS (3); and water
(6). The medium for patterns A, B, D, E consisted of 0.8% Noble agar,
0.5% SDS, and 1.0% sodium azide, whereas the medium for patterns
C and F contained only 0.8% Noble agar and 1.0% sodium azide. Note
that sufficient SDS is present in the agar to satisfactorily degrade the
virus, but not pinwheel inclusions, into diffusible antigens (B and E,
respectively). When SDS is also added to the sap, however, the pin-
wheel inclusions are more extensively disrupted and strong reactions
are obtained (D). Neither the virus nor inclusion antigens diffuse when
SDS is lacking from both the medium and the sap (C, F).


2. Salts
Sodium azide (17%) is the only salt used in the medium re-
ported by Gooding and Bing (22) and others (e.g., 35, 79).
Batchelor (5) found that as sodium azide concentration was re-
duced there was a reduction in the strength of TEV-antiTEV
precipitates. Fig. 3 illustrates the effects of sodium azide con-


A 72M







II i










A B C D

Fig. 3. Influence of sodium azide and SDS on precipitin formation
with TEV. Each of the top wells was charged with resuspended, freeze-
dried extracts from TEV-infected tobacco. The bottom wells were
charged with similarly prepared extracts from healthy tobacco. The mid-
dle wells were charged with antiserum to SDS-treated TEV. The media
contain 0.8% Noble agar with the following additives: A=0.5% SDS
and 1.0% sodium azide; B=0.5% SDS and 0.05% sodium azide; C=
0.5% SDS; and D=1.0% sodium azide. As sodium azide concentration
is diminished, the precipitin bands become much smaller. When SDS
is omitted, no precipitin lines are formed, presumably because TEV is
unable to diffuse into the agar medium. Photographs taken 22 hrs after
reactants were added.


centration on immunoprecipitation of TEV antigens in SDS-
containing gels.
Batchelor (5) reported that sodium chloride could be sub-
stituted for sodium azide. Tolin and Roane (121) have developed
a medium which contains both sodium chloride and sodium azide.

3. SDS
It may be necessary to include SDS in the agar either to
facilitate dissociation of antigens (Figs. 2, 3) or to minimize
problems with nonspecific precipitates (Section III. F. 1.). Our
experience has been mostly with agar media containing 0.5%
SDS, but several workers have used lower concentrations with
good results (21, 121). Manipulation of the SDS and salt con-
centrations may prove valuable if the standard 0.5% SDS-1.0%
sodium azide medium fails to perform well with a particular
system.







Preparations of SDS which contain insoluble impurities
may result in cloudy media, and should not be used unless the
impurities are removed.


D. Effects of SDS on Immunoprecipitin Formation
and on Serum Titers
1. Ephemerality of precipitin lines

With double-diffusion tests conducted in SDS-media under
conditions outlined in Section II, the precipitin lines are strongest
after about 24-48 hours. Then the lines gradually fade and usu-
ally disappear completely after 5-10 days. This is of little sig-
nificance if the plates can be photographed or recorded at the
appropriate time. We have found that if reactant wells are
emptied after incubation of the plates for 20-24 hours, and filled
with a slurry of Norit A charcoal (15%, w/v, in water), the
precipitin lines can be partially or completely maintained for
months.
If reactant wells (antigen and antibody wells) are too far
apart, no precipitin lines will form; consequently, well spacing
is important.

2. Effects of SDS on serum titers

Titers of antisera in SDS-immunodiffusion are markedly de-
pendent on the material used for dilution of antisera. Antisera
diluted in normal serum had titers 2-8 fold higher than the same
sera diluted with 0.05 M Tris, pH 7.2, containing 0.85% NaCI
(Fig. 4 and Table 3). Thus the titers reported previously for
antisera to certain viruses and virus-induced inclusions may not
accurately reflect the relative potencies of those sera (82).
The basis for the enhancement of titers by dilution in normal
serum is unknown, but possibly it has to do with the protection
by other serum proteins against effects of SDS on antibody (11,
38) or on antigen-antibody precipitation. Before normal serum
is used as diluent, however, it should be tested against each anti-
gen for nonspecific reactions and to make sure that the serum
does not already contain antibodies to that antigen.
Preliminary tests have indicated that sera diluted in 5%
bovine serum albumin prepared in Tris-buffered saline have
titers similar to those of sera diluted in normal serum. In cases
where there is a likelihood that normal sera will already contain































Fig. 4. Effect of diluent on serum titer in SDS-immunodiffusion tests. Note enhancement of titer by dilution with
normal serum compared to dilution with buffered saline. Center wells A, B, and C were filled with TEV antiserum diluted
1/, 1/, and 1/, respectively, with normal serum. Center wells D. E. and F were filled with the same TEV antiserum diluted
1/2, 1/4, and //, respectively, with 0.85% NaCI in 0.05 M Tris, pH 7.2. The peripheral wells contained: 1-Purified TEV,
500 t.g/ml; 2-Purified TEV, 50 /Ag/ml; 3-Purified TEV, 10 /_g/ml; 4-Sap from healthy tobacco; 5 and 6-Sap from
TEV-infected tobacco. Medium=0.8% Noble agar, 0.5% SDS, 1.0% sodium azide. Photos taken 26 hr after reactants
were added.







Table 3. Effect of diluent on serum titer as determined in SDS-immuno-
diffusion tests."
Titer of serum when diluted in
Virus-
Antiserum 0.85% NaCI in
0.05 M Tris, pH 7.2 Normal Serumd
PMV 2 8
PVX 4 16
TEV 2 16
TEV 2 8
TEV' 4 8
TEV-I 2 16

"Titer expressed as reciprocal of highest dilution which gave visible precipitate
in SDS-immunodiffusion tests.
'Refer to Table 1 for meaning of abbreviations.
'Three different TEV antisera.
'Normal serum alone gave no reactions with any of the test antigens.


antibodies to the antigen under study (e.g., with animal viruses),
bovine serum albumin might be preferable to normal serum for
use as diluent of antiserum.
Glycerin is sometimes mixed with antiserum (1:1, v/v) for
use as a preservative (3). We have found, however, that anti-
sera diluted with glycerin may give considerably weaker reac-
tions than antisera diluted with normal serum in SDS-immu-
nodiffusion tests (based on results with antisera to WMV and
SBMV). Therefore, the routine use of antisera diluted with gly-
cerin is not recommended.

E. Immunospecific Reactions of Normal Plant Proteins

As with other immunodiffusion systems (99, 129), virus
antisera that are contaminated with antibodies to normal plant
proteins may react with these proteins in SDS-immunodiffusion
tests. Such sera may be absorbed with healthy plant proteins
prior to use (63, 84). Appropriate controls from noninoculated
(healthy) plants should always be included in serodiagnostic
tests.

F. Nonspecific Precipitates

Nonspecific precipitates are considered to be those that are
not the result of specific antigen-antibody reactions. Use of ap-
propriate controls is essential for detecting nonspecific reactions.






Normal serum nonimmunee serum) should be tested against all
antigens, and extracts from healthy plants of each species should
be tested against all sera as minimal controls. Several types of
nonspecific reactions that have been noted are as follows.


1. Serum-SDS interactions

These types of nonspecific reactions have been commonly ob-
served when either SDS-treated antigen or SDS alone is tested
against sera in agar media which lack SDS (12, 58, 70; Fig. 5).
Sera which have been stored at 40C were reported to give
stronger nonspecific reactions with SDS than sera which were
stored frozen (58). The nonspecific precipitates can be very con-
fusing because they often are similar in appearance and location
to the antigen-antibody precipitates. If SDS is incorporated into
the agar medium, however, the nonspecific SDS-serum reactions
usually occur as a ring at the edge of the serum well (Fig. 5).
Under these conditions, the nonspecific reactions usually do not
seriously interfere with interpretation of double-diffusion tests.
In the case of one TEV antiserum, a confusing, nonspecific
SDS-serum precipitate was observed even though the medium
contained SDS. This nonspecific reaction was minimized by
adding 2% bovine serum albumin to the medium (Fig. 6). The
use of bovine serum albumin as an additive to the medium, how-
ever, needs further evaluation. In tests with an antiserum to
SDS-denatured SBMV, the immunospecific reactions were also
weaker in plates containing bovine serum albumin.
According to van Regenmortel, von Wechmar, and Lelarge
(personal communication), nonspecific serum-SDS interactions
could be prevented by using partially purified gamma globulins
rather than whole serum. They used globulins that were precipi-
tated twice with 4M ammonium sulfate solution (1 vol 4M solu-
tion with 1 vol antiserum).


2. Serum-plant sap interactions

Extracts from some plant species give nonspecific precipi-
tates with serum (99). For example, the healthy pepper extract
in Fig. 7 gave a precipitate with normal serum; dialysis of the
serum overnight against 0.05 M Tris-HC1, pH 7.2, containing
0.85% NaCI (98) was effective in preventing this type of pre-
cipitate.


























S H.




H H S






Fig. 5. Importance of adding SDS to agar media to minimize occur-
rence of confusing nonspecific precipitates (those which do not involve
specific antigen-antibody reactions) in immunodiffusion tests with SDS-
treated antigens. The center wells were charged with TEV-antiserum
(A) and normal serum (N). The peripheral wells were charged with:
1.5% SDS (S); extracts from TEV-infected tobacco (V) treated with 1.5%
SDS; or extracts from healthy tobacco (H) treated with 1.5% SDS. The
agar media for the upper patterns contained 0.8% Noble agar, 1.0%
sodium azide and 0.5% SDS, whereas the medium for the lower pat-
terns contained only agar and sodium azide. In media containing SDS
(upper patterns), the nonspecific reactions with either antiserum or
normal serum occur as rings at the edge of the serum wells, and the
virus specific reaction (between A and V) is easy to discern. In media
without SDS (lower pattern), the SDS diffusing out of the peripheral
wells precipitates serum components farther out in the agar, somewhat
complicating interpretation of the test.





.A
A7




1A Vr
Fig. 6. Effect of bovine serum albumin on nonspecific precipitates
with certain antisera. The peripheral wells contain: V=lyophilized ex-
tracts from TEV-infected tobacco in 1.5% SDS; H=lyophilized extracts
from healthy tobacco in 1.5% SDS; and S=1.5% SDS alone. The center
wells contain TEV antiserum. The agar medium in A contains Noble
agar-SDS-sodium azide. The agar medium in B is the same except that
2% bovine serum albumin has been added. Note the nonspecific precipi-
tates in A which are absent in B.















Fig. 7. Nonspecific precipitates (those which do not involve
specific antigen-antibody reactions) in SDS-immunodiffusion tests, due
to sap-serum interactions. The wells contain extracts from healthy pep-
per (P), normal serum (N), and normal serum after dialysis overnight
against buffered saline (Nd). The nonspecific reactions appear as bands
between the serum well and wells containing pepper sap; dialysis re-
moved the serum factor responsible for the pepper sap-serum precipi-
tate.






3. Plant sap-plant sap interactions
Precipitates sometimes form between wells which contain
crude plant extracts, particularly when the extracts are from
different species. This type of precipitate may occur, for exam-
ple, between adjacent wells containing pepper and tobacco ex-
tracts (Fig. 8). The types of precipitates observed in Figs. 7 and
8 were removed by soaking the agar plates for 1-2 days in a bath
of 0.02 M sodium phosphate, pH 7.4, containing 0.857% NaC1. On
the basis of other experiments, this treatment did not remove
specific immunoprecipitates.


A


Fig. 8. Nonspecific precipitates due to sap-sap interactions. The wells
contain extracts from healthy pepper (P) and healthy tobacco (T). Non-
specific reactions occur between wells containing heterologous plant
saps.

4. Chemical additives to media or to reactants
Nonspecific precipitation can occur with certain chemicals in
SDS-immunodiffusion tests. For example, Batchelor (5) noted
strong nonspecific reactions when urea was added to agar me-


'_ :ciii







dium containing SDS. If materials are to be added to the me-
dium (or substitutions made), it is suggested that the sensitivity
and specificity of tests in the modified media be compared di-
rectly to the sensitivity and specificity of tests in the standard
medium (0.5% SDS-1% sodium azide-0.8% agar).


IV. USES IN PLANT VIROLOGY FOR IMMUNODIFFUSION
TESTS WITH SDS-TREATED ANTIGENS
A. Diagnosis and Detection of Plant Viruses
SDS has proved very useful in facilitating diagnosis of vari-
ous viruses and for detection of viruses or inclusion body pro-
teins in crude extracts from over 30 species in 10 plant families
(Table 4). For the past five years we have used the SDS-im-
munodiffusion technique reliably for diagnosis of several potato
Y group viruses, potato virus X, and tobacco mosaic virus in
various crops or weeds. The method has given positive results
with each virus-host combination tested, although individual
samples have sometimes given negative results (e.g., lettuce mo-
saic virus in lettuce and endive). These failures were presumably
due to low virus concentration.
Although the SDS immunodiffusion tests have been reported
to work satisfactorily for 15 potyviruses (Table 1), unsatisfac-
tory results have been reported for at least one virus in this
group, the pea seed-borne mosaic virus (109). Cowpea aphid-
borne mosaic, another potyvirus, has been reported to give both
positive (18) and negative results (73) in SDS-immunodiffusion
tests. The reasons for negative results with those viruses are not
understood.

B. Relationships of Plant Viruses
Analyses of some serological relationships of SDS-denatured
viral antigens in immunodiffusion tests may be summarized as
follows. Virus strains may give reactions of identity, such as
with certain PVY strains (23) or TEV strains (79). Strains of
certain viruses, such as TuMV, have been reported to give either
reactions of identity or partial identity, dependent on the strain
(63). Different viruses within a taxonomic group, such as the
PVY group, may either cross-react to give spur formation (e.g.,
PeMV and PVY in Fig. 9), or give no cross-reaction (e.g., BMoV
and LMV in Fig. 10). Batchelor (5) found no cross reactivity
between three viruses (PVX, PMV, and CYMV) in the PVX























Fig. 9. Reactions of partial identity (spur formation in left pattern)
and identity (fused precipitin lines in right pattern) in SDS-immunodif-
fusion. The center wells were charged with PVY antiserum. The pe-
ripheral wells were charged with SDS-treated, freeze-dried extracts from
tobacco infected with PeMV (P) or PVY (Y) and healthy tobacco (H).
PeMV and PVY are immunochemically related but distinct, resulting in
spur formation in the left pattern.

group. Likewise, in the papers referenced in Tables 1 and 4 and
in our unpublished work, there is no evidence that viruses in
different morphological groups (e.g., PVX and PVY) cross-react
at all in SDS-immunodiffusion.
Some differences in the specificity of pyrrolidine-treated ver-
sus SDS-treated TuMV antigens have recently been reported
(34). McDonald (62) reported that strains which gave spur for-
mation when treated with SDS, failed to do so when tested by
immunodiffusion following treatment with pyrrolidine. He also
showed that in SDS-immunodiffusion, TuMV in freshly prepared
crude extracts spurred over partially purified preparations of
TuMV that had been stored for 15 months at 40C. Hiebert and
McDonald (34) showed that the loss of antigenic activity in
partially purified TuMV was due to proteolytic degradation of
the capsid protein. Differences between freshly prepared antigens
and virus with proteolytically degraded capsid protein were not
detected, however, when the antigens were treated with pyrroli-
dine rather than SDS.
The finding that antigenic differences can exist between dif-
ferent preparations of the same virus is a factor that should be
considered when making serological comparisons of PVY-group








Table 4. Host plants used as crude antigen sources for SDS-immunodiffusion
tests with plant viruses or virus-induced inclusions.

Genus and Species Family Antigena Citation


Arachis hypogea L.
Argemone mexicana L.
Bidens pilosa L.
Brassica perviridis Bailey

Capsicum annuum L.









Carica papaya L.
Cassia tora L.
Chenopodium quinoa Willd.
Cichorium endivia L.
Citrus medical L.



Cladrastis lutea Koch
Colocasia antiquorum Schott
Colocasia esculenta (L.)
Schott
Cucurbita pepo L.
Datura metel L.

Datura stramonium L.



Erechtites hieracifolia
(L.) Rab.
Erigeron canadensis L.
Glycine max (L.) Merr.


Helianthus annuus L.

Lactuca sativa L.


Lepidium virginicum L.


Leguminosae
Papaveraceae
Compositae
Cruciferae

Solanaceae









Caricaceae
Leguminosae
Chenopodiaceae
Compositae
Rutaceae



Leguminosae
Araceae
Araceae

Cucurbitaceae
Solanaceae

Solanaceae



Compositae

Compositae
Leguminosae


Compositae

Compositae


Cruciferae


PMoV 120
BMoV 84
BMoV 80,84
TuMV 5,34,63
TuMV-I 63,82
PeMV 5,66,79,85,
139,140
PeMV-I 5,82,85
PVY 66,79,85,
138,139,
140
TEV 66,79,85,
138,139,
140
PMV 5,79
TEV Unpublished
BMoV 84
BMoV 84
CVV Purcifull,
Garnsey, &
Gonsalves,
unpublished
BYMV 77
DMV 1
DMV 1


WMV
PVY-I
TEV-I
PVX
TEV
TEV-I
TEV-NI
BMoV

BMoV
BPMV
PMoV
SoyMV
BMoV
BMoV-I
BMoV
BMoV-I
LMV
BMoV


5
82
82
Unpublished
79
Unpublished
45
84

84
121
121
121
84
82
79,80,84,141
Unpublished
5,79,84,141
80


continued







Table 4. Host plants used as crude antigen sources for SDS-immunodiffusion
tests with plant Viruses or virus-induced inclusions. (Continued).

Genus and Species Family Antigena Citation


Lupinus angustifolius L. Leguminosae BMoV


Lycopersicon esculentum
Mill.



Nicotiana hybrid









Nicotiana tabacum L.











Petunia hybrida Vilm.

Phaseolus vulgaris L.

Philodendron selloum C.
Koch
Pisum sativum L.


Proboscidea jussieu
Keller
Vigna unguiculata (L.)
Walp
Zinnia elegans Jacq.


Solanaceae




Solanaceae









Solanaceae











Solanaceae

Leguminosae

Araceae

Leguminosae


Martyniaceae

Leguminosae

Compositae


PVY
PVY-I
TEV
TEV-I
TMV
BMoV
BMoV-I
PeMV
PeMV-I
PVMV
PVY
PVY-I
TEV
TEV-I
PeMV
PeMV-I
PVX
PVY

PVY-I
TEV

TEV-I
TMV
TVMV
PVY-I
TEV-I
BYMV
SBMV
DMV

BYMV
CYMV
LMV
BYMV

BICMV

BMoV
BMoV-I


Edwardson,
Christie, &
Purcifull,
unpublished
22
82
22
82
Unpublished
5,79,80,84
82
79
82
5
Unpublished
82
Unpublished
82
5,66,79
5,82
5,21,24,79
5,21,23,24,66,
79
5,82
5,13,21,22,24,
66,79
5,82
79
5,74,106,112
82
82
76
5
1

76,77
5,79
79
78,127

53

84
82


aSee Table 1 for meaning of abbreviations.
























Fig. 10. Use of SDS-immunodiffusion for distinguishing viruses in
crude plant extracts. The peripheral wells contain extracts that were
prepared in 1.5% SDS, freeze-dried and then resuspended in water just
prior to use:
1-BMoV in naturally infected lettuce
2-LMV in naturally infected lettuce
3-LMV in garden pea
4-Healthy lettuce
5-Healthy pea
6-BMoV in Nicotiana hybrid
The center wells contain lettuce mosaic virus antiserum (L) and bidens
mottle virus antiserum (B).


viruses by SDS-immunodiffusion (34). Other PVY-group viruses
also can show this phenomenon, as evidenced by results with a
TEV preparation provided by E. Hiebert (Fig. 11).

C. Analyses of Proteinaceous Inclusion Bodies Induced by
Potato Y Group (PVY) Viruses

The members of the PVY group of plant viruses character-
istically induce pinwheel inclusions in their hosts (17). The pin-
wheel inclusions have been isolated and are known to consist of
protein subunits with molecular weights of about 70,000 (33).
The principal findings about serological properties of the inclu-
sions, based on SDS-immunodiffusion tests, are these:
1) The inclusion body proteins are immunochemically distinct
from viral coat protein or host proteins (35, 63, 82).

2) Each of five different viruses (BMoV, PeMV, PVY, TEV,
TuMV) induces a type of inclusion protein that is immuno-






Fig. 11. Evidence for antigenic
difference between TEV in crude
plant extracts and purified prepara-
tions of TEV which contain de-
graded coat protein (e.g., as re- ( i
ported by Hiebert and McDonald,
1976). The center well contains
antiserum to SDS-denatured TEV;
the peripheral wells contain antigens
in 0.5% SDS as follows: Resus- -
pended, freeze-dried crude extracts
from TEV-infected tobacco (1 and
4); purified TEV, 0.5 mg/ml (2);
freshly prepared crude extracts from
TEV-infected tobacco (3); and ex-
tracts from healthy tobacco (5 and 6). Note that TEV in either fresh or
freeze-dried crude extracts apparently contains determinants that are
lacking in the purified TEV. Some purified TEV preparations, however,
may give reactions of identity with TEV in crude extracts, as shown in
Fig. 4.

chemically distinct from the others (82), although the in-
clusions induced by some of these viruses are serologically
related.

3) Three strains of TuMV induce immunochemically identical
inclusion proteins, even though the virus coat protein of
one of these strains is distinct from the others (62, 63).

4) The pinwheel inclusion proteins induced by a particular
virus in different plant species show reactions of serologi-
cal identity (63, 82 and Fig. 12).
5) Inclusion proteins of two different viruses (TEV and
PVY) can both be detected in extracts from doubly-in-
fected plants (Fig. 13, A, B). The coat proteins of both
viruses can also be detected in the same extracts (Fig. 13,
C, D).
6) PeMV inclusion protein and PeMV were produced in
plants that were inoculated with purified PeMV prepara-
tions which indexed negatively for PeMV-inclusion protein
(85).
These findings and cytological evidence (17) support the
proposal that the pinwheel inclusions are virus-specified (35, 82).
Although the inclusions are produced abundantly in infected
tissues and are apparently characteristic for the PVY group,
their functions are unknown.




















Fig. 12. Immunochemical specificity of pinwheel inclusion antisera.
Center wells were filled with antisera to TEV-I (A) and PVY-I (B). The
peripheral wells were filled with SDS-treated extracts from PVY-infected
petunia (1), PVY-infected tobacco (2), TEV-infected tobacco (3), TEV-
infected petunia (4), healthy petunia (5), and healthy tobacco (6). TEV-I
and PVY-I are serologically distinct, and specificity of the inclusions
induced by a particular virus is host-independent (e.g., based on re-
actions of identity with PVY-I from petunia and tobacco).


The crystalline nuclear inclusions induced by TEV have also
been partially purified and studied serologically in SDS immuno-
diffusion tests (45). That study provided evidence that the nu-
clear inclusions are serologically distinct from TEV coat protein,
host protein, and TEV pinwheel inclusion protein. TEV-induced
nuclear inclusions isolated from infected plants of three different
species were immunochemically identical. The results of Knuht-
sen, et al. (45) were based on studies with antisera obtained by
immunizing rabbits with untreated antigens (TEV pinwheel in-
clusions, nuclear inclusions, and coat protein). Batchelor (5)
obtained antisera to the SDS denatured antigens and confirmed
that the three proteins are immunochemically distinct.

D. Characterization of Viral Proteins
Polyacrylamide gel electrophoresis is a useful and popular
method for the characterization of SDS-treated viral proteins
(43, 71, 72, 130, 132). The proteins of several plant viruses or
virus-induced proteins have been specifically identified by sero-
logy following electrophoresis of SDS-treated preparations on
gels (5, 33, 44, 46, 82, 97, 100, 103, 137). In some cases antisera
have been prepared against the proteins following electrophoresis
(46, 100).









6 66




5 3 5




1 1





55 3



Fig. 13. Use of pinwheel inclusion and viral coat protein antisera
to distinguish TEV from PVY. Center wells contain: A-antiserum speci-
fic for TEV inclusions; B-antiserum specific for PVY inclusions; C-
antiserum specific for TEV coat protein; and D-antiserum specific for
PVY coat protein. The peripheral wells contain SDS-treated extracts
from Nicotiana hybrids as follows: 1 and 5-extracts from plants singly
infected with TEV; 2 and 4-extracts from plants doubly infected with
PVY and TEV; 3-extracts from plants singly infected with PVY; 6-ex-
tracts from healthy plants. Note that both TEV and PVY inclusion anti-
gens and coat proteins of both viruses are detected in extracts from
doubly infected plants.

V. CONCLUDING REMARKS
Successful use of the SDS-immunodiffusion methods can be
markedly affected by several characteristics of the system: (1)
SDS is an important component of the agar medium, not only for
promoting dissociation of proteins into diffusible antigens, but







also in helping to minimize occurrence of confusing nonspecific
precipitates; (2) immunoprecipitates formed in SDS gels usually
do not persist longer than a few days; (3) the concentrations of
sodium azide and SDS affect intensity of precipitates; (4) serum
titers are considerably higher when sera are diluted with normal
serum as compared with buffered saline; (5) in cases where
marked antigenic disparity exists between virus and its SDS-
denatured coat protein, an antiserum to the latter may be neces-
sary to achieve satisfactory results.
The immunodiffusion procedures described herein have been
applied successfully for detection of a wide variety of plant vi-
ruses and virus-induced inclusions. Further explorations of the
SDS-immunodiffusion system should include efforts to enhance
its reliability and to test its applicability for an even broader
range of viruses.


VI. REFERENCES ON TECHNIQUES AND ON THE EFFECTS
OF SDS ON VIRUSES AND PROTEINS
A. Immunodiffusion studies with SDS-treated plant viruses (see
Table I for references on specific viruses).
B. Serological studies with other SDS-treated viruses or proteins
(11, 31, 38, 41, 42, 48, 57, 92, 94, 95, 111, 134, 136).
C. Nature of SDS-protein interactions and binding of SDS to
proteins (14, 19, 26, 32, 37, 52, 65, 75, 86, 89, 90, 91, 93, 108,
110, 114, 115, 116, 117, 118, 119, 124, 131, 132, 135).
D. Separation of SDS-treated proteins (5, 14, 64, 132).
E. Methods for removing SDS from solution (5, 8, 51, 88, 105,
125, 128, 136).
F. Purification and/or assay of SDS (15, 30, 65, 87, 91, 119).




ACKNOWLEDGEMENTS
We appreciate the assistance of S. R. Christie and W. E. Craw-
ford. Grateful acknowledgement is made to E. Hiebert for the puri-
fled TEV used for the tests shown in Fig. 11, and to A. A. Brunt for
antiserum to pepper veinal mottle virus.
We thank the following persons for allowing us to cite their un-
published data: 0. W. Barnett, S. R. Christie, A. O. Jackson, R.
Koenig, J. A. Lima, and M. H. V. van Regenmortel.
This work was supported in part by Grants GB-32093 and BMS
75-14014 from the National Science Foundation.







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Institute of Food and Agricultural Sciences


TEACHING IFAS
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