INFECTION AND IMMUNITY, Feb. 1972, p. 227-231Copyright © 1972 American Society for Microbiology

Vol. 5, No. 2Printed in U.S.A.

Screening of Sera for Antibodies to Foot-and-MouthDisease Viral Antigens by Radial Immunodiffusion

G. G. WAGNER,1 K. M. COWAN, AND J. W. McVICAR

Plum Island Animal Disease Laboratory, Veterinary Sciences Research Division, Agricultural Research Service,U.S. Department of Agriculture, Greeniport, New York 11944

Received for publication 15 July 1971

Radial immunodiffusion procedures have been developed for testing bovinesera for the presence of antibody activity against three foot-and-mouth diseasevirus types and a virus infection-associated antigen. Reactions with virus antigenswere obtained as early as 4 days after infection, and the virus type causing the re-

sponse was identified. The procedure had the advantages of great sensitivity forthe detection of antibody and the ease with which antibodies to a variety of anti-gens could be detected.

A recent report (2) described the adaptationof radial immunodiffusion (RID) techniques fordetecting foot-and-mouth disease virus (FMDV)antigens and antibodies and the use of the methodfor antibody quantitation. The greatest sensitiv-ity for detecting antibody resulted when anti-serum was incorporated into the agar. Underthese conditions, as little as 1 ,ug of antibodyprotein per ml of serum-agar mixture could bedetected with virus as antigen. This sensitivity andthe ease of examining sera for antibodies to avariety of precipitating antigens suggested appli-cation of the technique as a diagnostic proce-dure. This report presents the results of using RIDfor examining various bovine sera to determinewhether the animal from which the serum wasobtained had antibody to FMDV.

MATERIALS AND METHODS

Antigens. The foot-and-mouth disease viruses usedwere: type A, subtype 24, strain Cruzeiro; type 0,subtype 1, strain Caseros; and type C, strain Resende.All were originally isolated from infected cattle inSouth America. The viruses were obtained as infec-tious bovine tongue epithelium and passaged sevento eight times in primary calf kidney cell cultures.They were then grown in baby hamster kidney-21,clone 13, cell cultures. Virus was concentrated fromthe harvested (at 24 hr) infectious tissue culture fluidsby precipitation with 6% (w/v) polyethylene glycol(Carbowax 20-M, Union Carbide Co. Inc., Chicago,111.) (4). The precipitate was collected by centrifuga-tion, resuspended to 0.1 original volume in 0.14 MNaCl and 0.02 M tris(hydroxymethyl)aminomethane(Tris) -chloride (Tris-NaCI), pH 7.6, and reprecipitatedwith 6% polyethylene glycol. The second precipitate

I Present address: East African Veterinary Research Organiza-tion, Muguga, P. 0. Kabete, Kenya, East Africa.

was resuspended to 0.01 original volume in Tris-NaCIand stored in 0.2-ml samples at -70 C. The prepara-tions were occasionally tested by Ouchterlony-typeimmunodiffusion analyses to detect the presence of12S virus protein subunits (a breakdown product ofthe virus) (1). The virus infection-associated (VIA)antigen was prepared from the infectious tissue culturefluids from cells infected with FMDV, type A, sub-type 12, strain 119, by procedures that bave beendescribed (1).

Sera. Blood samples from cattle were allowed toclot at room temperature, after which the serum wasremoved by centrifugation and stored at -20 C.Some samples had been heated to 56 C for 30 minbefore storage, but those not treated were neverheated before testing. Heating sera to 56 C had noeffect on subsequent reactions.

All sera were from steers experimentally infectedwith FMDV, type A, 0, or C, although not neces-sarily the same subtype or strain as those listed aboveas antigens. For infection, animals were exposed byeither upper respiratory instillation, injection, orcontact with diseased animals. The dates of serumcollection ranged from 4 days to 230 days postin-fection (DPI). Several sera from noninfected steerswere included as controls.RID techniques. A previous report (2) showed that

maximal sensitivity in the RID technique is attainedwhen the component to be detected is incorporatedinto the agar. The procedure reported here consistsof four steps: (i) preparation of a dilution set of eachunknown serum; (ii) addition of melted agar to eachserum dilution to prepare serum-agar mixtures; (iii)transfer of the mixtures to plates; and (iv) additionof a type-specific FMDV or VIA antigen suspensionto wells cut in the hardened serum-agar mixtures.The detailed procedures for preparing plates for RIDanalyses have been described (2).

Plates were incubated at room temperature in ahumidified chamber and checked periodically for thepresence of precipitin rings, with a final reading at

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1/5 1/10 1/20 I/ 40 I /8 0 1 /I 60 1 / 3 20

FIG. 1. Radial immunodiffusion reactions of various dilutions ofbovine aniti-FMDV type A serum tested againstdilutions ofFMDV type A antigen2. The position of tlhe antigen dilutionzs on all slides was as indicated in the left-hand photograph. The final dilutions ofantiserum are indicated above each slide.

TABLE 1. Radial immunodiffusion reactions betweenvarying concentrationis of FMD V type A

antigen and homologous anttiserum

Serum dilutionAnti,endilution-

1/5 1/10 1/20 1/40 1/80 1/160 1/320

1/1 3.Oa 3.2 4.2 4.9 16.0 8.3 10.91/2 b1b 3.4 4.0 4.9 6.0 8.21/4 - -- 3.2 3.9 4.7 6.01/8 - - - 3.2 4.0 4.7

a Diameter in millimeters of precipitin ring in-cluding well.

b Not measurable.

48 hr. The presence of a visible precipitin ring (seeFig. 1) surrounding the antigen well was considered apositive reaction of the antigen and serum. Thehighest serum dilution giving a positive reactiondetermined the type specificity of the original bovineinfection.

Plates were photographed unstained.

RESULTSThe virus antigens concentrated by precipita-

tion with polyethylene glycol and stored at-70 C were found to remain stable for pro-longed periods, as determined by virus antigentiters obtained by complement fixation, andinfectivity titers obtained by tissue culture assays.One preparation of FMDV, type A, remainedunchanged for over 14 months. The virus antigenstocks used in this study were stored approxi-mately 6 months with no detectable change.The amount of antigen to be used for detec-

tion of antibody was determined from the results

of a block, or two-dimensional, titration with asuitable antiserum.

Figure 1 shows a block titration with FMDV,type A, antigen and a 79-DPI homologousantiserum. The antiserum was incorporatedinto the agar at the dilutions shown along thetop of the figure. Samples (10 ,liters) of theantigen preparation were added to the wells atthe dilutions shown along the left side of thefigure. The reactions were recorded in Table 1 asprecipitin ring diameters, measured after 48 hrof incubation.The results in Table 1 demonstrated that high

concentrations of antibody, such as that repre-sented by the 1/5 and 1/10 serum-agar mixtures,kept the positive reactions pushed in towardthe well. Therefore, reactions of 3 mm or lesswere considered questionable unless the ringbecame measurable as the serum was diluted.The 1/2 antigen dilution was selected for usesince it permitted conservation of the virusreagent but gave a positive reaction with verylow antibody concentration, such as that repre-sented by the 1/320 serum-agar mixture. Suchoptimal conditions were established for eachantigen used.The survey experiments involved 150 serum

samples, 30 of which were from normal animals,and the balance from animals infected with oneor more types of FMDV. None of the sera fromnormal animals gave any evidence of reactionwith the antigen preparations used. Figure 2shows the reactions obtained with several repre-sentative sera which were positive for antibody.The reactions in Fig. 2 are summarized, alongwith several others, in Table 2. All of the serum

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DETECTION OF FMDV ANTIBODIES

FIG. 2. Radial immuntodiffusioni reactions of dilutionis of various bovine anitisera tested againist virus infectionl-associated (VIA) antigen and FMDV types A, 0 and C antigenis. The positioni of the anttigens in all wells was asinidicated in the upper left-hand photograph. The reactions are sutmmarized anid the sera are identified in Table 2.

samples collected 5 or more DPI gave positiveand specific virus reactions. Two of four samplesobtained 4 DPI gave positive reactions. The othertwo were retested; however, no positive reactionswere detected. The VIA-specific reactions wereobtained as early as 9 DPI and were present inall the samples tested through 142 DPI. Thelatest serum tested was obtained at 230 DPIfrom an animal infected with type A virus. Thevirus-specific reaction was detected but not aVIA reaction. The highest levels of precipitating

activity were usually obtained with sera fromtype C-infected animals.A double precipitin ring was noted with some of

the sera obtained from type 0-infected steers.The outer ring was from 12S antigen. Thisreaction remained specific and did not interferewith the typing of the serum.

Extensive cross-reactions were evident at thehigher serum concentrations, as illustrated inTable 2. Serum 118M, for instance, reacted withA, 0, and C virus antigens in the 11/5 and 1/20

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TABLE 2. Radial immunodiffusionantisera tested against virus infecti

(VIA) and virus antigen

Sample

104H

508K

77G

118M

4230

506E

505Q

506A

104E

Dilution

1/1c1/201/801/3201/51/201/801/3201/51/201/801/3201/51/201/801/3201/51/201/801/3201/51/201/801/3201/51/201/801/320'1/51/201/801/3201/51/201/801/320

Reaction against

VIA Aa 0o C«

.+

++++

+

+

+

+

+

+

+

+

+

+

+

+

+

+

++

+++

+

+

+

a FMDV type antigen.b Refers to the FMDV type the a

fected with and the day postinfectwhich the serum was collected.

c Indicates final dilution of serummixture.

dilutions. The specificity becameever, as the serum was diluted.

DISCUSSIONThe results of this investigatior

the applicability of RID procedurtermination of antibody resulting frThe main advantage of the meth

sensitivity for detecting antibody. )(2) demonstrated that the RID p

reactions of as that described in this report was approximatelyon-associated 10 times more sensitive than conventional doubleis immunodiffusion techniques, and approached the

sensitivity of the complement fixation test. InIdentityb the present study, positive and specific virus

reactions were obtained as early as 4 DPI andpersisted as long as 230 DPI. The intensity of

A, 21 DPI the reactions observed with the late sera indicatedthat specific reactions could be expected for anextended time after infection. In every case,type-specific reactions were obtained within 48

C&O, 26 DPI hr after adding antigen to the wells. Indeed,with potent antisera, specific reactions werereadable within 1 hr. It was of interest thatextensive cross-reactions were evident at the

A higher serum concentrations, but specificitybecame clearly evident as the sera were diluted.These observations indicated some very interest-

c, 14 DPI ing possibilities concerning antigenic relation-ships between the different types as well as therelationships of subtypes. These aspects are beingpursued. It was also noted that all of the precipi-

A, 230 DPI tin reactions tended to be faint with indistinctborders (Fig. 2), in comparison with the resultsobtained with guinea pig sera (2), where the

0, 7 DPI reactions were strong with distinct borders. Theexplanations for these differences are not yetclear, but may be related to the antibody class(es)involved.

C, 14 DPI Another advantage of the procedure is the easewith which sera can be tested for antibodies tovarious antigens. Thus, while emphasis in this

0, 7 DPI investigation was on the VIA antigen of FMDVand three South American FMDV types, onecan imagine the use of a standard petri platecontaining the serum-agar mixture and testing

A, 21 DPI with multiple antigens. Thus, it would be possibleto screen sera for antibody to all seven FMDVtypes as well as VIA antigen, or to several sub-types within a serotype. Additionally, the exami-nation of the sera for antibodies to any viralagents which also give precipitin reactions could

tionalDPI) on be considered. Thus, the method might haveapplication in the examination of sera from

in serum-agar domestic and zoological animals being imported,in epizootiological surveys, and in diagnosiswhere field outbreaks of the disease may occur.The importance of the use of VIA antigen in

evident, how- the detection of FMDV-infected animals wasstressed in a recent study (3) which described anOuchterlony-type immunodiffusion assay forVIA antibody in sera obtained during epizootio-

n demonstrate logical surveys. The VIA antigen is disease-es for the de- specific but not virus type-specific. Thus, the'om infections. antigen might be used advantageously to broadeniod is its great the scope of the test in case the correct virusA recent study type antigens had not been selected.Irocedure such The major difficulty of the RID procedure is

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DETECTION OF FMDV ANTIBODIES

that concentrated antigens are required. If tissueculture sources of virus are available, however,both the VIA antigen (1) and the virus antigenscan be readily prepared. The polyethylene glycolmethod (4) has been found to be a satisfactoryprocedure for the concentration and storage ofvirus antigens. The VIA antigen preparationshave been found to remain stable for at least 4months at 4 C, and the virus antigen preparationsare stable for at least 14 months at -70 C.

ACKNOWLEDGMENTS

The excellent technical assistance of Nicholas J. Shuot, Jr.,and Jay Card is acknowledged.

LIERATURE CITED

1. Cowan, K. M., and J. H. Graves. 1966. A third antigenic com-ponent associated with foot-and-mouth disease infection.Virology 30:528-540.

2. Cowan, K. M., and G. G. Wagner. 1970. Immunochemicalstudies of foot-and-mouth disease. VIII. Detection andquantitation of antibodies by radial immunodiffusion. J.Immunol. 105:557-566.

3. McVicar, J. W., and P. Sutmoller. 1970. Foot-and-mouthdisease: the agar gel diffusion precipitin test for antibody tovirus-infection-associated (VIA) antigen as a tool for epi-zootiologic surveys. Amer. J. Epidemiol. 92:273-278.

4. Wagner, G. G., J. L. Card, and K. M. Cowan. 1970. Immuno-

chemical studies of foot-and-mouth disease. VII. Charac-terization of foot-and-mouth disease virus concentrated bypolyethylene glycol precipitation. Arch. Gesamte Virusforsch.30:343-352.

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