Title: Production and characterization of polyclonal and monoclonal antibodies to three virus-induced proteins of papaya ringspot virus type W
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00102750/00001
 Material Information
Title: Production and characterization of polyclonal and monoclonal antibodies to three virus-induced proteins of papaya ringspot virus type W
Physical Description: Book
Language: English
Creator: Baker, Carlye Ann, 1948-
Copyright Date: 1989
 Record Information
Bibliographic ID: UF00102750
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: ltuf - AHH6778
oclc - 22711592

Full Text












PRODUCTION AND CHARACTERIZATION OF POLYCLONAL AND
MONOCLONAL ANTIBODIES TO THREE VIRUS-INDUCED PROTEINS OF
PAPAYA RINGSPOT VIRUS TYPE W







By

CARLYE ANN BAKER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1989















ACKNOWLEDGEMENTS


No endeavor is ever accomplished by any one person

without the help and support of other people. The completion

of this dissertation is no exception.

I would like to thank Dr. Dan Roberts, undergraduate

coordinator and my first teacher in the field of plant

pathology, for kindling my interest in plant pathology and

encouraging me to aim for a Ph.D., not just a second

bachelor's degree.

I would like to thank Dr. Charudattan, the graduate

coordinator during the midpoint of this endeavor, for never

losing faith or patience as I tried to redirect the focus of

my studies.

I owe the greatest debt of gratitude to Dr. F.W.

Zettler. He recognized my potential and not only believed in

me himself but encouraged others to believe in me as well. I

wish to express my deepest appreciation to Dr. Zettler for

giving me the chance to prove myself, for patiently putting

up with me as I learned to write scientific papers, and,

most of all, for his friendship and good humor.

I want to thank Richard Christie for teaching me the

skills that allowed me to publish my first paper and gave me










two trips to Mexico. I am always ready to carry on the

crusade.

To Dr. Dan Purcifull, my boss on the endless peanut

project and then chairman of my supervisory committee, I

offer sincere appreciation for his example of scientific and

personal integrity; his expertise; his uncompromising,

honest, and fair critiques of my work; and his patience, or

is that "pateince"?

I would like to thank Dr. Hiebert, Dr. Zam, and Dr.

Edwardson for serving on my committee and for your help and

suggestions throughout this work. Your belief in me, your

encouragement, and your support has been invaluable.

I am grateful to Kristin Figura, now Kristin Beckham

and Mom, Mark Elliott, Valerie Seay, Gail Wisler, and

especially Gene Crawford for their support, their friendship,

and their endless patience with a person who always wanted

things she couldn't find on her own RIGHT NOW!

Special thanks goes to the late Steve Christie, whom

I miss and who always was willing to give help and advice

when I needed it. I am sorry he missed the 1988 election; he

would have enjoyed it.

To my parents, Bob and Eva Baker, who were always

enthusiastic and supportive even though they had a hard time

explaining to friends why their eldest daughter was still in

school, I owe a great debt of gratitude. They gave me my

thirst for scientific knowledge which started me on this

career change and has kept me going throughout.










I would also like to thank Dr. J. Moore of the

University of Georgia, for his help with Fig. 2-1 and for

the use of his Macintosh II while we were in Georgia for 'the

book'.

Last but certainly not least, I want to thank Patrick

Colahan, my husband and my very best friend. There really

aren't words that can express my gratitude to him for his

constant and unfaltering support and encouragement.

Presents, I know, presents.


















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS.............................................ii

LIST OF FIGURES............................................... vii

LIST OF TABLES.................................... ...........xi

ABSTRACT ................ ..................................xiii

CHAPTER


1. INTRODUCTION...........................................1

2. PRODUCTION AND PARTIAL CHARACTERIZATION OF
POLYCLONAL ANTISERUM AND MONOCLONAL ANTIBODIES TO
THE CAPSID PROTEIN OF PAPAYA RINGSPOT VIRUS
TYPE-W..................................................... 14

Introduction.............................. ............14
Materials and Methods ................................ 18
Preparation of Immunogens ....................... 18
Polyclonal Antisera ............................. 19
Monoclonal Antibodies ........................... 20
Polyclonal Mouse Ascites ........................ 24
Virus Isolates .................................. 25
SDS Immunodiffusion ..............................28
Indirect ELISA ..................................29
Aphid Transmission .............................. 33
Protein Blotting.................................34
Results .............................................. 36
SDS-Immunodiffusion Tests ....................... 36
Indirect ELISA .................................. 38
Fusions, Screening, and Cloning ................. 52
Characterization of Six Monoclonal Antibodies
(MCAs) ....................................... 59
Discussion ........................................... 64










3. PRODUCTION AND PARTIAL CHARACTERIZATION OF
POLYCLONAL ANTISERUM AND MONOCLONAL ANTIBODIES TO
THE AMORPHOUS INCLUSION PROTEIN OF PAPAYA RINGSPOT
VIRUS TYPE-W ......................................... 90

Introduction ......................................... 90
Materials and Methods ................................ 95
Protein Purification ............................ 95
Polyclonal Antiserum ............................96
Production of Monoclonal Antibodies. ............ 97
Virus Isolates .................................. 99
Indirect ELISA ..................................101
Protein Blots ...................................101
SDS-Immunodiffusion................................ 101
Light Microscopy................................ 102
Results.................................. ...........102
Light Microscopy ................................ 102
Reactivity of Polyclonal Antiserum
1136 .............................................102
Results of Fusions and Primary Screening........ 111
Reactivity of Monoclonal Antibody (MCA) F10E-
A5..............................................112
Reactivity of MCA F19K-G3.......................116
Discussion...........................................121

4. PRODUCTION AND PARTIAL CHARACTERIZATION OF
POLYCLONAL ANTISERUM AND MONOCLONAL ANTIBODIES TO
THE CYLINDRICAL INCLUSION PROTEIN OF PAPAYA
RINGSPOT VIRUS TYPE-W ................................127

Introduction........................................ 127
Materials and Methods ................................131
Protein Purification.............................131
Polyclonal Antiserum.............................132
Monoclonal Antibodies ...........................133
Virus Isolates................................. 134
Serology............................................ 134
Results ..............................................135
Reactivity of Polyclonal Antiserum 1130..........135
Monoclonal Antibodies ...........................140
Discussion. ....... ...................................143

5. SUMMARY AND CONCLUSIONS..............................146

REFERENCES................................................151

BIOGRAPHICAL SKETCH......................................... 162















LIST OF FIGURES


Figure Page


2-1 Diagram of the steps used in two types of
indirect ELISA ....................................30

2-2 Serological cross-reactivity of seven
potyviruses with papaya ringspot
virus type-W in sodium dodecyl sulfate
(SDS)-immunodiffusion tests........................39

2-3. Serological assay for papaya ringspot virus
type-W (PRSV-W) and Trichosanthes virus (TV) in
manually and aphid inoculated Cucurbita pepo L.
'Small Sugar pumpkin' (ssp) ........................40

2-4. Detection of serological differences between
papaya ringspot virus type-W (PRSV-W) isolate
W-1A and two other isolates of PRSV-W in
sodium dodecyl sulfate (SDS)-immunodiffusion
tests ...............................................41

2-5. Serological reactivity of polyclonal antiserum
1125 (bleeding Feb.l, 1987) with papaya ringspot
virus (PRSV) isolates and other cucurbit
potyviruses in sodium dodecyl sulfate (SDS)
immunodiffusion tests ..............................42

2-6 Reactivity of polyclonal antiserum 1125 with
papaya ringspot virus isolates and six other
viruses in plate-trapped indirect ELISA test
E91 .................................................46

2-7 Reactivity of polyclonal antiserum 1125 with
papaya ringspot virus isolates and seven other
viruses in plate-trapped indirect ELISA test
E-94................................................47

2-8 Reactivity of monoclonal antibody F3C-C10
(MCA-5) and polyclonal antiserum 1125 with 22
papaya ringspot virus isolates in plate-trapped
indirect ELISA .....................................48


vii










2-9 Reactivity of polyclonal antiserum 1125 with
various potyviruses in plate-trapped indirect
ELISA tests (E-75 and E-77)........................ 49

2-10 Reactivity of polyclonal antiserum 1142 with
various potyviruses in two plate-trapped indirect
ELISA tests, E-75 and E-77......................... 50

2-11 Reactivity of polyclonal ascitic fluid to papaya
ringspot virus type-W (PCA-WV) and its capsid
protein (PCA-CP) in antibody-trapped indirect
ELISA with papaya ringspot virus and other
potyviruses which infect cucurbits .................54

2-12 Reactivity of polyclonal ascitic fluid to papaya
ringspot virus type-W (PCA-WV) and its capsid
protein (PCA-CP) in plate-trapped indirect ELISA
with papaya ringspot virus and other viruses
which infect cucurbits .............................57

2-13 Reactivity of tissue culture fluid to monoclonal
antibody F21D-E10 (MCA-1) against papaya ringspot
virus (PRSV) and other potyviruses in indirect
ELISA...............................................68

2-14 Reactivity of ascitic fluid to monoclonal
antibody F21D-E10 (MCA-1) to papaya ringspot
virus and other potyviruses in indirect
ELISA...............................................69

2-15 Reactivity of tissue culture fluid to monoclonal
antibody F22A-B9 (MCA-2) to papaya ringspot virus
and other potyviruses in indirect ELISA............70

2-16 Reactivity of ascitic fluid to monoclonal
antibody F22A-B9 (MCA-2) with papaya ringspot
virus and other potyviruses in indirect ELISA......71

2-17 Reactivity of monoclonal antibody-2 (MCA-2)
with watermelon mosaic-2 (WMV-2) in indirect
ELISA...............................................72

2-18 Reactivity of tissue culture fluid to monoclonal
antibody F22A-E8 (MCA-3) to papaya ringspot virus
and other potyviruses in indirect ELISA............73

2-19 Reactivity of tissue culture fluid to monoclonal
antibody F21C-E4 (MCA-4) with papaya ringspot
virus and other potyviruses in indirect ELISA......74


viii









2-20 Reactivity of ascitic fluid to monoclonal
antibody F21C-E4 (MCA-4) with papaya ringspot
virus and other potyviruses in indirect ELISA......75

2-21 Reactivity of tissue culture fluid to monoclonal
antibody F3C-C10 (MCA-5) with papaya ringspot
virus and other potyviruses in indirect ELISA......77

2-22 Reactivity of ascitic fluid to monoclonal
antibody F3C-C10 (MCA-5) with papaya ringspot
virus and other potyviruses in indirect ELISA......78

2-23 Reactivity of monoclonal antibody F3C-C10 (MCA-5)
with eleven potyviruses in plate-trapped indirect
ELISA...............................................79

2-24 Reactivity of monoclonal antibody F22A-C8 (MCA-6)
with papaya ringspot virus and other
potyviruses in indirect ELISA......................80

3-1 Serological detection in SDS-immunodiffusion
tests of the amorphous inclusion protein of
papaya ringspot virus (PRSV) ..................... 104

3-2 Serological detection in SDS-immunodiffusion of
cross-reactivity of polyclonal antiserum (1136)
to the amorphous inclusion protein of papaya
ringspot virus type-W (PRSV-W) ....................105

3-3 Reactivity of polyclonal antiserum 1136 with
papaya ringspot virus and six other viruses in
plate-trapped indirect ELISA......................106

3-4 Reactivity of monoclonal antibody (MCA) FO1E-A5
in protein blots ..................................114

3-5 Reactivity of monoclonal antibody (MCA) F10E-A5
with papaya ringspot virus and other potyviruses
in indirect ELISA .................................117

3-6 Reactivity of monoclonal antibody (MCA) F19K-G3
with papaya ringspot virus and other potyviruses
in indirect ELISA .................................118

4-1 Reactivity of polyclonal antiserum 1130 in plate-
trapped indirect ELISA with extracts of various
squash potyviruses................................ 136

4-2 Reactivity of polyclonal antiserum to papaya
ringspot virus type-W cylindrical inclusions
antiserumm 1130) with various antigens............137








4-3 Effect of absorption with host antigens on
reactivity of polyclonal antiserum to papaya
ringspot virus type-W cylindrical inclusions...... 138

4-4 Serological detection of the cylindrical
inclusion protein of papaya ringspot virus in
SDS-immunodiffusion tests..........................139















LIST OF TABLES


TABLE Page

2-1 Cross-absorption test with polyclonal antiserum
1142...............................................43


2-2 Plate-trapped indirect ELISA test to determine
the optimal dilutions F3C-C10 (MCA-5) and to
polyclonal antiserum 1125 (PCA-1125) ..............45

2-3 Use of plate-trapped indirect ELISA to assay
papaya inoculated with isolates of papaya
ringspot virus ....................................51

2-4 Indirect ELISA tests to determine the optimal
dilution of polyclonal ascitic fluid to papaya
ringspot virus type-W..............................55

2-5 Indirect ELISA tests to determine the optimal
dilution of polyclonal ascitic fluid to papaya
ringspot virus type-W capsid protein..............56

2-6 Summary of data from fusions 2, 3, and 4.........58

2-7 Cloning results for monoclonal antibodies (MCAs)
to papaya ringspot virus type-W or its capsid
protein ...........................................65

2-8 Isotyping of six monoclonal antibodies to papaya
ringspot virus type-W by indirect ELISA...........66

2-9 Indirect ELISA tests to determine the optimal
dilution of ascitic fluid to monoclonal antibody
F21D-E10 (MCA-1) ..................................67

2-10 Indirect ELISA tests to determine the optimal
dilution of ascitic fluid to monoclonal antibody
F21C-E4............................................... 76

2-11 Summary of the reactivity of six monoclonal
antibodies (MCA) with papaya ringspot virus and
other viruses that affect cucurbits...............81









3-1 Representative data of the reactivities of
polyclonal antiserum 1136 in plate-trapped
I-ELISA and of MCA F1OE-A5 in plate-trapped and
antibody-trapped I-ELISA with papaya ringspot
virus type-W (PRSV-W) and various other
potyviruses. ..................................... 107

3-2 Reactivity of monoclonal antibodies F3C-C10,
F1OE-A5, and F12A-B10 to three purified proteins
of papaya ringspot virus type-W (PRSV-W)..........115

3-3 Reactivity of monoclonal antibody F19K-G3 in
plate-trapped and antibody-trapped indirect
ELISA to papaya ringspot virus type-W (PRSV-W)
and ten other potyviruses........................119

3-4 Summary of the reactivity of polyclonal
antiserum 1136 (PCA-1136) and monoclonal
antibodies FO1E-A5 and F19K-G3 to papaya
ringspot virus type-W (PRSV-W) and 16
other potyviruses................................ 120

4-1 Reactivity of polyclonal antiserum 1130 and two
monoclonal antibodies to the cylindrical
inclusion protein of papaya ringspot type W
(PRSV-W) with various isolates of PRSV...........142


xii















Abstract of Dissertation Presented to the
Graduate School of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



PRODUCTION AND CHARACTERIZATION OF POLYCLONAL
AND MONOCLONAL ANTIBODIES TO THREE VIRUS-INDUCED PROTEINS
OF PAPAYA RINGSPOT VIRUS TYPE-W

By

Carlye Ann Baker

December, 1989


Chairman: Dan E. Purcifull
Major Department: Plant Pathology


Papaya ringspot virus type-W (PRSV-W) encodes capsid

(CP), amorphous inclusion (AIP), and cylindrical inclusion

(CIP) proteins which can be purified and are immunogenic.

The purpose of this study was to produce monoclonal

antibodies (MCAs) and polyclonal antiserum (PCA) to each of

these proteins and assess their potential for the detection

and differentiation of PRSV.

The MCAs were obtained after fusion of Sp2/O-Ag 14

myeloma cells with spleen cells of BALB/c mice immunized with

PRSV-W, its CP, AIP, or CIP. The PCA was obtained by

immunization of New Zealand white rabbits. All were assayed

in indirect enzyme-linked immunosorbent assay (I-ELISA)

and/or protein blots (PB) against 15 isolates of PRSV-W, one


xiii










isolate of PRSV from papaya (PRSV-P), the Tigre isolate of

PRSV (PRSV-T), the Moroccan watermelon mosaic virus, zucchini

yellow fleck virus (ZYFV), zucchini yellow mosaic, watermelon

mosaic virus-2 (WMV-2), and healthy squash.

Six MCAs to PRSV-W or its CP defined six epitopes of

PRSV. PCA to the CP of PRSV-W detected a seventh epitope in

SDS-immunodiffusion. Six of these epitopes varied among PRSV

isolates. Two of the MCAs cross-reacted with WMV-2. Three of

the six MCAs reacted in PB to a 34kD protein of PRSV-W

infected squash.

Two MCAs to PRSV-W AIP defined two epitopes of the

AIP. One MCA cross-reacted with ZYMV, ZYFV, and WMV-2. The

other cross-reacted only with ZYFV. Both MCAs reacted to a

51kD protein of PRSV-W extracts in PB.

Two MCAs to the PRSV-W CIP reacted with purified CIP

in I-ELISA and with a 69kD protein of PRSV extracts in PB.

They defined two epitopes of the CIP, one present on 15

isolates of PRSV-W and PRSV-P; the other on 12 of 15 PRSV-W

isolates, PRSV-P and PRSV-T. PCA reacted to all PRSV

isolates, WMV-M, and the other potyviruses.

The MCAs and PCAs in this study defined four types of

epitopes: specific PRSV epitopes, epitopes varying among

strains of PRSV, epitopes variable among PRSV-W isolates, and

epitopes shared with other potyviruses. These MCAs should be

useful in the detection and differentiation of PRSV and could

be useful in the study of potyvirus proteins in general.


xiv















CHAPTER 1
INTRODUCTION




Of all the recognized plant virus groups, the

potyvirus group is the largest (Matthews, 1982). Members of

this group are widespread throughout the world and cause

disease in many agriculturally important crops (Hollings and

Brunt, 1981). Because of their economic importance, much

effort has been expended in identification and

characterization of potyviruses.

Plant viruses recognized as members of the potyvirus

group are filamentous particles (680-900 nm in length)

containing (+)-sense, single-stranded RNA with a molecular

weight of 3.0-3.5 x 106. All reported viruses in this group

produce cylindrical inclusions in infected host cells

(Edwardson, 1974). These inclusions consist of a 67-70kD

protein monomer (Hiebert and McDonald, 1973), are virus

induced (Dougherty and Hiebert, 1980a), and are serologically

distinct from their respective capsid proteins (Hiebert et

al., 1971; Purcifull et al., 1973).

Some viruses of this group produce other

proteinaceous inclusions which are visible in the light

microscope and are distinguishable from their cylindrical

inclusions (Edwardson, 1974; Christie and Edwardson, 1977).









One type consists of the nuclear inclusions induced by such

viruses as tobacco etch (TEV), bean yellow mosaic, and clover

yellow vein The nuclear inclusion protein has been found to

be an aggregate of two proteins, one with a molecular weight

of 54-60kD and the other with a molecular weight of 49kD.

Using antisera to the specific protein monomers of this

inclusion it has been shown that these are also virus-coded

proteins which are distinct from each other and from their

respective capsid and cylindrical inclusion proteins (Knuhtsen

et al., 1974; Chang et al., 1988a). It has also been shown

that the 49kD protein is a protease (Carrington and Dougherty,

1987; Chang et al., 1988b) and that the amino acid sequence of

the 54K protein is similar to RNA-dependent RNA polymerases

associated with other viruses (Allison et al., 1986).

Another virus-induced nonstructural protein, which

forms inclusions in some potyviruses (e.g., PRSV and pepper

mottle virus) and has been isolated in soluble form for other

potyviruses, is the amorphous inclusion-helper component

protein (de Mejia et al., 1985a; Hellmann et al., 1983). The

soluble form of this protein has been shown to be involved in

aphid transmission (Govier and Kassanis, 1974a and 1974b;

Thornbury and Pirone, 1983) and it is serologically related to

the amorphous inclusion protein (de Mejia et al., 1985b).

More recently the carboxyl-terminal half of this protein has

been shown to also have proteolytic activity (Carrington et

al., 1989).









Serology has been one of the tools used to

differentiate the virus-induced proteins of potyviruses from

each other and to identify individual potyviruses. The

majority of serological identification and classification of

potyviruses, however, has been done with polyclonal antisera

to the capsid protein only. While extremely useful, there are

major drawbacks to reliance on polyclonal antisera to the

capsid protein and to using polyclonal antiserum in general.

One of these drawbacks is that the capsid protein represents

only a small percentage of the total gene products coded by

the viral RNA. According to the published genetic maps of the

potyvirus genome, the capsid protein represents only 10% of

the total genome ( Chang et al., 1988b; de Mejia et al.,

1985b; Hellman et al., 1986; Dougherty and Hiebert, 1980b). In

addition, the capsid protein may be the least conserved of all

the known potyvirus induced proteins. Chang et al. (1988a)

used polyclonal antisera to the coat protein, the cylindrical

inclusion protein, and the large and small nuclear inclusion

proteins of the PV-2 isolate of bean yellow mosaic virus and

the Pratt isolate of clover yellow vein virus to distinguish

these two viruses. They found that the nonstructural proteins

were antigenically more conserved among bean yellow strains

than was the capsid protein. Clearly, diagnosis and

classification of potyviruses can be enhanced by serological

studies using antisera to the nonstructural proteins in

conjunction with antisera to the capsid protein (Chang et al.,










1988a, Edwardson, 1974; Purcifull et al., 1973, Yeh and

Gonsalves, 1984; Alconero et al., 1986).

One drawback of the use of polyclonal antisera in

the past has been their heterogeneity. Only a small

percentage of the antibodies in any antiserum are to the

immunizing antigen, the remainder consisting of antibodies to

previous antigenic stimulation (Mason et al., 1983). The

percentage of antibodies that are homologous to the immunogen

vary in affinity and are directed to several different

epitopes on the immunizing antigen. Some of these epitopes

are virus specific while others are group specific (Shepard et

al., 1974). The amount of each in a given antiserum can vary

due to variations in immunization procedure and the state of

the immunogen (e.g., freshly purified vs frozen) (Shukla et

al., 1989a). In addition, the antiserum may contain

antibodies to host contaminants not removed in the

purification procedure, the amount of any particular antiserum

is limited and impossible to duplicate, and the reactivity can

differ from laboratory to laboratory, animal to animal, and

bleeding date to bleeding date.

The heterogeneity of polyclonal antisera reveals

itself in the serological cross-reactivity common among

potyviruses (Edwardson, 1974; de Mejia et al., 1984b; Hiebert

and Charudattan, 1984; Hiebert et al., 1984b; Chang et al.,

1988a). In immunodiffusion tests, the cross-reactivity

observed depends on the serum used (Quiot-Douine et al., 1986a

and b), the collection date of that serum (Purcifull and









Hiebert, 1979; Davis and Yilmaz, 1984; Purcifull et al.,

1984a) and whether whole protein or degraded protein is used

as the immunogen (Shepard et al.,1974). These data indicate

that while individual potyvirus capsid proteins possess

specific antigenic determinants which can distinguish them,

there are also shared antigenic determinants which can confuse

attempts to correctly identify closely related viruses,

particularly those with similar host ranges. Thus serological

data based on polyclonal antisera must be used only with

caution for potyvirus identification and classification

(Moghal and Francki, 1976).

One approach to solving the problem has been taken

by Shukla and co-workers. Their work has confirmed that the

N-terminus of potyvirus coat proteins is located on the

surface of the virus particle and has shown that the N-

terminus appears to be the most immunodominant and virus-

specific regions of the virus particle (Shukla et al., 1988).

By removing the N-terminal region of the coat protein with

mild trypsin treatment, attaching this core protein to

Sepharose, and passing polyclonal antisera through the column,

they obtained only those antibodies specific to the homologous

virus and its strains (Shukla et al., 1989a). Using this

technique they were able to demonstrate that 17 potyvirus

strains, thought to be strains of a single potyvirus, were

actually four distinct potyviruses (Shukla et al., 1989b).

While this approach can be used to overcome some of the









problems of cross-reactivity, it can not solve other problems

inherent with polyclonal antiserum.

Another approach being taken to overcome many of

these problems is the production and use of monoclonal

antibodies. Monoclonal antibodies are by definition

homogeneous. The antibodies produced by a single hybridoma

cell line have the same antigen-binding site directed to only

one epitope of the target immunogen. Because each antibody-

secreting hybridoma is made from the fusion of a single B-

lymphocyte and a myeloma cell, specific antibodies can be

isolated even if the immunizing antigen preparation was not

pure, they can be produced in unlimited amounts, and they

allow the researcher to investigate one epitope of a given

protein at a time (Mason et al. 1983; Goding, 1982; de St.

Groth, 1985).

Monoclonal antibodies have shown great potential for

use in plant virus detection, differentiation, and the

exploration of taxonomic relationships (Halk and De Boer,

1985; Hsu et al., 1984). The monoclonal antibodies that have

been made to the capsid protein of potyviruses (Dougherty et

al., 1985; Diaco et al., 1985; Gugerli and Fries, 1983; Hill

et al., 1984; Jordan and Hammond, 1988) are no exception. For

example, monoclonal antibodies to potato virus Y (Gugerli and

Fries, 1983) have been isolated which can distinguish strains

previously not consistently distinguishable with polyclonal

antiserum. Another monoclonal antibody to PVY was found that

reacted to 24 isolates of PVY, including the three most









frequently occurring strains. This antibody has been used

successfully to screen potato samples for PVY. Monoclonal

antibodies to tobacco etch virus (TEV) could be grouped into

two classes based on specificity (Dougherty et al., 1985).

One group was specific to TEV. These antibodies appeared to

recognize epitopes on the virion surface. The second group

recognized epitopes on TEV and heterologous potyviruses. The

epitopes of the latter were localized in the interior of the

virions. Jordan and Hammond (1988) found one monoclonal

antibody which has reacted to every potyvirus tested. This

monoclonal antibody has recently been marketed for the

diagnosis of general potyvirus infections (Agdia, Inc. 30380

Country RD. 6, Elkhart, IN 46514).

Papaya ringspot virus type-W (PRSV-W)(Purcifull et

al., 1984b), previously known as watermelon mosaic virus-1

(Adlerz, 1969; Webb and Scott, 1965; Purcifull and Hiebert,

1979), is one of many potyviruses whose identification could

be enhanced by the production of monoclonal antibodies. In

addition, PRSV-W has three virus-induced proteins which are

easily purified and are known to be antigenic (Purcifull, et

al. 1984b; de Mejia et al. 1985a).

PRSV-W is one of several potyviruses recognized

throughout the world as an important pathogen of squash,

cantaloupe, cucumber, and watermelon (Lovisolo, 1980).

Others include watermelon mosaic-2 (Purcifull et al., 1984c)

and zucchini yellow mosaic virus (Lisa and Lecoq, 1984;

Provvidenti et al., 1984).









Clear distinction between these three potyviruses

using polyclonal antisera to the protein coat has not always

been possible, and in the case of PRSV-W (=WMV-1) and

watermelon mosaic virus-2 (WMV-2), it has been controversial

(Webb and Scott, 1965; Milne and Grogan, 1969). The most

recent evidence confirms that certain isolates of PRSV-W can

be differentiated serologically from WMV-2 (Purcifull and

Hiebert, 1979; Russo, et al., 1979). However, cross-reactions

between PRSV-W and WMV-2 have been observed in immunodiffusion

and ELISA depending on the antiserum used (Purcifull and

Hiebert, 1979; Dodds et al., 1984). In addition, antisera to

a serologically distinct strain of PRSV-W from Guadeloupe

(PRSV-T) reacted with partial identity to PRSV-W, PRSV-P (the

papaya strain of PRSV) and a Moroccan isolate of watermelon

mosaic virus (WMV-M) (Quiot-Douine et al., 1986a). No spur

was formed between PRSV-W and PRSV-P, which differ

biologically in that PRSV-P infects papaya whereas PRSV-W does

not infect papaya. WMV-M, which did not react with capsid

protein antiserum of either PRSV-W or WMV-2 in one report

(Purcifull and Hiebert, 1979), reacted with spur formation to

an antiserum to the capsid protein of PRSV-W in the Quiot-

Douine et al. (1986a) study.

Polyclonal antisera made to the cylindrical inclusion

proteins of PRSV-W, WMV-2, and ZYMV have shown clear antigenic

distinctions (Yeh and Gonsalves, 1984; Provvidenti et al.,

1984; Baum and Purcifull, 1981). However, these data

represent a small number of bleedings from only two research









laboratories. In addition, antisera to WMV-M cylindrical

inclusions cross-reacted with both PRSV-W and WMV-2 (Baum and

Purcifull, 1981). Cross-reactions were also found when

polyclonal antisera to the cylindrical inclusions of PRSV-P,

WMV-M and PRSV-T were compared (Quiot-Douine et al., 1986a).

In 1976, Martelli and Russo reported the presence of

aggregates of amorphous, electron dense material in leaves of

squash inoculated with several Mediterranean isolates of

watermelon mosaic virus. While there was some confusion as to

the identity of the virus inducing these aggregates, it was

concluded that these bodies were different from the

cylindrical inclusions and were composed of protein and RNA.

Similar inclusions were reported in Cucurbita pepo infected

with PRSV-W and PRSV-P (Edwardson, 1974). Further study of

the Mediterranean isolates, together with known isolates of

PRSV-W and WMV-2 from the United States, demonstrated that the

isolates reported to induce amorphous inclusion bodies were

PRSV-W, not WMV-2 ( Purcifull and Hiebert, 1979; Russo et al.,

1979).

The amorphous inclusions of PRSV-W have been

partially purified and specific polyclonal antisera have been

produced to the amorphous inclusion protein (de Mejia et al.,

1985a). In SDS-immunodiffusion tests these antisera did not

cross-react with extracts from plants infected with WMV-2,

ZYMV (Purcifull, unpublished data) or WMV-M (Quiot-Douine et

al., 1986a). The antiserum also did not react with extracts

from pepper mottle virus-infected plants (de Mejia et al.,









1985a), which produces amorphous inclusions similar in

morphology in light microscopy to those of PRSV-W (Edwardson,

1974; Christie and Edwardson, 1977). The antisera to PRSV-W

amorphous inclusion protein reacted homologously with PRSV-T

(Quiot-Douine et al., 1986a) and all other isolates or strains

of PRSV tested (de Mejia, 1984a; Purcifull and de Mejia,

unpublished data).

A homologous reaction with these polyclonal antisera

and the presence of the amorphous inclusion in epidermal

strips are considered definitive evidence for the diagnosis of

strains of papaya ringspot virus (Purcifull and Hiebert, 1979;

Russo et al., 1979). However, some isolates of PRSV-W, which

react homologously to the amorphous inclusion antisera, do not

produce typical amorphous inclusion aggregates in situ

(Purcifull et al., 1984b; Purcifull and de Mejia, unpublished

data). In addition, another cucurbit virus, zucchini yellow

fleck virus (Vovlas et al., 1981), reacted with these antisera

in SDS-immunodiffusion tests (Baker and Purcifull, 1987).

Using an indirect immunofluorescent staining technique (Chang

et al., 1988a), the amorphous inclusion protein antisera

reacted with WMV-2, ZYMV,and ZYFV (Baker and Purcifull, 1987,

and unpublished) as well as with the amorphous inclusions of

PRSV-W.

It is clear from the above data that while polyclonal

antisera can be used to serologically distinguish PRSV-W from

other potyviruses infecting cucurbits, the proteins induced by

PRSV-W share enough antigenic determinants with other cucurbit









potyviruses to complicate diagnosis. This is further

complicated by the finite nature of polyclonal antisera as no

one antiserum, regardless of its specificity, can be

maintained indefinitely. This makes comparisons over time and

space inconsistent and in many cases impossible.

Monoclonal antibodies, on the other hand, can be

selected for specificity or cross-reactivity, and with

appropriate care, the hybridomas which produce the antibodies

are essentially immortal. With proper selection, it is

hypothesized that monoclonal antibodies can be found which are

specific to each of the proteins of PRSV-W. In addition, it

is hypothesized that monoclonal antibodies to each of the

three PRSV-W proteins can be found which cross-react with the

respective proteins of other potyviruses. Based on the

information using polyclonal antiserum, some cross-reactive

antibodies that might be expected include 1) antibodies common

to the protein coat of PRSV-W and WMV-2 (Purcifull and

Hiebert, 1979; Dodds et al., 1984), 2) antibodies common to

the cylindrical inclusions of PRSV-W, WMV-2, PRSV-T,and WMV-M

(Baum and Purcifull, 1981; Quiot-Douine et al., 1986a), 3)

antibodies common to the capsid proteins of PRSV-W and ZYFV

(Baker and Purcifull, 1987), 4) antibodies common to

cylindrical inclusion proteins of PRSV-W and ZYFV (Baker and

Purcifull, 1987), and 5) antibodies common to the amorphous

inclusions of PRSV-W, and similar non-aggregating proteins of

WMV-2, ZYMV (Baker, unpublished) and the amorphous inclusion

of ZYFV (Baker and Purcifull, 1987). It should also be









possible to find antibodies which can differentiate the known

strains of PRSV (e.g. PRSV-W from PRSV-T) and which can define

heretofore unknown differences in PRSV proteins.

In addition to diagnosis and strain differentiation

of PRSV, monoclonal antibodies to the proteins of PRSV-W can

be used as immunological probes to study the epitope diversity

and similarity among potyviruses other than those infecting

cucurbits. For example, polyclonal antiserum to the helper

component of tobacco vein mottling virus (TVMV) reacted with

the cell-free translation products of PRSV-P, PRSV-W WMV-2,

WMV-M, ZYMV, and several other potyviruses (Hiebert et al.,

1984b). Therefore it should be possible to isolate a

monoclonal antibody to the amorphous inclusion of PRSV-W which

will also react with some of these potyviruses. Some of these

monoclonal antibodies could also be useful in study of the

function of the helper component-amorphous inclusion protein.

While the development of monoclonal antibodies has

its own set of drawbacks (i.e. time, expense, and over-

specificity), once antibodies are selected and characterized,

their use as diagnostic tools or as serological probes for

other uses are invaluable. Used individually or with others

as part of a synthetic polyclonal antiserum with known

reactivities, monoclonal antibodies offer a consistency of

reactivity which can help solve some of the problems inherent

in the study of potyvirus relationships to date.

The development and characterization of monoclonal

antibodies to the structural protein and two nonstructural






13



proteins of PRSV-W will help in the consistent identification

of this virus in field samples, help define differences among

isolates and between strains of this virus, and give us

additional tools for the general study of potyviruses.















CHAPTER 2
PRODUCTION AND PARTIAL CHARACTERIZATION OF
POLYCLONAL ANTISERUM AND MONOCLONAL ANTIBODIES TO THE
CAPSID PROTEIN OF PAPAYA RINGSPOT VIRUS TYPE-W


Introduction


Papaya ringspot virus type-W (PRSV-W) (Purcifull

et al., 1984b) formerly known as watermelon mosaic

virus-i (WMV-1) (Webb and Scott, 1965; Adlerz, 1969;

Purcifull and Hiebert, 1979) is one of several

potyviruses recognized throughout the world as important

pathogens of squash, cantaloupe, cucumber, and

watermelon (Lovisolo, 1980). Two other important

potyviruses which infect cucurbits and have a wide

distribution are watermelon mosaic virus-2 (WMV-2)

(Purcifull et al., 1984c) and zucchini yellow mosaic

virus (Lisa and Lecoq, 1984). A fourth potyvirus,

zucchini yellow fleck, has been recognized in Italy

(Vovlas et al., 1981) and Greece (Volvas et al., 1983).

Other potyviruses known to infect members of the

Cucurbitaceae include clover yellow vein and bean yellow

mosaic viruses (Lovisolo, 1980).

While there has been some confusion about the

identities of PRSV-W (= WMV-1) and WMV-2 in the past

(Webb and Scott, 1965; Milne and Grogan, 1969), more










recent evidence confirms that PRSV-W can be

differentiated serologically from other potyviruses

infecting cucurbits (Purcifull and Hiebert, 1979; Russo

et al., 1979; Makkouk and Lesemann, 1980; Vovlas et al.,

1981; van der Meer and Garnett, 1987; Quiot-Douine et

al, in press; Nameth et al., 1985; Provvidenti et al.,

1984). However, serological cross-reactivity has also

been observed between cucurbit potyviruses (Purcifull

and Hiebert, 1979; Dodds et al., 1984; Quiot-Douine et

al., 1986a; Huang et al., 1986; Lisa et al., 1981; Baker

and Purcifull, 1987; Davis and Yilmaz, 1984; Purcifull

et al., 1984a). In SDS-immunodiffusion tests, the

homologous antigen (i.e. PRSV) spurs over the

heterologous antigen (i.e. WMV-2) and the two reactions

are therefore distinguishable. In ELISA, however,

cross-reactivity may result in a difference of

absorbance levels between homologous and heterologous

antigens. Such differences are difficult to distinguish

from differences caused by factors such as low virus

titers. Consequently, clear distinctions between

cucurbit potyviruses using polyclonal antiserum to the

virus or its purified coat protein can depend on the

antiserum and serological test used.

PRSV isolates have been separated into three

groups based on serological similarities and differences

(Quiot-Douine, et al., in press). One group includes

isolates which are serologically identical to PRSV-W.










This group includes the papaya strains of PRSV (PRSV-P),

which differ from the watermelon strains by their

ability to infect papaya (Carica papaya L.) (Yeh et al.,

1984; Purcifull et al., 1984b). Group two includes the

Moroccan isolate of watermelon mosaic virus (WMV-M)

which has been found in Morocco (Fischer and Lockhart,

1974), Spain (Lecoq, unpublished), and South Africa (van

der Meer and Garnett, 1987). The third group includes

the Guadeloupe strain, PRSV-T (Quiot-Douine et al.,

1986a). Previous work with different antisera to the

virus coat protein had indicated that WMV-M (Purcifull

et al., 1979; van der Meer and Garnett, 1987) and PRSV-T

(Quiot-Douine et al., 1986b) might be separate viruses.

Therefore, establishing clear relationships between

strains of PRSV also depends on the polyclonal antiserum

used. In the case of PRSV-W and PRSV-P, inoculation to

papaya is currently the only way to differentiate these

two strains of PRSV.

Clearly the heterogeneous nature of polyclonal

antiserum, the cross-reactivity common to potyviruses,

and the lack of correlation between biological

differences and serological similarities in the case of

PRSV-P and PRSV-W, can be hindrances to consistent

diagnosis and serotyping of PRSV. An additional

hindrance to this consistency is the finite nature of

polyclonal antisera. Once a specific polyclonal









antiserum is depleted it can never be duplicated. This

makes comparative research over time impossible.

Monoclonal antibody technology (Kohler and

Milstein, 1975) can be used to overcome many of these

problems. First, the hybridomas which produce

individual antibodies can be frozen in liquid nitrogen,

making these hybridomas essentially immortal. They are

theoretically available to produce more antiserum with

consistent and known reactivity at any time. Second,

monoclonal antibodies are by definition homogeneous.

Monoclonal antibodies are antibodies produced by clones

of a single hybridoma cell and therefore they all have

the same antigen-binding site specific to one epitope of

the immunogen.

It has been recognized for a long time that

potyviruses have epitopes on their coat protein which

are virus specific and epitopes which are group

specific. Polyclonal antiserum consists of a mixture of

antibodies to these two types of epitopes, hence its

inconsistencies. This conclusion is drawn in part from

data which showed polyclonal antiserum to the whole

virus was usually more specific than antiserum to the

degraded virus (Shepard et al., 1974).

Monoclonal antibodies made to potyvirus coat

proteins and to whole virus particles should be able to

sort out virus specific epitopes from group specific

epitopes. In fact, monoclonal antibodies to the tobacco










etch potyvirus were able to define individual epitopes,

some of which were located on the exterior of the virus

and were TEV specific, and some of which were located in

the interior of the virions and were cross-reactive

(Dougherty et al., 1985). In addition, monoclonal

antibodies to potato virus Y (PVY) were able to define

epitopes that could differentiate PVY strains and other

epitopes which were common to all the PVY strains tested

(Gugerli and Fries, 1983).

Thus, given the data on other potyviruses, it

should be possible using monoclonal antibody technology

to make antibodies to PRSV which can be used to

differentiate PRSV from other potyviruses infecting

cucurbits, to differentiate the strains of PRSV, and to

add consistency to the diagnosis and study of this

virus.



Materials and Methods



Preparation of Immunoaens

The Florida isolate of PRSV-W used for virus

purification was originally obtained from W.C. Adlerz

and is hereafter called W-1A (Purcifull and Hiebert,

1979). It was maintained in Cucurbita pepo L. 'Small

Sugar' pumpkin (ssp) by manual inoculation. The virus

was purified from ssp three weeks after inoculation

using the procedure described by Purcifull et al.









(1984b). Purified virus from several purifications were

dissociated in an equal volume of Laemmli dissociating

solution (Laemmli, 1970), heated, and electrophoresed in

10% sodium dodecyl sulfate preparatory polyacrylamide

gels (SDS-PAGE). The protein bands were detected using

cold 0.2 M KC1, excised, and crushed with a mortar and

pestle. The proteins were removed from the gel using

methods for the amorphous inclusion protein of PRSV-W

described by de Mejia et al. (1985). Protein yields

were estimated by spectophotometry at an absorbance of

280 nm (Hiebert et al., 1984a) and the protein purity

was determined by analysis in SDS-PAGE. The dialyzed

protein was freeze-dried and stored at -17 C. Purified

virus from other purifications was stored at -80 C.



Polvclonal Antisera

Two polyclonal antisera, one to the capsid

protein and one to the whole virus of W-1A, were made by

immunizing New Zealand white rabbits.

Rabbit 1125 was initially immunized with 1 mg/ml

(1 O.D. at 280 nm) of purified capsid protein emulsified

with 1 ml of Freund's complete adjuvant (Difco

Laboratories, Detroit MI 48232). A week later, a second

injection (1 mg/ml of capsid protein) was given in 1 ml

of incomplete adjuvant (Difco Laboratories, Detroit MI

48232) followed the next week by a third injection also

in incomplete adjuvant. For each injection, 0.15 ml was









given in a toe pad and the remainder was injected into a

thigh muscle. The first bleeding was taken from an ear

vein one month after the first injection and the rabbit

was bled on a weekly interval for several months. The

rabbit was given a 1 mg/ml booster in Freund's

incomplete adjuvant eight months after the first

injection and again bled weekly for several months.

Rabbit 1142 was injected with whole virus as

above except that the toe pad injection was omitted and

no booster was given after 8 months. These injections

were done soon after purification so the virus was not

frozen for longer than three weeks.



Monoclonal Antibodies

Three immunization procedures were originally

tried for the generation of monoclonal antibodies to the

capsid protein. An in vitro technique was used in

fusion 1 (Fl) and fusion 2 (F2) and was dropped for lack

of success. Thereafter, two immunization procedures

were used for the generation of monoclonal antibodies to

the whole virus and the capsid protein of W-1A. A pre-

immunized in vitro technique (Wiegers et al., 1986),

modified (S. Zam, personal communication) from that

described by Boss (1986), was used for fusion 3 (F3) and

fusion 22 (F22). This procedure was used to generate

hybridoma cells F3C-C10, F22A-B9, F22A-E8, and F22A-C8.

Fusion 4 (F4), fusion 5 (F5) and fusion 21 (F21) were









done using the more common in vivo technique (Galfre and

Milstein, 1981) and generated MCAs F21D-E10 and F21C-E4.

For the pre-immunized in vitro technique used in

F3 and F22, BALB/c mice were immunized subcutaneously

(SQ) with 50 jg (in 200 il warm water) of capsid protein

(F3) or whole virus (F22) mixed 1:1 with Freund's

complete adjuvant, followed in 1-2 weeks by an

intraperitoneal injection (IP) in Freund's incomplete

adjuvant (also 1:1). Three weeks after the first

injection, spleen cells of the mouse used for F3 were

boosted in vitro and fused. The mouse used in F22 was

reinjected with 50 pg of whole virus in PBS IP three

months after the first injection and boosted in vitro

three days later.

The in vitro boost given to the spleen cells

prior to fusions Fl, F2, F3, and F22, was done in the

following manner. Each mouse spleen was removed and the

spleen cells were suspended in 5 ml of serum-free

Dulbecco's modified eagle medium (Gibco Laboratories,

Life Technologies Inc. Grand Island, NY 14072) (DMEM).

The spleen cells were added to an in vitro medium

containing the following: 1) 25 pg of antigen capsidd

protein for the mouse used for F3 and whole virus for

the mouse used for F22), 2) 0.3-0.4 ml of adjuvant (N-

acetyl muramyl-L-alanyl-L-isoglutamine, Sigma Chemical

Co. St. Louis, MO 63178), 3) 4 ml of fetal calf serum

(F3) or horse serum (F22), and 4) 11 ml of DMEM










supplemented with 10% serum, 1% penicillin-streptomycin

(Sigma Chemical Co., St. Louis, MO 63178) and 0.1 jg/ml

amphotericin B (Sigma Chemical Co. St Louis, MO 63178)

(DMEM-F) to which 5mM 2-mercaptoethanol was added. The

spleen cells were incubated in a CO2 incubator (6.0%

CO2, 37 C and 90% humidity). Following three days in

culture, the cells were centrifuged, put into 5 ml of

serum free DMEM, counted using a hemocytometer, and

fused.

The mouse used for F21, whose previous injection

schedule was the same as that for the mouse used for

F22, was boosted IP with 50 pg of virus in PBS three

days before the fusion. After removal from the mouse,

the spleen was infused with DMEM and gently mashed

though an 80-mesh sieve. The spleen cells were

centrifuged, counted, and fused.

For each fusion the spleen cells were fused with

Sp2/0-Agl4 (Sp2/O) (Shulman et al., 1978) myeloma cells

which were grown to mid-logarithmic phase in DMEM-F.

Like the spleen cells, the Sp2/0 cells were washed by

centrifugation at 800-900 g for 5-7 min in DMEM. They

were combined (1 part Sp2/0 cells to 2 parts spleen

cells) and recentrifuged. The combined cells were

resuspended in 1 ml of 50% polyethylene glycol (PEG)

(3000-3700 MW, Sigma Chemical Co., St, Louis, MO 63178)

in 75mM HEPES (pH 8.0) over a period of 15 sec, swirled

in a water bath (37 C) for 30 sec, and incubated in the










water bath for 90 sec. One ml of DMEM was added over 30

sec, the solution was swirled for 30 sec, an additional

10 ml of DMEM was added over 30 sec, and the solution

was incubated for 5 min in the water bath. The cells

were centrifuged and the pellet was resuspended in HAT

selective media. This medium contained DMEM-F with 20%

serum, and 1 ml HAT/50 ml of media (5 x 10-3 M

hypoxanthine, 2 x 10-5 M aminopterin, and 8 x 10-4 M

thymidine, Sigma Chemical Co., St. Louis, MO. 63178).

The cells (5x105 cells/ml) were plated (100 pl/well)

into 96 well, sterile polystyrene flat bottom plates

with lids (Cell Wells TM, Corning Glass Works, Corning,

NY 14831) and placed in a CO2 incubator.

As growing colonies of primary hybridoma cells

developed they were fed with HT media (DMEM-F which

contains hypoxanthine and thymidine but not aminopterin,

Sigma Chemical Co., St. Louis, MO 63178) and their

supernatants were tested for antibody production by

plate-trapped and antibody-trapped indirect ELISA

They were tested against purified capsid protein and/or

extracts of healthy pumpkin (Hssp) and extracts of ssp

infected with W-1A. Antibody producing primary

hybridomas of F3 and F4 were frozen in liquid nitrogen

(1 x 106 cells) in HT freezing media containing 10% DMSO

and 20% serum. Eleven primary hybridomas, eight from F3

and three from F4, were thawed at a later date. The

vials were removed from the freezer and immediately put










into a water bath (37 C). The cells were put into 10 ml

of HT, centrifuged, resuspended in 2-3 ml of HT, put

into 96 well plates, and into the CO2 incubator. The

supernatants from the cells that were viable were tested

for antibody production in I-ELISA. Those that produced

antibodies were cloned twice by limiting dilution (Zola,

1987) and the supernatant of each clone was tested for

antibody production. The antibody producing hybridomas

from F21 and F22 were cloned by limiting dilution

immediately. Three of the secondary clones, three of the

primary clones, and the primary hybridomas of each were

frozen in liquid nitrogen. One of the secondary clones

from each primary hybridoma (except for F22A-E8 and

F22A-H9) was increased and injected IP (1 x 107 cells in

sterile PBS) into BALB/c mice for ascites production.

These mice were injected IP with 0.5 ml 2,6,10,14

tetramethyl pentadecane (pristane-primed) 1-2 weeks

before the cells were injected. The supernatant from the

injected cells was used to isotype each monoclonal

antibody using a MonoAb EIA kit (Zymed Lab., Inc., San

Francisco, CA 94080)



Polyclonal Mouse Ascites

To obtain a polyclonal mouse antiserum for use

in antibody-trapped I-ELISA, BALB/c mice were immunized

with capsid protein or whole virus like those used for

fusion, pristane-primed, and injected IP with non-fused









Sp2/O cells for the production of polyclonal mouse

ascitic fluid (Scott, et al., 1969).

Nonimmunized, pristane-primed, BALB/c mice were

also injected with nonfused Sp2/O cells to obtain an

ascitic fluid with no PRSV-W specific antibodies.

Ascitic fluid was removed from the mice,

centrifuged to remove the cells, and frozen.



Virus Isolates

Primary virus isolates. W-1A and fourteen other

isolates of PRSV-W were used throughout this study. An

isolate from Greece and an isolate from Jordan came from

G. Martelli, an isolate from California came from R.

Webb, an isolate from New York came from R. Provvidenti,

one isolate was obtained from the American Type Culture

Collection (ATCC PV-23), and the remaining isolates were

collected in Florida (Purcifull et al., 1988; Purcifull

and Simone, unpublished). The Florida isolates 2052 (in

zucchini) and 2040 (in yellow squash) were collected in

Jan. 1987 from Dade County. Isolate 2030 (in zucchini)

and 2038 (in watermelon) were collected in Collier

County at the same time. Isolates 2201 and 2207 (both

in pumpkin) and 2169 (in watermelon) were collected in

Alachua County in the fall of 1987. Isolates 1870 (in

yellow squash) and 1637 (in zucchini) were both

collected in Dade County in April 1986 and January 1985,

respectively. Also included in this study were an









isolate of PRSV-T from Guadeloupe from L. Quiot-Douine

(Quiot-Douine et al., 1986a), an isolate of PRSV-P

(Hawaii) from D. Gonsalves, and an isolate of watermelon

mosaic virus from Morocco (WMV-M), originally from B.

Lockhart. Virus isolates either from live cultures or

from dried leaves were ground in 0.02 M potassium

phosphate, pH 7.5, with the addition of carborundum and

were inoculated to ssp and to papaya using sterilized

cheesecloth pads.

Single isolates of six other cucurbit viruses

were used throughout this study: zucchini yellow mosaic

virus (ZYMV) (isolate 1119 of Purcifull et al.,1984a),

watermelon mosaic virus-2 (WMV-2) (isolate 1656 of

Purcifull, unpublished), squash mosaic virus (SqMV)

(ATCC isolate PV-36), cucumber mosaic virus (CMV)

(isolate 2147 of Purcifull, unpublished), Trichosanthes

virus (TV) (isolate 1860 of Purcifull et al., 1988,

presumed to be a potexvirus), and an isolate of zucchini

yellow fleck virus (ZYFV) originally from C. Vovlas

(Vovlas et al., 1981). Later in this study, an

additional 19 isolates of WMV-2 and 12 of ZYMV (Wisler

and Purcifull, unpublished) were also tested in I-ELISA.

Additional PRSV-W isolates. Forty cucurbit

samples, collected in the spring of 1988 during a survey

of the distribution of cucurbit viruses in Florida

(Purcifull et al., 1988), were also tested for

reactivity to the MCA F3C-C10 (MCA-5) and polyclonal









antiserum 1125 (PCA-1125). Samples 2514, and 2246 to

2276 were collected from Dade County, and samples 2277

to 2283 were collected from Collier County. Sixteen of

the forty samples and two other isolates, 2387

(collected in North Fl.) and 2514A (aphid transmission

from a mixed infection of PRSV-W and TV), were

inoculated to ssp for further characterization. These

18 isolates were retested against MCA-5 and PCA-1125 in

plate-trapped I-ELISA. The remaining sap from these

tests was frozen and tested at a later date against MCAs

F21C-E4 (MCA-4) and F22A-C8 (MCA-3). Based on the

results of the latter test, isolate 2254 was

reinoculated to ssp from dried material and retested

using freshly ground material against MCA-3, MCA-4, and

MCA-5. Isolates 2040, 2030, 2052, 2169, 2207, PRSV-P,

and Hssp were also included in this plate-trapped I-

ELISA test.

Additional viruses. The potyviruses, WMV-2,

tobacco vein mottling, clover yellow vein, cowpea aphid-

borne mosaic, soybean mosaic, Bidens mottle, PVY, peanut

stripe, peanut mottle viruses and the cucumovirus, CMV,

were inoculated either from dried material or from

infected plants to healthy plants of Nicotiana

benthamiana Domin. Pepper mottle and tobacco etch were

propagated in N. tabacum L. var. 'Samsun NN' (NN

tobacco), bean yellow mosaic virus 204-1 (Jones and

Diachun, 1977) was propagated in Pisum sativum










L.'Alaska' (Alaska pea), and wheat streak mosaic virus

was propagated in Triticum vulqare L.(wheat). Three

weeks after inoculation, samples of each plus their

healthy counterparts were tested in SDS-immunodiffusion

and I-ELISA.



SDS Immunodiffusion

SDS immunodiffusion tests were done using the

procedure described by Purcifull and Batchelor (1977).

Each isolate in the primary study was tested against the

homologous antigen (W-1A) and Hssp with antiserum to the

capsid protein (PCA-1125), the whole virus (PCA-1142),

and normal serum. Field collections 2246-2283, 2387,

and 2514 were tested with antiserum to the capsid

protein of PRSV-W (PCA-1125), ZYMV, WMV-2, SqMV, CMV,

TV, and normal serum. All the potyviruses in this study

were tested against their homologous antiserum in

reciprocal tests with PCA-1125.

Intragel cross-absorption tests were done using

crude extracts as above (Purcifull and Batchelor, 1977).

The absorbing antigen was placed in the center well for

16-20 hours. The absorbing antigen was removed, freshly

ground antigen was put in the outside wells, and PCA-

1142 was put in the center well. The plates were

incubated for 48 hr and read at least twice during that

time period. Three cross-absorption tests were done









with PRSV-W isolates W-1A, 2201, 2169, 2207, 2040, 2030,

2052, the New York isolate, and Hssp.



Indirect ELISA

Two types of indirect ELISA (I-ELISA) were used

in this study, plate-trapped and antibody-trapped

(Fig.2-1). In plate-trapped I-ELISA, the antigen is

applied first and is immobilized to the plastic. In

antibody-trapped I-ELISA, the antigen is applied second

and is trapped by polyclonal antibodies.

Plate-trapped I-ELISA was done in the following

manner. One gm of tissue was ground in a mortar and

pestle in 10 ml of sodium carbonate buffer (pH 9.6).

The extracts were strained through cheesecloth and 100

pl was added to the appropriate wells of Immulon II flat

bottom plates (Dynatech Laboratories Inc,. Chantilly, VA

22021). The antigens were incubated at 37 C for 1-2

hours. The plates were washed four times in phosphate

buffered saline (PBS), pH 7.4, with 0.05% Tween-20

(PBST). Fifty (50) pl of antibody was added to each

well. For screening, the fluid in which the hybridomas

were growing, called tissue culture fluid, was either

added full strength or was diluted 1:1 in 0.2 M Tris-HCL

buffer (pH 7.2) with 0.15 M NaC1. Ascitic fluid and

polyclonal antiserum were also diluted in this buffer.

The plates were incubated for one hour at 37 C

and again washed 4 times in PBST. Fifty pl of goat anti-









Plate-Trapped
Indirect ELISA


Antibody-Trapped
Indirect ELISA


Step 1
Antigen


Step 1
Rabbit
Antibody





Step 2
Antigen


:yme


Step 3
Enzyme
Conjugated
Antibody


Step 3

Monoclonal
Antibody


Substrate


Step 4

Substratel


Substrate
]Enzyme
J Steps 4 & 5
Enzyme
Conjugated

/ Substrate
Dr. J. Moore, Jr.


Fig. 2-1. Diagram of the steps used in two types of
indirect ELISA. Each step, except for the final step,
was incubated 1-2 hrs at 37 C followed by 3 washes with
phosphate buffered saline-Tween 20. The final step was
incubated at room temperature and the plates were read
on a Biotek automated plate reader three to four times
at 15 min intervals following substrate addition.









rabbit IgG (for the polyclonal controls), goat anti-

mouse IgG and/or IgM alkaline phosphatase conjugate (for

the MCAs), each diluted 1:1000 in PBS (pH 7.2) with 2.0%

polyvinyl pyrrolidone (Sigma Chemical Co., St Louis, MO

63178) and 0.2% ovalbumin (grade V, Sigma Chemical Co.,

St. Louis MO 63178), were added to the appropriate

wells. The plates were incubated for one hour at 37 C or

overnight at 4 C and washed 5 times in PBST. Fifty (50)

pl of substrate (p-nitrophenyl phosphate, disodium, 1

mg/ml) (Sigma Chemical Co., St Louis, MO 63178) in 9.7%

diethanolamine buffer (pH 9.8) (Fisher Scientific, Fair

Lawn, NJ 07410) was added to the plates and incubated at

room temperature. Absorbance readings (405 nm) were

taken on a Biotek automated microplate reader, model EL

309 (Bio-Tek Instruments Inc., Winooski, VT 05401) at

fifteen minute intervals for 1-2 hr.

For antibody-trapped I-ELISA, antiserum 1125

(bleeding Feb.5, 1987) was used as the trapping antibody

for most of the tests. It was diluted 1:1000 in Na

carbonate buffer and 100 pl was added to each well.

Polyclonal antiserum to WMV-2 (1134, bleeding Jan. 21,

1988) and ZYMV (1028, ammonium sulfate precipitated IgG

from bleeding Feb. 18, 1983) were also used as trapping

antibodies in later tests in which the antigens of

interest were WMV-2 or ZYMV isolates, respectively.

These antisera were also used at a 1:1000 dilution in

carbonate buffer. The trapping antibodies were










incubated overnight at 4 C or for 1-2 hr at 37 C. The

plates were washed with PBST and 50 il of antigen (1 gm

of tissue ground in 10 ml of 0.25 M potassium phosphate

buffer, pH 7.2) (Gonsalves and Ishii, 1980), was added

to the appropriate wells. The remainder of the steps

was the same as those described above for the plate-

trapped test.

Each MCA and PCA-1125 were tested in I-ELISA

against sap from plants infected with the immunogen, W-

1A, and Hssp at various dilutions to determine the

dilution with the lowest background and highest

reactivity. Once the best dilution was determined,

extracts from each of the 17 original isolates of PRSV,

plus WMV-M, ZYFV, ZYMV, WMV-2, SqMV, CMV, TV in ssp were

tested in plate-trapped and antibody-trapped I-ELISA

against all six MCAs and the polyclonal ascites to the

capsid protein, the whole virus, and Sp2/O cells. All

isolates were tested in plate-trapped I-ELISA with PCA-

1125. The additional PRSV-W isolates were tested only

in plate-trapped I-ELISA. The other potyviruses were

tested in plate-trapped I-ELISA with PCA- 1142 and PCA-

1125, ascites of MCA-5, and tissue culture fluid of the

other MCAs. Sap from the inoculated papayas and a

noninoculated papaya were tested for virus infection in

plate-trapped indirect ELISA using PCA-1125 and normal

serum. Every I-ELISA test was repeated at least once.










MCAs to WMV-2 and ZYMV (Wisler et al., in press)

were used as positive controls when the 19 additional

isolates of WMV-2 and the 12 additional isolates of ZYMV

were tested against the six MCA to PRSV-W and their

homologous MCA. In these antibody-trapped tests the

WMV-2 and ZYMV isolates were trapped with their

respective homologous polyclonal antisera.



Aphid Transmission

Since isolate 2514 proved to be infected with

both PRSV-W and TV, an aphid transmission test was done

with isolate 2514 in an attempt to separate these

viruses. The TV is presumed to be a potexvirus and

potexviruses typically are not aphid transmitted. The

transmissibilities of isolate 2207 (PRSV-W alone), and

isolate 2499 (TV alone) were also tested The aphids,

Mvzus persicae (Sulz.), maintained on pepper (Capsicum

annuum L. 'Cal Wonder') and moved to mustard (Brassica

perviridis L.) two weeks before the test, were starved

for two hours and then placed on an infected leaf for

30-40 seconds. The aphids were then moved to Hssp. Each

treatment consisted of one pot containing five plants,

and 10 aphids were placed on each test plant. Aphids

which had no access to infected tissue were placed on

healthy plants in one pot and one pot received no

treatment. After 1-2 hours the plants were sprayed

with insecticidal soap to kill the aphids. The plants










were inspected for live aphids and the plants were

resprayed as needed. Manual inoculations of 2207, 2499,

and 2514 were also done. Two weeks later, composite leaf

samples from each pot were tested in SDS-immunodiffusion

with PCA-1125 and antiserum to TV (Purcifull et al.,

1988).



Protein Blotting

Polyclonal antiserum 1125, Sp2/O ascites and each

MCA was tested for reactivity in protein blots. They

were tested against the homologous antigen (W-1A in ssp)

and Hssp. One gram of each tissue was ground in 1 ml of

distilled water, 1 ml of 3.0 % SDS was added, and the

mixture was expressed through cheesecloth. Five hundred

il were put into a microcentrifuge tube and centrifuged

for 10 min at 10,000 rpm in a Beckman Microfuge 12

Centrifuge (Beckman Instruments, Inc. Palo Alto, CA

94304). One hundred pl of the supernatant was removed,

100 pl of Laemmli dissociation solution (LDS) (0.1 M

Tris-HCl, pH 6.8, 2.5% SDS, 5% 2-mercaptoethanol and 5%

sucrose) (Laemmli, 1970) was added, and the mixture was

heated in a boiling water bath for 2-5 min. Fifteen pl

of each extract was added to the appropriate wells of the

5.6% stacking gel. Five pl of high range (200-14.3kD)

pre-stained molecular weight standards (Bethesda Research

Lab., Inc., Gaithersburg, MD 20877) in 10 pl of LDS were

added to at least one lane of each gel. The 10% SDS-









polyacrylamide gel was electrophoresed at a constant

voltage of 200 V for 45 minutes using a Bio-Rad Mini-

Protean II (Bio-Rad, Richmond, CA 94804-9989). The gel

was removed and the proteins were transferred to

nitrocellulose using a Poly Blot Transfer System, model

SBD-1000 (American Bionetics, Emeryville, CA 94608) at a

constant 250V for 30 min. The molecular weight bands

were marked with pencil, the nitrocellulose sheet was cut

into pieces, and individual pieces were incubated 30-45

min at room temperature in blocking solution with a

1:2000 dilution of polyclonal antiserum, ascitic fluid,

or a 1/2 dilution of tissue culture fluid as appropriate.

The blocking solution consisted of Tris buffered saline

(TBS) (0.02 M Tris-HCl and 0.15 M NaC1, pH 7.2), 5.0 %

Carnation nonfat dry milk, and 1.0 % Tween-20. The

nitrocellulose sheets were rinsed 3 times for 5 min each

in TBS. Then, as appropriate, the sheets were incubated

for 30-45 min with goat anti-rabbit IgG, goat anti-mouse

IgG, or goat anti-mouse IgM (Sigma Chemical Co., St.

Louis, MO 63178) diluted 1:1000 in blocking solution.

This solution was removed, the sheets were washed in TBS

three times, and the sheets were incubated for 5 minutes

in developing buffer (0.1 M Tris-HCl, 0.1 M NaC1, pH

9.5). The developing buffer was removed, and 10 ml of

developing buffer with 20 il of MgC12, 22 pl nitroblue

tetrazolium chloride (NBT) and 22 pl 5-bromo-4-chloro-3-

indolylphosphate p-toluidine salt (BCIP) (Bethesda










Research Laboratories, Life Technology, Inc.,

Gaithersburg, MD 20877) was added. After 5-15 minutes

the developing solution was removed and the reaction was

stopped with distilled water.



Results



SDS-Immunodiffusion Tests

Representative bleeding dates of PCA-1125, made

to the capsid protein of W-1A, were tested against SDS-

degraded sap from ssp infected with W-1A, WMV-2, ZYFV,

or ZYMV and with Hssp. Cross-reactivity was seen with

ZYFV (Baker and Purcifull, 1987) and WMV-2. The reaction

with ZYFV was seen in earlier bleeding dates (i.e.,

Oct.1,1986 and Jan.7,1987) and was generally a stronger

reaction than that of WMV-2. The reaction with WMV-2

(bleeding Feb.26,1987) was seen after the rabbit was

given the final booster injection (Jan.29,1987). No

reaction was seen with ZYMV or Hssp with any of the

bleeding dates tested.

Using a single bleeding date of 1125 taken one

month after the booster injection (Feb.26,1987), cross-

reactivity (W-1A spurring over other antigens) was seen

with Bidens mottle virus, peanut mottle virus, potato

virus Y, soybean mosaic virus, tobacco vein mottling

virus, (Fig.2-2), and pepper mottle virus (not shown).

Very faint reactions were also seen with bean yellow









mosaic 204-1 and tobacco etch viruses (Fig.2-2). There

were no reactions with clover yellow vein virus, cowpea

aphid-borne mosaic virus, peanut stripe virus, wheat

streak mosaic virus, CMV, healthy Nicotiana benthamiana,

healthy NN tobacco, healthy wheat, healthy Alaska pea,

or Hssp. All viral antigens in this test reacted with

their respective homologous antiserum and extracts from

non-inoculated plants were negative.

SDS-immunodiffusion tests of field samples 2246-

2283 showed that 36 of the 38 samples were infected only

with PRSV-W. Two of the samples did not react with any

of the antisera used. Field sample 2387 was also singly

infected with PRSV-W and 2514 was doubly infected with

PRSV-W and TV. After aphid transmission, 2514A was

found to be singly infected with PRSV-W (Fig.2-3)

Using early bleeding dates (1-2 mo after the

second injection) of PCA-1125, PRSV-W isolate 2201 and

the New York PRSV-W isolate were spurred over by the

homologous antigen, W-1A (Fig.2-4). The precipitation

lines of the other PRSV-W isolates coalesced with the

homologous antigen. W-1A also spurred over PRSV-T and

early bleedings did not react with WMV-M. When later

bleedings were used (e.g., those after the last booster

injection), spurs were no longer seen with 2201 and the

New York isolate but were seen with WMV-M and PRSV-T

(Fig.2-5).










Using the second bleeding (July 27,1988) of PCA-

1142, made to the whole virus of W-1A, the homologous

antigen spurred weakly over 2207, 2169, 2201, New York,

and strongly over 2040, 2030 and 2052. This antiserum

did not react with PRSV-T, WMV-M, ZYMV, ZYFV, WMV-2, or

any of the other potyviruses in SDS-immunodiffusion. It

also did not react to SqMV, CMV, TV, or Hssp.

In the intragel absorption tests, when the

absorbing antigen was W-1A, absorption was complete and

no precipitation lines were seen. When the absorbing

antigens were 2207 or 2169, precipitation lines were seen

only with W-1A. When the absorbing antigens were 2201 or

the New York isolate, precipitation lines were seen with

W-1A, 2207, and 2169 in one test (the precipitation lines

appeared after 48 hours, they were not visible at 24

hours) and only W-1A in the other two tests. When the

absorbing antigens were 2040, 2030, or 2052,

precipitation lines were visible for W-1A, 2207, 2169,

2201 and the New York isolate but not for 2030, 2040, or

2052. When Hssp was the absorbing antigen, precipitation

lines were seen for all isolates tested. Table 2-1

summarizes the results of these tests.



Indirect ELISA

Polvclonal antiserum. All of the isolates of

PRSV which reacted with PCA-1125 in SDS immunodiffusion

also reacted with PCA-1125 (used at a dilution of







































Fig. 2-2. Serological cross-reactivity of seven
potyviruses with papaya ringspot virus type-W in sodium
dodecyl sulfate (SDS)-immunodiffusion tests. Central
wells contain polyclonal antiserum 1125 made to the
capsid protein of PRSV-W isolate W-1A. The peripheral
wells contain SDS-treated sap from: A, W-1A in
Cucurbita Depo L. 'Small Sugar pumpkin' (ssp); B,
watermelon mosaic virus-2 in Nicotiana benthamiana (Nb);
C, tobacco vein mottling virus in Nb; D, cucumber
mosaic virus in Nb; E, healthy ssp; F, cowpea aphid-
borne mosaic virus in Nb; G, soybean mosaic virus in
Nb; H, Bidens mottle virus in Nb; I, potato virus Y
in Nb; J, peanut stripe virus in Nb; K, peanut mottle
virus in Nb; L, zucchini yellow mosaic virus in ssp;
M, healthy ssp; N, clover yellow vein virus in Nb; O,
healthy Pisum sativum 'Alaska' (Alaska pea); P, bean
yellow mosaic virus 204-1 in Alaska pea; Q, tobacco etch
virus in N. tabacum "NN (NN tobacco); R, healthy NN
tobacco; S, wheat streak mosaic virus in Triticum
vulaare (wheat); and T,healthy wheat.






































Fig. 2-3. Serological assay for papaya ringspot virus
type-W (PRSV-W) and Trichosanthes virus (TV) in manually
and aphid inoculated Cucurbita peDo L. 'Small Sugar
pumpkin' (ssp). Isolate 1860 (TV, homologous antigen to
TV antiserum 1129) and W-1A (PRSV-W, homologous antigen
to PRSV-W antiserum 1125) were also included in this
test. Center wells contain: P, antiserum 1125, and T,
antiserum 1129. Peripheral wells contain SDS-treated
sap from: cn, TV isolate 2499, manually inoculated;
dn, isolate 2514, a mixed infection of TV and PRSV-W,
manually inoculated; en, isolate 2500, a mixed
infection of TV and PRSV-W, manually inoculated; fn,
PRSV-W isolate 2207, manually inoculated; g, PRSV-W; hn,
mock inoculated ssp; ca, isolate 2499, aphid
transmission; da, isolate 2514, aphid transmission
(2514-A); ea, isolate 2500, aphid transmission; fa,
2207, aphid transmission; ha, healthy ssp treated with
non-viruliferous aphids; W, W-1A; V, isolate 1860, and
va, isolate 1860, aphid transmission.









































Fig. 2-4. Detection of serological differences between
papaya ringspot virus type-W (PRSV-W) isolate W-1A and
two other isolates of PRSV-W in sodium dodecyl sulfate
(SDS)-immunodiffusion tests. W-1A spurs over PRSV-W
isolate 2201 and the New York isolate (see arrows), but
not over PRSV-W isolates 2169 and 2207. Center well
contains polyclonal antiserum 1125 made to the capsid
protein of W-1A, bleeding 7-23-86. Peripheral wells
contain SDS-treated sap from: A, W-1A; B, PRSV-W isolate
2201; C, the New York isolate of PRSV-W; D, PRSV-W
isolate 2169; E, PRSV-W isolate 2207.







































Fig. 2-5. Serological reactivity of polyclonal
antiserum 1125 (bleeding Feb.12, 1987) with papaya
ringspot virus (PRSV) isolates and other cucurbit
potyviruses in sodium dodecyl sulfate (SDS)-
immunodiffusion tests. This collection of antiserum
1125 weakly spurred over the Tigre isolate of PRSV
(PRSV-T) and the Moroccan isolate of watermelon mosaic
virus (WMV-M) (see arrows). Central wells contain
antiserum 1125. Peripheral wells contain SDS-treated
sap from: A, PRSV-W isolate W-1A; B, PRSV-W isolate
1637; C, PRSV-W isolate 2201; D, PRSV-W isolate 2207;
E, PRSV-W isolate 2030; F, PRSV-W isolate 2038; G,
PRSV-W isolate 2040; H PRSV-W isolate 2052; I, PRSV-W
isolate 2169; J, the papaya isolate of PRSV, PRSV-P;
K, the PRSV-W isolate from Jordan; L, the PRSV-W isolate
from Greece; M, healthy sap; N, PRSV-T; O, PRSV-W
isolate 1870; P, the PRSV-W isolate from California;
Q, the PRSV-W isolate from New York; R, PRSV-W isolate
PV-23 from the American Type Culture Collection; S,
watermelon mosaic virus-2; T, zucchini yellow mosaic
virus; U, WMV-M.






43













TABLE 2-1. Cross-absorption test with polyclonal
antiserum 1142.



Absorbing antigens


Test
antigens W-1A 2201 N YORK 2169 2207 2030 2040 2052 HssP
W-1A a +a + + + + + + +
2201 + + + +
NEW YORK + + + +
2169 +/b +/- + + + +
2207 +- +/- +/- + + + +
2030 +
2040 +
2052 +
Hssp -


a
Positive (+) results represent visible precipitation
seen 24-48 hr after addition of test antigens
and antiserum. Negative (-) results were recorded
when no precipitation lines were seen after 48 hr.
The cross-absorption study was done three times.
b
In the case of test antigens 2169 and 2207, different
results were obtained in the first one of the three
tests. This probably reflects differences in virus
titer from one test date to the other. With all other
test antigens the results were the same in all tests.










1:100,000, Table 2-2) in plate-trapped I-ELISA (Figs.2-

6, 2-7, and 2-8). None reacted with normal serum. The

bleeding date of PCA-1125 used (Feb.5, 1987) also

reacted with WMV-M, ZYMV, ZYFV, and WMV-2 but did not

react with SqMV, CMV, TV, or Hssp (Fig.2-6 and 2-7).

In plate-trapped I-ELISA, antiserum 1125 also

reacted with Bidens mottle, peanut mottle, potato Y,

soybean mosaic, and tobacco veinal mottle viruses. It

reacted weakly with cowpea aphid-borne mosaic virus and

peanut stripe virus (Fig.2-9). Antiserum 1142 (used at a

dilution of 1:10,000) also reacted with Bidens mottle

virus, peanut stripe virus, potato virus Y, soybean

mosaic virus, tobacco vein mottling virus, ZYMV, and

WMV-2 (Fig.2-10) in plate-trapped I-ELISA. It did not

react to cowpea aphid-borne mosaic virus, and only

weakly reacted with peanut mottle virus. Neither

antiserum reacted with wheat streak mosaic virus,

healthy tobacco, or healthy wheat. In a later test,

PCA-1125 was found to also react with bean yellow mosaic

virus 204-1, clover yellow vein virus, and pepper mottle

virus (E-90).

In the I-ELISA test of the inoculated papayas,

only the positive controls (W-1A and PRSV-P in ssp) and

the papaya inoculated with PRSV-P reacted with PCA-1125

(Table 2-3). None reacted with normal serum.














TABLE 2-2. Plate-trapped indirect ELISA test to
determine the optimal dilutions of ascitic fluid to
monoclonal antibody F3C-C10 (MCA-5) and to polyclonal
antiserum 1125 (PCA-1125).


Dilution

1 x 10-2


1 x 10-3


1 x 10-4


1 x 10-5


1 x 10-6


1 x 10-7


Antigen


W-1A
Hssp

W-1A
Hssp

W-1A
Hssp

W-1A
Hssp

W-1A
Hssp

W-1A
Hssp


MCA-5a

0.957
0.042

1.015
0.001

0.962b
0.000

0.424
0.000

0.151
0.000

0.098
0.000


PCA-1125a


2.251
0.739

2.034
0.207

1.877
0.050

1.215c
0.000

0.410
0.000

0.153
0.000


Absorbance readings
substrate addition.
wells.


(405 nm) taken at 1/2 hr
Readings are an average


Dilution selected as the optimum for MCA-5.

Dilution selected as the optimum for polyclonal
antiserum 1125.


after
of two










E-91
1637 7 1.092
1870 0 .316
2030 1.218
2038 0.904
2040 2.05

2052 1.933




[ 2207 1.593
ATCC 0.753
CALIF .. .......... .... 1.514
O
M NEW YORK 0.725
JORDAN 0 222

5Q GREECE 0.255
PRSV-P 0.173
S PRSV-T o.327

WMV-M 0.165
WMV-2 0.042
ZYMV o 0.09
ZYFV B 0.068
CMV 0.003
SqMV 0.014
TV 0.002

Hssp 0.001

0.0 1.0 2.0 3.0

ABSORBANCE (405 nm)


Fig. 2-6. Reactivity of polyclonal antiserum 1125 with
papaya ringspot virus isolates and six other viruses in
plate-trapped indirect ELISA test E-91. Absorbance
readings (405 nm) were made 30 min after addition of
substrate and are an average of two wells. Antiserum
1125 was used at a dilution of 1:100,000. All antigens
but CMV, SqMV, TV, and Hssp are considered positive.











W-1A 1.053
1637 0.861
1870 1.079
2030 1 .o05
2038 1.05
2040 1.001
Fi 2052 0.975

[-4 2169 4 1.021
2201 1.076
1.-i
0 2207 0.97
ATCC 0.91s
CALIF 1.013
NEW YORK 1.016
JORDAN 1.012
S GREECE 0.99
PRSV-P 0. 539
PRSV-T o 1 0.745
WMV-M 0.45
WMV-2 o.154
ZYMV 0.067
ZYFV 0.216
CMV 0.000
SqMV 0.0oo
TV 0.000 E-94

Hssp 0.000

0.0 0.2 0.4 0.6 0.8 1.0 1.2

ABSORBANCE (405 nm)


Fig. 2-7. Reactivity of polyclonal antiserum 1125 with
papaya ringspot virus isolates and seven other viruses
in plate-trapped indirect ELISA test E-94. Absorbance
readings (405 nm) were made 30 min after the addition of
substrate and are an average of two wells. Antiserum
1125 was used at a dilution of 1:100,000. All antigens
but CMV, SqMV, TV, and Hssp are considered positive.










111.366 1.3662.281
1 0.027


2.232
2.192


W-1A
2030

2040

2052

Hssp



2246

2247

2249

2251

2252

2254

2260

2262

2263

2264

2266

2267

2268

2269

2272

2283

2287

2514A


2.314


0.052

0.036

0.018
0.027
0.02

0.02
0.025
nlllllumfffsmwssfsmmujlll


2.171


S 0.826
. .... 2.564
1.409


0.039

1.319

1.428

0.038

0.053

0.59

0.019

0.035
2.0
1 -co


S0.061

S1.452
~sslrzzzzzzzzzzzzzzz zzZZE


1.347


1.359

p 1.067
IsHsfflM^..^^L


I
0.0


I
1.0


2.292

2.242

2.359

2.361

2.312

2.438

2.362

2.474


58


2.136

2.134

g 2.319

2.189

2.161

2.061


2.0
2.0


POSITIVE
SMCA-5

M PCA-1125
NEGATIVE

*


I
3.0


ABSORBANCE


(405 nm)


Fig. 2-8. Reactivity of monoclonal antibody F3C-C10
(MCA-5) and polyclonal antiserum 1125 with 22 papaya
ringspot virus isolates in plate-trapped indirect
ELISA. Absorbance readings (405 nm) were taken 1 hr
after substrate addition and were an average of two
wells. MCA-5 was used at a dilution of 1:10,000.
Polyclonal antiserum 1125 was used at a 1:100,000
dilution.


2.298


I











papaya ringspot

zucchini yellow
mosaic

healthy ssp





Bidens mottle


2.521


1.201


0.772


0.228
0.013
0.015


1.629


0.701


cowpea aphid-borne 0.061
mosaic 0.123


S peanut mottle


(D peanut stripe


potato virus Y


soybean mosaic

tobacco vein
mottling

watermelon mosaic-2

healthy
N. benthamiana




wheat streak mosaic


healthy wheat


1.215


r ~0.232
10.149


00.411
0.492


1.338


0.642


W 0.389
0.319

0.027
0.005


0.031
0.01

0.023
0.008


POSITIVE
E-75

0 E-77

NEGATIVE

*


0 1 0 1 5 I I
0.0 0.5 1.0 1.5 2.0 2.5 3.0


ABSORBANCE


(405 nm)


Fig. 2-9. Reactivity of polyclonal antiserum 1125
with various potyviruses in plate-trapped indirect
ELISA (tests E-75 and E-77). Absorbance readings
(405 nm) were taken 1 hr after substrate addition and
readings are an average of two wells. Antiserum 1125
was used at a dilution of 1:100,000.


I


0.312













papaya ringspot


zucchini yellow
mosaic

healthy ssp





Bidens mottle





peanut mottle


peanut stripe


potato virus Y


I soybean mosaic

tobacco vein
mottling




healthy
N. benthamiana



wheat streak
mosaic


healthy wheat


a .


0.0 0.5 1.0 1.5 2.0 2.5 3.0


ABSORBANCE (405 nm)


Fig. 2-10. Reactivity of polyclonal antiserum 1142
with various potyviruses in two plate-trapped
indirect-ELISA tests, E-75 and E-77. Absorbance
readings (405 nm) were taken 1 hr after substrate
addition and are an average of two wells. Antiserum
1142 was used at a dilution of 1:10,000.


0.486
0.808

0.642
1 .,

0.353
















TABLE 2-3. Use of plate-trapped indirect ELISA to assay
papaya inoculated with isolates of papaya ringspot
virus.



PRSV isolates in papaya PRSV-W and PRSV-P in ssp

Virus Absorbance Virus Absorbance

PRSV-W
W-1A 0.000a W-1A 1.059
1637 0.000 PRSV-P 1.049
2207 0.015 Hssp 0.000
2040 0.000
2030 0.000
2052 0.000
2038 0.000
2169 0.000
1870 0.000
2201 0.000
Jordan 0.000
Greece 0.000
New York 0.001
Calif. 0.000
ATCC 0.000
PRSV-P 0.315
PRSV-T 0.000
WMV-M 0.000
H. papaya 0.000

a
Absorbance values (405 nm) are an average of 2
wells.Only PRSV-P in papaya and W-1A and PRSV-P in
squash were positive in this test. Antiserum 1125 was
used as the detecting antiserum in this test (E64).









Polvclonal ascitic fluid. In antibody-trapped

I-ELISA, the polyclonal ascitic fluid to W-1A reacted

with all the PRSV-W isolates and PRSV-P, and weakly with

PRSV-T. They did not react with any of the other squash

viruses tested or with Hssp (Fig.2-11).

In antibody-trapped I-ELISA, the polyclonal

ascitic fluid to the the whole virus (used at a dilution

of 1:2000, Table 2-4) did not react with WMV-M. The

polyclonal ascitic fluid to the capsid protein (used at

a dilution of 1:5000, Table 2-5) reacted weakly with

WMV-M.

In plate-trapped I-ELISA, the polyclonal ascitic

fluid to the capsid protein reacted stronger to PRSV-T

and WMV-M than in antibody-trapped I-ELISA and it cross-

reacted with WMV-2, ZYMV, and ZYFV (Fig.2-12).

The ascitic fluid made to Sp2/0 cells was used

at 1:10,000 and did not react with anything It was

used as a negative control in both types of I-ELISA

throughout this work.



Fusions, Screening, and Cloning

Six fusions were done using spleen cells which

had been exposed to the structural protein of W-1A.

Four were exposed to the purified capsid protein (Fl-4)

and two (F21-22) were done with spleen cells exposed to

the whole virus.









F1 produced only 5 hybridomas from 4 plates (96

well plates). None produced specific antibodies.

Fusions 2, 3, and 4 were done at the same time using the

three methods described in the Materials and Methods

section. Table 2-6 summarizes the results of these

three fusions. The nonimmunized in vitro fusion (F2)

gave no antibody producing hybridomas and it was decided

to drop this method in future fusion attempts. The

fusion, F3 (preimmunized in vitro), gave the highest

percentage of hybridomas and the highest percentage of

W-1A specific antibody producing hybridomas. The fusion

efficiency of both F21 and F22 was extremely low. Only

five hybridomas were produced from F21 (10 plates) and

two of these produced antibodies. Twelve hybridomas

were produced from F22 (one plate). Eleven of these

were antibody positive in the first test but only four

remained viable after manipulation. Of the eight

primary hybridomas from F3 and three from F4 taken out

of liquid nitrogen, eight were viable, three produced

antibodies, and only F3C-C10 retained antibody producing

capabilities throughout cloning. The two antibody

producing hybridomas from F21, C-E4 and D-E10 and the

four antibody producing hybridomas from F22, A-C8, A-B9,

A-E8, and A-H9, were tested further and cloned. Since

the secondary clones of A-H9 lost antibody producing

capabilities and it had been determined that the

reactivity of this MCA was the same as that of C-E4












W-1A

1637

1870

2030

2038

2040

2052

2169

2201

2207

ATCC


CALIF

NEW YORK

JORDAN

SGREECE


PRSV- P

PRSV-T
WMV-M

WMV-2

ZYMV

ZYFV

Hssp


0.973
1.288
0.642
0.849
0.619
0.787
0.563
0.665
0.595

0.607
0.703
0.536
0.616
0.523
0.655
0.528

0.536
0.741
0.583
0.626
0.689
0.778
0.709
0.824
0.516
0.682
0.663
0.791
0.55
0.655


_ 0.193
0.135
0.116
0.081
0.071
0.065
0.077
0.076
0.088
0.07
S0.035
0.049


0.0
0.0


0.5


I
1.0


POSITIVE
M PCA-CP
E PCA-WV

NEGATIVE

*


1.5


ABSORBANCE


(405 nm)


Fig. 2-11. Reactivity of polyclonal ascitic fluid
to papaya ringspot virus type-W (PCA-WV) and its
capsid protein (PCA-CP) in antibody-trapped
indirect-ELISA with papaya ringspot virus and
other potyviruses which infect cucurbits.
Absorbance readings were taken 30 min after the
addition of substrate. PCA-WV was used at a
dilution of 1:2000. PCA-CP was used at a dilution
of 1:5000.


2.0



















TABLE 2-4. Indirect ELISA tests to determine the
optimal dilution of polyclonal ascitic fluid to papaya
ringspot virus type-W.


Dilution Antigen Antibody-trapped


30 45 60


1 X 10-3

b
2 X 10-3


5 X 10-3


1 X 10-4


5 X 10-4


1 x 10-5


W-1A
Hssp


Plate-trapped

30 45 60


0.449 0.868 1.193 0.265 0.560 0.713
0.016 0.040 0.060 0.017 0.027 0.032


W-1A 0.326 0.634 0.879 0.204 0.416 0.562
Hssp 0.029 0.038 0.046 0.010 0.019 0.018

W-1A 0.178 0.335 0.473 0.140 0.284 0.370
Hssp 0.013 0.031 0.042 0.011 0.017 0.016

W-1A 0.116 0.215 0.293 0.071 0.146 0.193
Hssp 0.011 0.025 0.032 0.007 0.037 0.017

W-1A 0.058 0.111 0.155 0.033 0.069 0.090
Hssp 0.024 0.023 0.052 0.010 0.013 0.015

W-1A 0.039 0.073 0.098 0.030 0.056 0.077
Hssp 0.019 0.019 0.030 0.008 0.024 0.016


Absorbance values


(405 nm) taken at 30,


minutes after substrate addition.


45, and 60


The dilution chosen for subsequent I-ELISA tests.
















TABLE 2-5. Indirect ELISA tests to determine the


optimal dilution of polyclonal ascitic
ringspot virus type-W capsid protein.


fluid to papaya


Dilution


1 X 10-3


2 X 10-3


5 X 10-3


1 X 10-4


5 X 10-4


1 X 10-5


Antigen


W-1A
Hssp


Antibody-trapped

30 45 60a

0.570 1.097 1.459
0.043 0.084 0.119


W-1A 0.462 0.911 1.226
Hssp 0.037 0.071 0.102


W-1A
Hssp

W-1A
Hssp

W-1A
Hssp

W-1A
Hssp


0.241 0.474 0.652
0.026 0.041 0.057

0.184 0.362 0.495
0.017 0.025 0.033

0.079 0.167 0.235
0.006 0.013 0.019

0.076 0.167 0.184
0.013 0.019 0.021


Plate-trapped

30 45 60

0.414 0.847 1.072
0.013 0.050 0.046

0.500 0.982 1.263
0.007 0.014 0.022

0.234 0.481 0.649
0.009 0.010 0.011

0.129 0.262 0.354
0.006 0.011 0.011

0.080 0.165 0.221
0.008 0.010 0.010


0.047 0.093
0.008 0.010


0.12
0.010


a
Absorbance values (405 nm) taken at 30, 45, and 60
minutes after addition of substrate.
b
The dilution selected for subsequent indirect
ELISA tests.











WMV- 1A

1637

1870

2030

2038

2040

2052
2169

2201

2207

ATCC

CALIF


NEW YORK

JORDAN


GREECE

PRSV-P

PRSV-T
WMV-M

WMV-2


ZYMV

ZYFV

SqMV

Hssp


1.469
1 .383

1.06
y^^ ^ ^.;r 1 06
1.031
1.002
0.886
l l l l l 0.73
1.019
.975
1.017
0.818
0.962
0.961
1.009
0.892
0.884
0.866
0.855
0.727
1.057
0.898
1.252
1 .202
1.218
1.124
1.176
1.215
1.212
1.063
0.99
0.908
0.716


- 0 .12
0.191

o 0.156
0.031
0.304

S0.013
0 0.01
0.009
0.006


0.0


0.437


0.5


1.0
1.0


POSITIVE
* PCA-CP
v PCA-WV
NEGATIVE
M


1.5


ABSORBANCE


(405 nm)


Fig. 2-12. Reactivity of polyclonal ascitic fluid to
papaya ringspot virus type-W (PCA-WV) and its capsid
protein (PCA-CP) in plate-trapped indirect-ELISA with
papaya ringspot virus and other viruses which infect
cucurbits. Absorbance readings were taken 45 min after
the addition of substrate. PCA-WV was used at a
dilution of 1:2000. PCA-CP was used at a dilution of
1:5000.


2.0


r






















TABLE 2-6. Summary of data from fusions 2, 3, and 4.


Fusion 2a Fusion 3a Fusion 4a

Total number of wells 152 384 960

Total number of hybridomas 15% 65% 22%
(fusion efficiency)

Total hybridomas tested b 5 169 100

Specific hybridomas 0 24 6

Specific hybridomas/ 0% 14% 6%
total hybridomas tested

a
Fusion 2 was a non-immunized in vitro fusion, fusion 3
was a pre-immunized in vitro fusion, and fusion 4 was
an in vivo fusion.
b
Only those hybridoma wells in which growth was
prolific were tested. The others either did not
continue to grow or grew too slowly to be useful.










except that it was an IgGl,k, nothing further was done

with this hybridoma beyond freezing it. The remainder of

the MCAs appeared to be different and each hybridoma was

cloned twice (Table 2-7).



Characterization of Six Monoclonal Antibodies (MCAs)

Six MCAs, obtained from the six fusions

described above, were characterized for their reactivity

in two types of I-ELISA to 15 isolates of PRSV-W, PRSV-

P, PRSV-T, WMV-M, ZYMV, ZYFV, and WMV-2, and for their

ability to react in protein blots.

MCA-1 (F21D-E10). MCA-1, an IgGl,K (Table 2-8),

only reacted in antibody-trapped I-ELISA (Fig.2-13 and

2-14). In antibody-trapped I-ELISA, it reacted

positively with all 15 isolates of PRSV-W tested so far,

with PRSV-P, and with PRSV-T; it did not react with WMV-

M, WMV-2, ZYMV, ZYFV, or Hssp (Fig.2-13 and 2-14). It

also did not react with CMV, SqMV, or TV. The ascitic

fluid, used at a dilution of 1:2000 (Table 2-9), gives

low absorbance readings and has a high background

(Fig.2-14). MCA-1 did not react in protein blots.

MCA-2 (F22A-B9). MCA-2, an IgM,K (Table 2-8),

reacted best in antibody-trapped I-ELISA (Fig.2-15 and

2-16). It reacted with all 15 PRSV-W isolates tested in

antibody-trapped I-ELISA and with PRSV-P; it did not

react to PRSV-T, WMV-M, ZYMV, ZYFV, or Hssp (Fig.2-15

and 2-16). It also did not react with CMV, SqMV, or TV.










When WMV-2 was trapped with PCA-1125, this MCA did not

react with WMV-2. However, when WMV-2 was trapped by its

homologous polyclonal antiserum, MCA-2 did react with

WMV-2 isolates and the absorbance values were

approximately equal to those of PRSV-W trapped with PCA-

1125. Sixteen of the twenty isolates of WMV-2 tested

reacted positively with this MCA when trapped with

homologous antiserum (Fig.2-17). Four isolates of WMV-

2 appeared to be negative as their absorbance values

were similar to that of the healthy tissue. This MCA

also reacted with cowpea aphid-borne mosaic virus and

potato virus Y in an antibody-trapped I-ELISA test (E-

77) with PCA-1125 as the trapping antibody. MCA-2 did

not react in protein blots.

MCA-3 (F22A-E8). MCA-3, an IgM,K (Table 2-8),

reacted in both plate-trapped and antibody-trapped I-

ELISA (Fig.2-18). This MCA also reacted to isolate

2254 (data not shown) but it did not react to PRSV-W

isolates 2207 and 2169, PRSV-P, PRSV-T,or WMV-M, ZYMV,

ZYFV, WMV-2, or Hssp (Fig. 2-18). It also did not react

to SqMV, CMV, or TV. MCA-3 did not react in protein

blots.

MCA-4 (F21C-E4). MCA-4, an IgG2a,K (Table 2-8),

reacted strongly in both plate and antibody-trapped I-

ELISA (Fig.2-19 and 2-20) The ascitic fluid was used

at a dilution of 1:10,000 (Table 2-10). Both the tissue

culture fluid and the diluted ascitic fluid reacted with










all but two (2207 and 2169) of the PRSV-W isolates

tested in the main study (Figs.2-19 and 2-20). This MCA

also did not react with isolate 2254 (data not shown).

It reacted with PRSV-P but it did not react with PRSV-T,

WMV-M, WMV-2, ZYMV, ZYFV, or Hssp (Figs.2-19 and 2-20).

It did not react with any of the other potyviruses

tested, SqMV, CMV, or TV. MCA-4 did react in protein

blots to a protein in infected tissue extracts at the

approximate molecular weight of the capsid protein of

PRSV. It did not react with healthy tissue extracts.

MCA-5 (F3C-C10). MCA-5, an IgGl,K (Table 2-8),

reacted in both plate-trapped and antibody trapped I-

ELISA (Figs.2-21 and 2-22). However, its reactivity in

plate-trapped I-ELISA was stronger than that in

antibody-trapped I-ELISA and the antibody-trapped test

tends to have higher background levels. Of the 33

isolates shown to be infected with PRSV in SDS-

immunodiffusion and I-ELISA tests using PCA-1125, eleven

(2040, 2052, 2030, 2246, 2247, 2252, 2262, 2263, 2266,

2267, and 2269) did not react with this monoclonal

antibody in either type of I-ELISA (Figs. 2-8, 2-21, and

2-22). The remaining isolates of PRSV-W reacted

positively with this antibody as did PRSV-P and PRSV-T.

WMV-M, ZYMV, ZYFV, and Hssp did not react with this

monoclonal antibody (Fig.2-21 and 2-22). SqMV, CMV, and

TV also did not react with this MCA. MCA-5 reacted in

protein blots to extracts from PRSV-W infected tissue










but not to extracts from healthy tissue. The molecular

weight of the reactive band was approximately that of

the capsid protein.

This MCA was tested in plate-trapped I-ELISA

against 8 isolates of ZYMV in addition to isolate 1119.

Four of these were from France (Leqoc, unpublished) and

the remainder from Florida. The mean value of the

absorbance readings 1 hour after the substrate was added

was 0.009 (Hssp =.002, W-1A = 1.394). The mean value of

their reaction with polyclonal antiserum 1125 was 0.119

(Hssp =.007, W-1A = 0.859). In another plate-trapped I-

ELISA test, this MCA was tested with sap from 12 plants

singly infected with isolates of ZYMV (one from Italy

and the remainder from Florida). Again there was no

reactivity with this MCA (mean = 0.047 at 60 min). The

mean absorbance values for W-1A and Hssp in this test

were 2.153 and 0.032, respectively. Rabbit polyclonal

antiserum to ZYMV reacted with all 12 of the ZYMV

isolates (mean = 1.575 at 60 min).

There also was no reactivity with this MCA to

the same 12 isolates of ZYMV used above (mean = 0.083)

when polyclonal antiserum to ZYMV was used as the

trapping antibody in an antibody-trapped test. The

absorbance values 60 min after substrate addition for

Hssp and W-1A (trapped with 1125) were 0.062 and 0.337,

respectively. A MCA to ZYMV (Wisler et al., 1989) did










react with all 12 ZYMV isolates (mean=1.768 at 30 min)

in this test.

WMV-2 reacted weakly with this MCA in plate-

trapped I-ELISA but not in antibody-trapped I-ELISA

(Fig.2-21 and 2-22). The absorbance value for the WMV-2

reaction in plate-trapped I-ELISA was always lower than

that of PRSV-W and sometimes appeared to be negative.

The results in antibody-trapped I-ELISA were the same

whether polyclonal antiserum 1125 or a polyclonal

antiserum to WMV-2 was used as the trapping antibody.

Similar results were seen with the 20 WMV-2 isolates

tested.

Other potyviruses which reacted with this MCA in

plate-trapped I-ELISA include: Bidens mottle virus,

peanut mottle virus, peanut stripe virus, potato virus

Y, and soybean mosaic virus. Except for Bidens mottle

virus, this cross-reactivity, like that of WMV-2, was

seen only in plate-trapped I-ELISA and not in antibody-

trapped I-ELISA (PCA-1125 was the trapping antibody).

It did not react with tobacco veinal mottle, cowpea

aphid-borne mosaic, wheat streak mosaic viruses, or with

healthy N. benthamiana or healthy wheat (Fig.2-23). In

another test, not shown here, it also did not react with

pepper mottle virus.

MCA-6 (F22A-C8). MCA-6, an IgM,K (Table 2-8),

reacted strongly in both I-ELISA tests but it reacted

only with the immunogen, W-1A (Fig.2-24). Like MCA-4 and










MCA-5, this MCA reacted in protein blots with infected

tissue but not with healthy extracts.

Table 2-11 summarizes the reactivities of the six

MCAs to 17 isolates of PRSV, WMV-M, six other squash

viruses, and Hssp.



Discussion



This study provides evidence for at least seven

different epitopes of PRSV-W; it shows the existence of

serological variability heretofore unknown in Florida

isolates of PRSV-W; and it shows that monoclonal

antibodies can be used successfully to distinguish PRSV-

W from its variants PRSV-P and PRSV-T and from other

cucurbit viruses.

MCAs 1 and 2 appear to react to two different

epitopes that are present on all the PRSV-W isolates

tested so far and on PRSV-P. The two epitopes are

differentiated by the fact that MCA-1 reacted with PRSV-

T while MCA-2 did not react with PRSV-T.

MCA-3 and MCA-4 recognize epitopes of PRSV which

appear to be altered in PRSV-W isolates 2207 and 2169.

These MCAs differ from MCA-1 and MCA-2 because they

react in both types of I-ELISA tests. They differ from

each other in that MCA-3 reacted with isolate 2254 but

not with PRSV-P; MCA-4 does react with PRSV-P but










TABLE 2-7. Cloning results for monoclonal antibodies
(MCAs) to papaya ringspot virus type-W or its capsid
protein.


MCAs

F21D-E10 F3C-C10 F21C-E4

Cloning 1st 2nd 1st 2nd 1st 2nd

+/# tested 15/15 6/7 26/26 19/20 15/16 14/14
b c b c b c
Clones used F3 H8 C12 C11 B3 D6



MCAs

F22A-B9 F22A-E8 F22A-C8

Cloning 1st 2nd 1st 2nd 1st 2nd
a
+/# tested 11/11 6/8 9/9 16/16 24/26 10/10
b c b c b c
Clones used G3 E2 E7 F12 F11 G5


a
Number of positive clones (+) over the number of
clones tested. Wells with only one dividing cell 24 hr
after dilution were selected. When the selected wells
contained 200 il of media the tissue culture fluid was
tested in plate-trapped and antibody-trapped I-ELISA
against extracts from W-1A infected tissue and Hssp.
b
These primary clones were used for the second cloning.
c
Except for F22A-C8-Fll-G5, these secondary clones were
increased and injected into pristane-primed BALB/c
mice for the production of ascitic fluid. They were
selected based on their high absorbance with infected
sap and low background with healthy sap.




















TABLE 2-8. Isotyping of six monoclonal antibodies to papaya
ringspot virus type-W by indirect ELISA.



Isotypinq antibody
a
Antibody I G1 IqG2a G2b IG3b IQA IM K A N
b
F21D-E10 0.716 0.052 0.085 0.041 0.126 0.049 1.125 0.070 0.028
F22A-B9 0.075 0.032 0.081 0.051 0.087 0.275 0.228 0.057 0.039
F22A-E8 0.066 0.096 0.090 0.088 0.111 1.402 1.162 0.110 0.064
F21C-E4 0.041 0.863 0.044 0.025 0.072 0.051 1.182 0.045 0.022
F22A-C8 0.052 0.078 0.074 0.054 0.097 1.346 1.031 0.110 0.036
c
F3C-C10 1.065 0.008 0.118 0.125 0.039 0.003 0.968 0.037 0.012

a
Isotyping was done using a MonoAb EIA Kit (ZYMED LAB,
Inc., San Francisco, CA) and tissue culture fluid from
the clone used for ascites production. IgG1, IgG2a,
IgG2b, IgG3b, IgA, and IgM represent the heavy chain
isotypes. The light chain isotypes are represented by
K and A. N stands for normal serum which was the
negative control.
b
Antibody-trapped test (E-89). Absorbance readings (405
nm) were read 1 hr after substrate addition.
c
Plate-trapped test (E-19) read 20 min after substrate
addition.





















TABLE 2-9. Indirect ELISA tests to determine
the optimal dilution of ascitic fluid to monoclonal
antibody F21D-E10 (MCA-1).


Dilution
1 x 10-3
2 x 10-3 a
5 x 10-3
1 x 10-4
5 x 10-4
1 x 10-5


Antibody-trapped

W-1A Hssp
0.388 b 0.094
0.241 0.068
0.179 0.044
0.169 0.032
0.239 0.022
0.136 0.015


Plate-trapped


W-1A
0.132
0.062
0.028
0.020
0.021
0.121


Hssp
0.035
0.018
0.011
0.009
0.012
0.015


a
Dilution used in subsequent tests.

b
Absorbance reading (405 nm) were taken 1 hr after
substrate addition.











W-1A

1637

1870


2030

2038

2040

2052

2169

2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


0.001
'r/rrrrrrrrrrlumjfjf llfwwwwmwwfllj


0.508


- 0.0460.5
/0.535
S10.106
0.408

0.565
~ 0.043 0.475

-I 0.059
0.564
--, 0.051


--- 0.052

1 0.008


0.591


0.429


0.468


- 0. 037


0.52


-- 0.058

.049


0.535


0.538


0.598


- 0.04

- 0.058


0.536

0.537


S0.048


0.503


0.006 74
-11 ........ 0 .301

O.n 0.081 PLA'
_0.051
0.083
--0.044
0.69 ANT

9O03 0.055
-no 0.044
'0.064


I I
0.0 0.1


TE-TRAPPED (E-91)
POSITIVE
[] NEGATIVE
[BODY-TRAPPED (E-90)
POSITIVE
[ NEGATIVE


0.2 0.3 0.4 0.5
0.2 0.3 0.4 0.5


' I I
0.6 0.7
0.6 0.7


ABSORBANCE (405 nm)



Fig. 2-13. Reactivity of tissue culture fluid to
monoclonal antibody F21D-E10 (MCA-1) against papaya
ringspot virus and other potyviruses in indirect ELISA.
ELISA 90 (E-90) and ELISA 91 (E-91) were done with
undiluted tissue culture fluid. Absorbance readings were
taken 30 min after substrate addition and are an average
of two wells.


~777-Mf lIU72l 7


I










1 0 nnA


i) 0.167
- n m )


1 0.008


W-1A

1637

1870

2030

2038

2040

2052

2169


I n 0.0


S0.006

0 009o


2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


0.0


n-i 0017


0.243


10.006

- n0 11


0.235


0.200


-1 0.015


0.215


0.011
l lllJl 0.259
S0.012
.......... 0.171
S0.01
S.0.277
0.000
, ,, , ,, 0 .14


I 0 003mm


0.000
7-7-7-7-77-7=


0.116


0.000
= I 0.067
0.000
=* 0.071


0.000
T f /7


0.075


0.063


' I
0.1


0.2
0.2


0.221


PLATE-TRAPPED
POSITIVE
] NEGATIVE
ANTIBODY- TRAPPED


0


0.3
0.3


ABSORBANCE


(E-94)


(E-93)


POSITIVE
NEGATIVE


0.4


(405 nm)


0.5
0.5


Fig. 2-14. Reactivity of ascitic fluid to monoclonal
antibody F21D-E10 (MCA-1) to papaya ringspot virus and
other potyviruses in indirect ELISA. ELISA 93 (E-93)
and ELISA 94 (E-94) were done with ascitic fluid
diluted 1:2000. Absorbance readings were taken 30 min
after substrate addition and are an average of two
wells.


0.355


1 0.01


0.287


0.279


0.466


0.409


0.339


0.272


-










0.00

0. 0.489

z0 0.359


W-1A

1637

1870

2030

2038

2040

2052

2169


2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


i 0.014
Z 0.482

S0.624

0 4 0.311

zz z 0.455
0 097
.ZZZZZZZZ 00.36

7_9 0.526

-W 0.348
- 0.088 0.602
'____iyyiwgzninm 0. 602


0.022
0.045
0.000
3 0.042
S0.02
0.043
10.032
a 0.041
0.000
2 0.062
0.00
300.035


0.0


0.5


PLATE-TRAPPED (E-91)
POSITIVE
5 NEGATIVE

ANTIBODY-TRAPPED (E-90)
[ POSITIVE
0 NEGATIVE


1.0


1.5


ABSORBANCE


(405 nm)


Fig. 2-15. Reactivity of tissue culture fluid to
monoclonal antibody F22A-B9 (MCA-2) to papaya ringspot
virus and other potyviruses in indirect ELISA. ELISA
90 (E-90) and ELISA 91 (E-91) were done using undiluted
tissue culture fluid. Absorbance readings were taken
30 min after substrate addition and are an average of
two wells.


1.329


--i 0.135

I 0.05
Zzz 0.432
-, 0.225

S.00.436

- 0.125


1.147


1.151


0.809


2.0











W-1A

1637

1870

2030

2038

2040

2052


2169

2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


-----, 0.06


0.172
WMM MW MM ff/ W If


0.068


0.086


0 U U0 7
- 0u.ubo

- 0.077


0.272


0.262


0.246


0.067
ZrMZlZVZ 0 0.278

M0.091 0.251
0.0710.293
0.293


~wnmM 0.294

.m 0.251

- 0.255

-m 0.241


0.067

0.12 0.127
0.132 0.204
1 0.132


i 0.105


0


I~I fl fl i; r r~llllllrr rr


-IT o o50.041

0.052
..-777.J 0.061


I 5 0.049
0.052

71 0.107
S0.046
^ ^'y^IJ 0.071
0.048
0. 0 .074

0 0.064


*1 A.


0.0


' I
0.1


0.277


0.308


.231

0.25


0.167


PLATE-TRAPPED


(E-94)


POSITIVE
NEGATIVE


ANTIBODY-TRAPPED (E-93)

H POSITIVE
]0 NEGATIVE


' I
0.2


ABSORBANCE


' I

0.3


(405 nm)


0.4


Fig. 2-16. Reactivity of ascitic fluid to monoclonal
antibody F22A-B9 (MCA-2) with papaya ringspot virus and
other potyviruses in indirect ELISA. ELISA 93 (E-93)
and 94 (E-94) were done using ascitic fluid at a
dilution of 1:2000. Absorbance readings were taken 30
min after addition of the substrate.


r~m~0.086


r////////////~sr/r/l/////////~r/~rr/////










1656

2005

2159

2235

2295

2315


2325

2328

2390

2400
2421


2567
ATCC PV-27

ATCC 619

ADLERZ

CALIF

NEW YORK

NEW ZEALAND

WEBB

628
Hssp


.752


0.35


0.562

0.490

0.415

0.388

0.490

0.514

0.306
0.328

0.382

0.454


0.073

Z 0.052
S0.126

0.309
-0.198


0.446


0.151



S--- 0.091


' I
0.2


0.267 U POSITIVE

D NEGATIVE


' I
0.4


I
0.6


ABSORBANCE


(405 nm)


Fig. 2-17. Reactivity of monoclonal antibody-2 (MCA-2)
with watermelon mosaic-2 (WMV-2)in indirect ELISA. In
this test polyclonal antiserum to WMV-2 was used as the
trapping antibody and MCA-2 was used at a dilution of
1:2000. Absorbance readings were taken 45 min after
addition of the substrate and are an average of two
wells.


0.8
0.8


0


I I


4.










W -1A 0 4527

1637 0 0.258 0.77

1870 0.258 0.614
0.421 0.549
2030 ......

2038 0 .255 0.727
20382
2040 .---------- 0.815

2052 0 m.2m 7 0.646
2169 20..o292
0.219
2201 0.243
0.052
C 2207 0.051
A C 0.416
[-H ATCC .... 0.248
CALIF 0.639
iI CALIF .... - . 02
0 NEW YORK 0.42
0.327
H JORDAN .. . .. 1.038
0.378
GREECE 0.393

PRSV-P 8:
H 0.021
PRSV-T 10.033
0.017 PLATE-TRAPPED (E-95)
WMV-M 0.031 POSITIVE
POSITIVE
0.024
WMV-2 0.029 O NEGATIVE
S 0.023
ZYMV 0.026 ANTIBODY-TRAPPED (E-96)
0. 029
ZYFV 0 .029 POSITIVE
0.036 NEGATIVE
Hssp 0.0226 1 NEGATIVE

0.0 0.2 0.4 0.6 0.8 1.0 1.2

ABSORBANCE (405 nm)



Fig. 2-18. Reactivity of tissue culture fluid to
monoclonal antibody F22A-E8 (MCA-3) to papaya ringspot
virus and other potyviruses in indirect ELISA. Tissue
culture fluid was diluted 1:5 in ELISA 95 (E-95) and
ELISA 96 (E-96). Absorbance readings were taken 45 min
after the addition of the substrate and are an average
of two wells.










W-1A 0.8 1.054
1637 1.585

1870
0.886 i.935
2030 ... -- ... 0883 1.578
2030

2038 0.922
2040 .961 1.535
2052 .9 1.409

S2169 047
2201 1.364
0.061
S2207 0.071
S ATCC T2 1.645
H 0.927
CALIF 1.557
1.488
D NEW YORK ... 0. 96 2488
Pm m 0.327
H JORDAN 0.783
GREECE 0.935
PRSV-P 110.8
PRSV-T 00.082
PLATE-TRAPPED (E-91)
0 049
WMV-M 0.057 POSITIVE

WMV-2 0.0 NEGATIVE
ZYMV ; 0.059 ANTIBODY-TRAPPED (E-90)
ZYFV 0.013 POSITIVE
ZYFV 0.046
0.057 NEGATIVE
Hssp 0.032


0.0 0.5 1.0 1.5 2.0

ABSORBANCE (405 nm)

Fig. 2-19. Reactivity of tissue culture fluid to
monoclonal antibody F21C-E4 (MCA-4) with papaya ringspot
virus and other potyviruses in indirect ELISA. ELISA
test 90 (E-90) and ELISA test 91 (E-91) were done with
undiluted tissue culture fluid. Absorbance readings were
taken 30 min after addition of the substrate.










W-1A

1637

1870

2030

2038

2040

2052

W 2169

2201

-21 2207
0
UC ATCC

CALIF

n NEW YORK


0.984


I 0.925


1 .1


0.785
1
0.907

0.815

0.884

0.032
0.038
1.016
i.
0.03
0.035


I Ul
m 1.
199
-m 1.1
1.013
'f/MM222*1


1.289

1.293

96

.252

1.264

.222

1.267



231



196

183

1.292


JORDAN A 0.741

GREECE 1.308
0.569
PRSV-P P.. 6 1.056
0.011
PRSV-T i 0.029
0.003 PLATE-TRAPPED (E-94)
WMV-M o 0.018
0.007 POSITIVE
WMV-2 a 0.035 NEGATIVE
0.005
ZYMV 3 0.041 ANTIBODY-TRAPPED (E-93)
0.004
ZYFV 0.026 0 POSITIVE

Hssp 0.027 NEGATIVE


0.0 0.5 1.0 1.5 2.


ABSORBANCE (405 nm)


Fig. 2-20. Reactivity of ascitic fluid to monoclonal
antibody F21C-E4 (MCA-4) with papaya ringspot virus and
other potyviruses in indirect ELISA. ELISA test 93
(E-93) and ELISA test 94 (E-94) were done with ascitic
fluid at a dilution of 1:10,000. Absorbance readings
were taken 30 min after addition of the substrate.


0


H


bra~r~n~ra~














TABLE 2-10. Indirect ELISA tests to determine the
optimal dilution of ascitic fluid to monoclonal
antibody F21C-E4.


Dilution


Antigen


Antibody-trapped
a
30 45 60


Plate-trapped

30 45 60


1 X 10-3


2 X 10-3


5 X 10-3


1 X 10-4


5 X 10-4

b
1 10-5


W-1A
Hssp


1.115 2.091 2.672 0.849 1.572 1.976
0.034 0.078 0.113 0.018 0.035 0.049


W-1A 0.919 1.707 2.221 0.656 1.263 1.598
Hssp 0.024 0.057 0.080 0.013 0.020 0.026


W-1A
Hssp


0.609 1.253 1.662 0.488 0.945 1.222
0.024 0.046 0.064 0.005 0.009 0.013


W-1A 0.618 1.235 1.639 0.463 0.869 1.128
Hssp 0.017 0.033 0.047 0.012 0.016 0.017


W-1A
Hssp


0.551 1.131 1.500 0.440 0.850 1.118
0.007 0.016 0.024 0.007 0.006 0.008


W-1A 0.428 0.913 1.182 0.342 0.662 0.875
Hssp 0.001 0.009 0.016 0.008 0.008 0.009


Absorbance values


(405 nm)


taken


min after addition of substrate.

The dilution selected for use in
tests.


at 30, 45, and 60


subsequent ELISA











W-1A

1637

1870

2030

2038

2040


2052

2169

2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV- P

PRSV T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


0.945
0. 636
0.928
0.301


1.493


0.555
0.000
0.051


1.242


0.634
0.000
Z] 0.095
0.000
S0.033


1.413


raa z 0.394


1.499


0 .408


"m 0.5


1.134


51 .274
0.519


vy..i..... 0.511


1.178

g- 1.012
564


1ena a 0.46 0.805
~0.456


0.479
JI- 0.327
0.278


- 0.358
0.014
0.007
0.005
0.228
0.002
0.003
0.000
0.011
0.011
0.005


_______________________ .1


0.5


0.0


0.965


1.249


PLATE-TRAPPED
POSITIVE
O NEGATIVE
ANTIBODY-TRAPPED


(E-91)



(E-90)


POSITIVE
NEGATIVE


1.0


1.5


ABSORBANCE


(405 nm)


Fig. 2-21. Reactivity of tissue culture fluid to
monoclonal antibody F3C-C10 (MCA-5) with papaya ringspot
virus and other potyviruses in indirect ELISA.. ELISA
test 90 (E-90) and ELISA test 91 (E-91) were done with
undiluted tissue culture fluid. Absorbance readings
were taken 30 min after addition of the substrate and
are an average of two wells.


2.0


- ---











W-1A

1637

1870

2030

2038

2040

2052


2169

2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE

PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


0.0


iasM 0.39

0.303


0.636


0.881


0.847

0.861



0.842


0.577


01

0.011
0.067


0.918


0.522

0.424


0.422

0.605


0.908

0.928


0.953

0.783
0.876


0.52

0.676


0.555


0.913

0.934


0.257
0.27


0.481


0.001
0.054
0.41
0.059
0.001
- 0.069

083064
0.001
0.043


1
0.2


0.631


PLATE-TRAPPED
POSI
0 NEGA


(E-94)
TIVE
TIVE


ANTIBODY-TRAPPED (E-93)
M POSITIVE
E NEGATIVE


' I I
0.4 0.6


' I
0.8


' I
1.0


ABSORBANCE


(405 nm)


Fig. 2-22. Reactivity of ascitic fluid to monoclonal
antibody F3C-C10 (MCA-5) with papaya ringspot virus
and other potyviruses in indirect ELISA. ELISA test
93 (E-93) and ELISA test 94 (E-94) were done with
ascitic fluid at a dilution of 1:10,000. Absorbance
readings were taken 30 min after addition of the
substrate and are an average of two wells.


I ILIIIII~PIIIIIII~













papaya ringspot

zucchini yellow
mosaic

Healthy ssp





Bidens mottle

cowpea aphid-borne
mosaic


1. 125
1.053
0.051
0.101

0.041
0.06


1.762


M1.178
0.036
0.058


peanut mottle


Z
W peanut stripe


potato virus Y

soybean mosaic
i soybean mosaic


tobacco vein
mottling

watermelon mosaic-2

healthy
N. benthamiana




wheat streak mosaic


healthy wheat


1.112


kwh- 0.715


0.762


0.065
0.047

0.204


0.042
0.062


0.724


0.036
0.051

0.065
0.047


0.0


0.5


1.0


1.494
1.205

- 1 .447
T1.297


1.203






POSITIVE

SE-75
SE-77

NEGATIVE
D E-75

3 E-77


1.5


2.0


ABSORBANCE (405 nm)



Fig. 2-23. Reactivity of monoclonal antibody F3C-C10
(MCA-5) with eleven potyviruses in plate-trapped
indirect ELISA. Absorbance readings for ELISA test 75
(E-75) were taken 2 hr after substrate addition and are
an average of two wells. Absorbance readings for ELISA
77 (E-77) were taken 2 hr after substrate addition and
are an average of 2 wells.


a











W-1A

1637

1870

2030

2038

2040

2052

2169

2201

2207

ATCC

CALIF

NEW YORK

JORDAN

GREECE


PRSV-P

PRSV-T

WMV-M

WMV-2

ZYMV

ZYFV

Hssp


1 458


0.067
0.046
0.006
0.03
0.022
0.05
0.022
0.035
0.018
0.054
0.036
0.068
0.013
0.036
0.022
0.05
0.028
0.039
0.02
0.052
0.025
0.023
0.025
0.05
0.021
0.044
0.02
0.046
0.015
0.035
0.018
0.031
0.014
0.023


0.01
S0.034
0.012
0.037
0.015
0.03


I I
0.0 0.5


PLATE-TRAPPED

U POSITIVE
O NEGATIVE

ANTIBODY-TRAPPED

[ POSITIVE
0 NEGATIVE


1.0


1.5 2.0


2.34


(E-94)


(E-93)


' I
2.5 3.0


ABSORBANCE


(405 nm)


Fig. 2-24. Reactivity of monoclonal antibody F22A-C8
(MCA-6) with papaya ringspot virus and other potyviruses
in indirect ELISA. Absorbance readings were taken 30 min
after addition of substrate and are an average of two
wells. ELISA test 93 (E-93) and ELISA test 94 (E-94)
were done with ascitic fluid at a dilution of 1:10,000.

















TABLE 2-11. Summary of the reactivity of six monoclonal
antibodies (MCA) with papaya ringspot virus and other viruses
that affect cucurbits.


ANTIGEN MCA-1 MCA-2 MCA-3 MCA-4 MCA-5 MCA-6 M/Ra
PRSV-W
Florida
W-1A +/ b +/- +/+ +/+ +/+ +/+ +/+
1637 +/- +/- +/+ +/+ +/+ -/- +/+
2207 +/- +/- -/- -/- +/+ -/- +/+
2040 +/- +/- +/+ +/+ -/- +/+
2030 +/- +/- +/+ +/+ -/- /- +/+
2052 +/- +/- +/+ +/+ -/- / +/+
2038 +/- +/- +/+ +/+ +/+ +/+
2169 +/- +/- /- /- +/+ -/- +/+
1870 +/- +/- +/+ +/+ +/+ /- +/+
2201 +/- +/- +/+ +/+ +/+ -/- +/+
Calif. +/- +/- +/+ +/+ +/+ -/+/+
Jordan +/- +/- +/+ +/+ ++ /+ -/ +/+
Greece +/- +/- +/+ +/ + /+ -/- +/+
N.York +/- +/- +/+ +/+ +/+ -/- +/+
ATCC +/- +/- +/+ +/+ +/+ /- +/+
PRSV-P +/- +/- +/+ +/+ -/- +/+
PRSV-T +/- -/ -/- +/+ -/- +/+
WMV-M -/- -/- -/- -/- / / -/+
WMV-2 -/- c/_ -/- -/- -/+ -/ -/+
ZYMV - -/- -/- -/- / / -/+
ZYFV -/- / -/+
SqMV -/- -/- -/- -- -
CMV / /- -//-
Hssp /- -/- -/- /-
a
Polyclonal antiserum: M = mouse ascitic fluid to the
capsid protein of PRSV-W. R = rabbit antiserum 1125 also
to the capsid protein of PRSV-W.
b
The symbol (+ or -) to the left of the / represents the
results of the antibody-trapped tests. The symbol to the
right of the / represents the results of the plate-
trapped tests.

c
Antiserum 1125 was used as the trapping antibody in
these antibody-trapped tests. When WMV-2 is trapped
by its homologous antiserum MCA-2 reacts positively
(see text).










not with isolate 2254. Hence, these MCAs recognize two

additional epitopes of PRSV.

MCA 5 defines a fifth epitope which varied on

33% of the PRSV-W isolates tested but is present on both

PRSV-P and PRSV-T. MCA-6, which reacts only with the

homologous antigen in both I-ELISA tests, recognizes a

sixth epitope.

The results with PCA-1125 in SDS immunodiffusion

defined a seventh epitope variation of the capsid

protein of two additional isolates. The epitope

variation of 2201 and the New York isolate was not

detected by any of the MCAs and is therefore different

from the serological differences found among Florida

isolates with the six MCAs in this study.

In SDS immunodiffusion, antiserum 1142 detected

epitope variations on seven PRSV-W isolates (2201, 2207,

2169, 2030, 2040, 2052, and the New York isolate). It

is not known if this variation is new or if PCA-1142 is

just particularly sensitive to normal epitope

differences of PRSV-W not detected previously. This is,

of course, an argument for the use of MCAs over

polyclonal antiserum in virus identification since MCAs

eliminate serological variation due to the response of

different animals and to differences in antisera

collected from the same animal over a period of time.

None of the MCAs reacted with WMV-M, indicating

that the capsid protein of PRSV-W has at least 5










epitopes not present on the capsid protein of WMV-M.

Two of the MCAs reacted with PRSV-T, indicating that

PRSV-T has at least three epitopes that differ from

PRSV-W. Four of the five MCA-defined epitopes, which

were present on the majority of PRSV-W isolates, were

also present on the virus of PRSV-P. This confirms the

close relationship of PRSV-P with PRSV-W but also shows

that there is a serological difference of at least one

epitope in the capsid proteins of PRSV-P and majority of

PRSV-W isolates tested in this study. However, more

isolates of PRSV-P will have to be tested. The epitope

difference detected by this MCA may simply not be

present in all isolates of PRSV-P as it is not present

in all isolates of PRSV-W.

While MCA-1 and MCA-2 reacted with all the PRSV-

W isolates tested, their positive absorbance values are

too low and their background readings are too high for

use in routine diagnosis. However, a combination of MCA-

4 and MCA-5 has been successfully used in antibody-

trapped I-ELISA together with MCAs specific to WMV-2 and

ZYMV (Wisler et al., in press) to distinguish cucurbit

potyviruses in Florida (Wisler, unpublished).

Besides defining two epitopes of PRSV, MCAs 2

and 5 defined two different epitopes of WMV-2 and one of

these epitopes is not present on all isolates of WMV-2

indicating variability in Florida isolates of WMV-2.










Since the absorbance values obtained in plate-

trapped I-ELISA for MCA-5 with WMV-2 are much weaker

than those with PRSV-W, it is possible that this epitope

on WMV-2 is more internally located than it is on PRSV-

W, is similar enough for binding but is not identical,

is affected by the pH of the sample buffers, or is

affected by some protein structure near the epitope

which interferes with antibody binding. Any of these

explanations could also explain inconsistency of

reactivity seen with this MCA to WMV-2 and why the

cross-reactions are only seen in plate-trapped I-ELISA

even though this MCA does react with PRSV in antibody-

trapped I-ELISA.

On the other hand, the absorbance values of MCA-

2 with WMV-2 trapped with WMV-2 antiserum and with PRSV-

W trapped with 1125 are essentially identical. This

suggests that MCA-2 is probably reacting to an epitope

common to PRSV-W and WMV-2.

Assuming that the reactivity in plate-trapped I-

ELISA is to dissociated virus particles (Dore et al.,

1988), the fact that the results of MCAs 3, 4, 5, and 6

in both plate and antibody-trapped I-ELISA were the same

(except for the lower reactivity of MCA-5 in antibody-

trapped I-ELISA) indicates that the epitopes recognized

by these MCA are present and exposed on the intact virus

and the dissociated virus particle of most PRSV-W

isolates. However, since plant sap was used as the










antigen and not purified virus as was done in the Dore

et al. (1988) study, this conclusion is tentative

because both assembled virus particles and unassembled

capsid protein presumably would be present in infected

plants and both would be trapped by PCA-1125. The

epitopes recognized by MCA-1 and MCA-2 appear to be

present only on the virus particle since these MCAs

react only in antibody-trapped I-ELISA.

Both PCA-1125 and PCA-1142 cross-reacted in

plate-trapped I-ELISA with other potyviruses. Although

this cross-reactivity can be overcome by conjugating the

polyclonal antiserum and using it in direct ELISA (data

not shown), using MCAs eliminates the problem of having

to reconjugate and retest each new conjugate. Using

virus specific MCAs or combinations of MCAs in I-ELISA

can save time and gives more continuity to virus

identification over time.

In this study, the preimmunized in vitro fusions

gave a higher ratio of PRSV-specific antibody producing

hybridomas even when the fusion efficiency was low.

Most reports of monoclonal antibody production to

antigens of plant viruses have been done using in vivo

immunization only. Therefore, it is hard to compare

these results with those in the literature, and the

sample size is too small to know if the difference in

techniques is significant. However, studies with animal

viral antigens have shown similar results. Collen et










al. (1984) compared the antibody production of non-

immunized vs presensitized mouse spleen cells during in

vitro stimulation with the foot-and-mouth disease virus.

Virus-specific antibody production was detected in the

culture supernatants of the previously immunized mouse

cells but not in the nonimmune mouse cell cultures. Fox

et al. (1981) found that spleen cells from preimmunized

mice, which were cultured with antigen prior to

hybridization, gave a 10 fold increase in the percentage

of positive wells per fused cell over those used in

direct hybridization (in vivo). Weigers et al. (1986)

increased antigen-specific hybridomas to poliovirus or

its purified peptides 6-20 times by using spleen cells

primed in vivo and stimulated in vitro. Another

advantage of the in vivo-in vitro immunization technique

is that there are fewer hybridomas to screen since there

are fewer viable cells after culture. This advantage

coupled with an increase in antigen-specific antibody

producing hybridomas make the preimmunized in vitro

technique potentially useful in the generation of MCAs

to plant virus antigens.

There are many factors involved in the success

or failure of the monoclonal antibody cell fusions. Some

of these include: 1) the immunization regime (amount of

immunogen per immunization, time between injections, the

amount and number of injections); 2) the cell ratios

(ratio of spleen cells to myeloma cells vary from 10:1




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - Version 2.9.7 - mvs