Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis

Research in Veterinary Science /987, 43, 225-232

Detection and typing of foot-and-mouth disease virus by enzymelinked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis P. L. ROEDER*, P. M. LE BLANC SMITH, CSIRO, Australian Animal Health Laboratory,

PO Bag 24, Geelong, Victoria 3220, Australia

Constraints placed on the use of live FMDV precluded the development of the test at the Australian Animal Health Laboratory and the work was performed at the Animal Virus Research Institute in Britain which is host to the World Reference Laboratory for Foot-and-Mouth Disease (WRL). The present report describes the formulation, characterisation and validation of the assay which was developed.

A highly sensitive indirect sandwich enzyme-linked immunosorbent assay suitable for adoption as the routine diagnostic and typing test for foot-and-mouth disease virus of all seven serotypes is described. The assay uses rabbit and guinea pig antisera raised against inactivated 146S virus antigens. Strong homotypic and minimal heterotypic reactions with both whole virion 146S and derived virion subunit US antigens achieved a detection sensitivity approximately 125 times that of the complement fixation test. When applied to diagnostic material with positivenegative threshold criteria generated from testing a large number of negative samples, a positive result was obtained on 83·3 per cent of original viruspositive epithelia, three times the rate for the complement fixation test, and all were typed after one passage in culture.

Materials and methods

HAMBLIN et al (1984) discussed the potentially valuable role of enzyme-linked immunosorbent assay (ELISA) in the diagnosis of foot-and-mouth disease virus (FMDV) infections. They had evaluated the indirect sandwich ELISA based on the method of Crowther and Abu Elzein (1979) and concluded that it had no great advantage over the complement fixation (CF) test. The use of anti-146S rabbit immunoglobulin (Ig) as opposed to convalescent bovine Ig as capture antibody resulted in increased sensitivity in ELISA (Ouldridge et al 1982) but again no clear advantage resulted from its use as a diagnostic procedure (Have et al 1984). Despite this the advantages of reagent economy, irrelevance of pro- and anti-complementary effects and ease of performance suggested that further developmental work was merited to explore the use of high-titred anti-146S rabbit and guinea pig sera for antigen capture and detection respectively (J. R. Crowther and C. Hamblin, personal communication). "Present address::':entral Veterinary Laboratory, Weybridge, Surrey KT;S 3NB

New

Haw,

Standard procedures with minor modifications were used to prepare the reagent antisera and reference antigens; their production and characterisation for use in ELISA will be described in a forthcoming paper and only a brief description is included here.

Reference viruses The low passage virus strains, selected for their relationship to current field strains and currently used vaccine virus strains and by reference to data presented by Ferris and Donaldson (1984), are listed in Table I.

Whole virion antigens: 146S Viruses were grown in rolled baby hamster kidney (BHK) monolayer cultures, inactivated with acetyl-

ethyleneimine (Wellcorne Foundation), purified and the mass quantified by techniques essentially the same as those described by Ferris and Donaldson (1984). After harvesting 146S antigens were stored in siliconised glass vials in liquid nitrogen vapour.

Virion subunit antigens: 12S The serial action of heat and acidification was used to degrade purified 146S antigens. Preparations of

225

P. L. Roeder, P. M. Le Blanc Smith

226

TABLE 1: Reference virus types used to raise antisera and to produce standard virus antigen preperations

System

WRL designation

o

0, BFS 1860/Great Britain 1967 As Allier / France 1960 A22 Iraq 24/1964 A 24 Cruzeiro/Brazil 1955 C, Noville/Switzerland 1965 Botswana 1/ 1968 Kenya 183/1974 Bechuanaland 1/1965 Pakistan 1/1954

A

C SATl SAT2 SAT3 ASIA1

146S antigens, diluted appropriately in ELISA diluent, were heated to 56°C and maintained at that temperature for I· 5 hours. The solution was then acidified to pH 6'4 by the addition of 12'5 J.l1 ml- I of molar hydrochloric acid. After 10 minutes the addition of a similar volume of molar sodium hydroxide returned the solution to pH 7·4 for subsequent use.

tion; these are termed 'blocked' sera. For routine use sodium azide was added to a concentration of 20 mM and working stocks were maintained at 4°e.

Test sample preparation Weighed portions of epithelium were ground with sterile sand and an appropriate volume of 0·04 M PBS pH 7· 4 was added to prepare standard 10 per cent suspensions which were clarified by centrifugation before use. ELISA

procedures

Optima for the test in terms of choice of plate, reagent volumes, coating conditions, diluent formulation, sera and conjugate working tit res and reaction times were all determined by trial. The final formulation of the assay is described here. All ELISA

Unblocked

Reference antigens for

The high titre virus content of culture medium harvested from BHK cell cultures was inactivated with binary ethyleneimine (BEl) and then stored in bulk at - 70°e. For this purpose BEl was produced by the cyclisation of 0·1 M bromoethylamine hydrobromide (Koch Light. Laboratories) in 0·2 M sodium hydroxide for one hour at 37°C (Bahnemann 1975) and added to a final concentration of 0·006 M. Uninfected cultures were sonicated and processed similarly to provide a negative antigen. For routine use one aliquot was thawed, preserved by the addition of sodium azide to a final concentration of 20 mM and dispensed in I ml aliquots all but one of which were returned to -70°C until required. The working stock was maintained at 4°e.

Antisera were prepared in rabbits (for capture) and in guinea pigs (for detection) by the inoculation of 146S antigens emulsified in Freund's complete adjuvant. After 28 days guinea pigs were bled out whereas rabbits received another inoculation of 146S antigen in Dulbecco phosphate buffered saline (PBS) containing 0'25 mg rnl "! saponin (Sigma Chemical, product S1252) before bleeding 10 days later. Antisera for detection of the A serotype of FMDV were raised against 146S antigens of the three subtypes contained in a combined inoculum. Sera were pooled and stored in bulk at - 20°e. When required for use an aliquot was thawed and heated to 56°C for 30 minutes. Guinea pig sera were mixed with an equal volume of normal bovine serum (NBS) before inactiva-

Uninfected

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ELISA

Infected

ELISA

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2

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ELISA

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~ o i i i

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2 4 8 16 OS 2 4 8 Reciprocal of epithelium suspension dilution

i

16

FIG 1: Optical density values obtained by titrating infected and uninfected epithelium suspensions in unblocked and blocked detection systems. The infected bovine tongue epithelium was collected 21 hours after the intradermolingual inoculation of FMDV type 0, BFS 1860

FMD

diagnosis by

tests were performed in 50 J.l1 volumes in polystyrene plates (Nunc Immunoplate 1). Washing with Dulbecco PBS pH 7·4 was performed with automated equipment (Titertek Microplate Washer, Flow Laboratories) using a five wash cycle with a 30-second soak after the third wash. All reactions with the exception of the final substrate reaction were enhanced by agitation during incubation (Rotatest Rocker, Luckham) and heating to 37°C. ELISA

diluent

Purified virus antigens, epithelium suspension, rabbit and guinea pig sera and the conjugated antiserum were all diluted in Dulbecco PBS pH 7·4 to which was added sodium chloride (20 g litre : '), bovine serum albumin (30 g litre-I) (Sigma Chemical, product A7906), polyethylene sorbitan monolaurate (0'05 per cent; Tween 20 Sigma Chemical, product P1379) and 1 M sodium hydroxide (approximately 2 mllitre - I) to bring the pH to 7·4.

Colour reaction The substrate was a solution of orthophenylenediamine (400 mg litre-I) (Sigma P9029) in citrate phosphate buffer pH 5, O. Samples were stored at - 20°C and warmed to room temperature in the dark before use. Immediately before use the substrate was activated by the addition of 12· 5 J.l1 of 30 per cent hydrogen peroxide 25 ml : I. Colour development was terminated by the addition of an equal volume of 1·25 M sulphuric acid.

Indirect

ELISA

jor anti-Ig activity

Plates were coated overnight at 4°C with a DE52 (Whatrnan). separated solution (5 J.lg ml : I) of bovine

IgO (Johnstone and Thorpe 1982) in coating bu ffer. After washing, a fourfold dilution series of the test sera from a starting dilution of 11400 were applied to the plates and allowed to react for 30 minutes at 37°C. Washing was repeated and bound rabbit and guinea pig Ig was detected with swine anti-rabbit (Miles Laboratories) and rabbit anti-guinea pig (OAKOimmunoglobulins) sera, respectively, conjugated to horseradish peroxidase and both used at a dilution of 112000 for 30 minutes at 37°C. After washing, colour

227

ELISA

reactions resulting from the application of substrate were then determined with reference to control wells which received no test sera.

Indirect sandwich

ELISA

jor detecting and typing

FMDV

In the final test protocol adopted the plates were coated for one hour at 37°C with the seven rabbit antisera and a normal rabbit serum all diluted to 115000 in coating buffer (0'05 M carbonate/bicarbonate buffer pH 9' 6) and dispensed to the wells of rows A to H. After washing the plates, two successive twofold dilutions of the neat (l in 10) test samples and the reference antigens (starting from a I in 50 dilution) were dispensed to the appropriate wells of columns 1 to 12. ELISA diluent alone was added to the plate background control columns 4, 8 and 12. The plates were incubated for one hour at 37°C and washed again before receiving the seven 'blocked' guinea pig antisera and a 'blocked' normal guinea pig serum diluted in ELISA diluent. All were used at a dilution of 11500 (that is, 111000 of 'unblocked' serum) with the exception of the type 0 antiserum which was diluted 1/1000 (that is, 112000 of 'unblocked' serum). Immediately the plates were set to incubate at 37°C for 30 minutes the required volume of rabbit antiguinea pig serum conjugated to horseradish peroxidase (OAKO immunoglobulins) to prepare a working 112000 dilution was mixed with an equal volume of NBS and allowed to react in the dark at room temperature. Following washing of the plates, the conjugated antiserum was diluted in ELISA diluent, dispensed to all wells and incubated for 30 minutes at 37°C. A final wash was followed by the addition of activated substrate solution to all wells and to the first column of wells of a fresh plate. The reaction was allowed to proceed for an accurately timed 15 minutes in the dark at room temperature and was then stopped by the addition of acid stopper solution to each well. Optical densities (00) were read visually as well as spectrophotometrically at 492 nm using a Titertek Multiskan (Flow Laboratories) blanked against the plate receiving substrate and acid alone. Individual well 00 values were corrected by subtracting from them the average 00 of the three appropriate control wells (which had received all reagents but no antigen preparation) for each test system.

TABLE 2: Positive-negative threshold criteria calculated from negative samples tested as 10 per cent suspensions (P=0·OOO5)

Species Cattle/sheep Pigs

n

0

A

263 100

0·00 0·09

0·08 0·09

OD value in each detection system SAT1 SAT2 C

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ASIA'

Control

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0·07 0·08

0·05 0·09

0·07 0·07

0·08 0'08

0·07 0·08

3-6

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1

2

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1·8

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1·2

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0·6

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-0-0

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~A

-0-0

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Concentration of 1465 or equivalent 125 antigen: logn ng ml- 1

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FIG2: Titration in ELISA of 1465 and derived 125 subunit antigens of six representatives of four FMDV types. Antigens were titrated in all seven detection systems but for clarity only the strongest heterotypic reactions are indicated

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diagnosis by ELISA

test

Samples were examined routinely in the WRL by the microtitre method of Casey (1965) with minor modifications as described by Hamblin et al (1984). Results

From the earliest stages of development of the assay systems it was evident that although the reagents used in the ELISA were capable of detecting FMOV at very low concentration and of identifying the serotype correctly the apparent degree of cross reactivity was unacceptably high and false positive reactions occurred. While the majority of virus-containing epithelium samples tested as a standard 10 per cent suspension gave clear typings a number of known FMov-negative samples gave unacceptably high 00 values particularly in the serotype 0, ASIAl and SAT3 detection systems (decreasing in that order) and this confounded the interpretation of test results for positive samples of low virus antigen content. By chequerboard titration of all trapping and detection sera with purified 146S antigen preparations and negative and positive epithelium suspensions it was possible to derive detection systems using low dilutions of reagent sera (1/10,000 to 1120,000 for capture sera and 1/4000 to 1/8000 for detection sera) which minimised the false-positive reactions. However, sensitivity was compromised by this procedure and the resulting assay was less sensitive than the conventional CFtest used for diagnosis. The cause of the false-positive reactions was identified as reactivity of the reagent sera to bovine serum components by demonstrating the binding capacity of the selected rabbit and guinea pig serum pools for bovine IgG attached to the solid phase of microtitre plates in an indirect ELISA. Understandably the double-inoculation rabbit antisera had higher reactivity than the single-inoculation guinea pig antisera but the activity in some of the latter sera was still evident at the working dilutions used in the assay systems. The hierarchy of reactivity was identical to that of the apparent cross reactivities and falsepositive reactions observed. The conjugated rabbit antiserum to guinea pig Ig also cross reacted to low levels with bovine Ig. The problem could be reduced by including bovine serum in the diluent used to prepare working solutions of detection sera and conjugate thus explaining its inclusion by other workers (Have et al 1984). However, it was considered desirable to prepare standardised reagents with the unwanted activity blocked at source to facilitate the use of the assay in countries where obtaining bovine sera free from antibodier to FMOV might be difficult. A large pool of NBS was obtained from British cattle and the

229

blocking effect of its addition to the detection sera at equal or lower proportions was examined in the assay against bovine Ig by indirect ELISA. The activity was virtually abrogated by combining guinea pig antisera and NBS in equal proportions. Optimal specific anti-rvov reactions in the indirect sandwich ELISA were achieved by blocking both the guinea pig detection sera and the conjugated antiserum but not by its addition to the rabbit capture serum coated to the plates where its inclusion resulted in unacceptably high plate background 00 values. Blocking the unwanted activity allowed the use of each serum at saturation for maximum sensitivity as determined by the earlier chequerboard titrations and the test formulation described in the materials and methods section was adopted for all further work. The improvement in test function is illustrated in Fig 1 which compares the 00 values obtained in each assay system using either blocked or unblocked detection sera and conjugate when examining an infected epithelium sample (type 0) and a negative sample from Britain which gave initially the strongest false-positive reaction encountered.

Estimation ofspecificity and sensitivity Aziridine-inactivated sucrose density gradientpurified 146S antigens and derived 12S antigens of each reference virus type were tested in all seven test systems over a range of concentrations from 4· 0 IJg to 3'9 ng ml- 1 (or an equivalent mass for 12S) in twofold dilution steps. Consistently high homotypic reactions were obtained with minimum detection levels of less than 7· 8 ng ml- 1 of 146S antigen for the homologous reaction in each system. At high concentrations of 146S antigens significant heterotypic reactions were observed but these presented no 2

Degraded

Untreated

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P. L. Roeder, P. M. Le Blanc Smith

230

difficulty for the interpretation of the typing results as illustrated by the graphs of Fig 2 which depict the results obtained with types 0" As, A 22 , A w C, and ASIA I antigens. The results for the SAT serotypes were essentially the same. Virion subunit antigens (presumably 12S) were also detected very efficiently by all homologous test systems. The antigen was detected with approximately eightfold lower sensitivity than the intact 146S virion antigen. Heterotypic reactions were minimal or very low (Fig 2). The ability to detect degraded virus antigen was examined further by testing the standard type epithelium suspension before and after degradation by heating and acidification. A clear typing was obtained even after degradation with this sample of high antigen content (Fig 3).

°

Validation of the

ELISA

test

Determination of positive-negative thresholds.

Threshold 00 values above which samples were taken to have given positive results were evaluated for each system from the spread of 00 values obtained by testing a large number of known FMDv-negative tongue, epithelium samples. It was not possible to obtain sufficient samples from material submitted to the WRL from Britain, Ireland and New Zealand and the samples available were augmented by obtaining specimens from abattoirs in Victoria, Australia. In total 113 cattle, 150 sheep and 100 pig specimens were examined. The test systems performed differently with ruminant and pig materials and the two sets of data were therefore analysed separately. The ranges of the 00 values for each assay system were very close to being normally distributed with only a slight tendency to skewness in some instances and this appeared to be a random effect. Upper threshold values were calculated using a one-tailed t value and the results given in Table 2 are for P=0·0005 for samples tested as a 10 per cent suspension in PBS, that is, there is only a 112000 chance that the values quoted could be obtained from a negative sample. The low values for ruminant samples in the system resulted from the fact that such samples consistently gave 00 readings slightly below that of the plate background (measured from wells not receiving the test sample) and, thus, a negative value after correction.

°

Routine application and comparison with the CF test. Specimens submitted routinely to the WRL were

examined by ELISA in parallel with standard techniques. During the developmental stages a large number of specimens was tested but only those examined after the final formulation of the test are described here. In total 70 specimens which yielded virus in culture were examined (Table 3) and of these 53 (75' 7 per cent) were typed successfully on original

TABLE 3: Relative efficiency of ELISA and CF in detecting and typing FMDV in diagnostic material. Percentage figures are given in brackets ELISA

Number tested 28 42

Total 70

as

18 (64·3) 35t (83·3)

53 (75'7)

as

CF

ELISA OS+TC

CF OS+TC 18 (64·3)

10 123·8)

28 (100) 42 11(0) 70 (100)

52 (74·3)

34 (81·0)

Virus types included: 0:42, A:10, SAT1,3, ASIA1:15 Sample origin: cattle, 65; buffalo, six; pigs, eight; antelope, two • Not all samples examined by CF t Five of six samples anticomplementary in CF were typed by ELISA as Original epithelium suspension TC First passage in culture

epithelium suspension (os), the majority of typings being obvious by visual inspection alone. It was possible to compare the relative efficiencies of the CF and ELISA tests on 42 virus-positive samples where both tests were performed on both os and the supernatant fluid derived from the first passage of the virus in bovine thyroid cell cultures (or IBRS2 cells in a few cases for pig samples), ELISA gave a typing on 35 (83' 3 per cent) os samples, considerably more than the 10 (23' 8 per cent) achieved by CF. After one passage in culture all samples had been identified by ELISA but 19 per cent (or nearly 26 per cent of all samples examined) required further passages in culture before an unequivocal typing was obtained by CF. All samples typed by CF were typed by ELISA and the results were in complete agreement. In addition 23 samples were examined which did not yield virus in culture. All were negative by CF but two pig epithelium samples from the Philippines gave weak but unequivocal type C reactions in ELISA.

Serial sampling of an experimentally infected calf.

Tongue and foot lesion material was collected daily after the first appearance of lesions from a calf which had been infected by aerosol inhalation of type 0 1 BFS 1860 (kindly supplied by Dr R. Oliver and Dr A. 1. Donaldson). Clarified standard suspensions were tested in ELISA with the results shown in Fig 4. Clear typings were obtained with all samples even the last tongue sample which was negative by CF.

Spectrum of subtypes detectable by ELISA, To examine the detection of virus serotypes not covered in recent submissions to the WRL and to explore the spectrum of antigenically different strains detectable a large number of cell culture-grown viruses derived over many years from many regions of the world was tested; their number was augmented by testing stored epithelium specimens. The WRL sample references

FMD

diagnosis by

and subtype designations, where known, are given in Table 4. All virus strains were detected by ELISA and typed correctly.

231

ELISA Day 1

2

Day 2 Mouth lesions

Day3 -0- SAll

4A -0-0

Discussion The significant contributions to the development of for the diagnosis of foot-and-mouth disease E made by Crowther and Abu Elzein (1979), Hamblin et ~ o 0---0------0 0------0-----0 ~ i i i i i i al (1984) and Have et al (1984) have been extended ~ and the test refined by the present study. ;; Have et al (1984) stated that 'maximum sensitivity .~ and type specificity cannot both be achieved within ~ -0 SAll Foot lesions the same diagnostic test' but clearly they can be if the ~ 2 .",.C appropriate reagents and test formulation are 4A -0-0 selected. The sensitivity of the assay developed was four to eight times higher than that described by Hamblin et al (1984) being comparable to that evident in the report of Have et al (1984), whose assay of a restricted number of serotypes lacked specificity, while retaining the specificity of the former multio 0------0---0 0---..0-----0 ~ serotype test. The residual heterotypic reactivity os 2 4 os :2 4 os :2 4 observed with higher concentrations of 146S antigens Reciprocal of epithelium suspension dilution may represent the detection of conserved epitopes FIG 4: Detection of FMDV antigen in tongue and foot epithelium particularly in the representatives of the A 22 , C 1 and samples collected serially from a calf experimentally infected with FMDV type 01 BFS 1860. For clarity only the strongest heterotypic ASIA I serotypes used to produce the reagent sera but caution is necessary in drawing this conclusion since reactions are indicated the observed result could be a manifestation of serum quality, anti-BHK cell activity or residual anti-bovine serum activity, Ferris and Donaldson (1984) showed from viruses grown in serum-free medium and subsethat antisera produced to different representatives of quently purified they must, none the less, have conthe same serotype differed markedly in their hetero- tained significant amounts of bovine Ig and possibly typic reactivity in CF and batch to batch variation may other bovine serum components. Whether aggregated Ig cosediments with 146S antigen during sucrose also occur. The identification in rabbit and guinea pig sera of density gradient centrifugation or the 19 adheres to the anti-bovine Ig activity and its negation in guinea the virion is unknown but the problem could probably pig antisera and conjugate was an important factor in be avoided by growing the cell substrate for virus improving the assay. Although antigen preparations production in media supplemented with horse serum used to immunise laboratory animals were derived or synthetic growth promoters. One area in which the results presented here differ markedly from those of other workers is in respect of the detection of 12S virus subunits being intermediate TABLE 4: Spectrum of stock viruses examined in ELISA in between the two extremes reported. Abu Elzein and addition to the reference types used to raise antisera Crowther (1979) reported that 12S antigen did not WRL designation indicating origin Type react in their system whereas Have et al (1984) ---------------estimated that 12S antigen titrated approximately Kenya 77 187, Thailand 1/80,0, India 53/79, 02 Brescial o Italy/1939, o, India 1/62 four times more efficiently than an equivalent mass of A Ethiopia 2/79, Kenya 5/80, Kenya 35/80, Venceslaul 146S antigen. Have et al (1984) also described strong Brazi1/1976, A lO Holland 1/81, A 22 MahmatlilTurkeyl heterotypic reactions between 12S preparations of the 1965 European serotypes 0, A and C, which gave only (Morocco 4/83,5/83,9/83), Kenya 267/67, India 51/79, C C3 Resende/Brazil/1955, C31ndaiai/Brazil/1971 minimal reactions for Hamblin et al (1984) although SAT1 Turkey 323/62, Tanzania 155/71, Botswana 1/77, the latter did observe both strong homotypic and Nigeria 4/81 heterotypic reactions with 12S antigen preparations (South Africa 12/83, 13183, 16/83) SAT2 of other serotypes. There is no doubt that the (Zimbabwe 4/83,2/841 SAT3 India 8/79, Cambodia 9/80, Burma 8/82, Greece 1/84, ASIA1 stringent procedures of heating and acidification used Iran 1/73 in the present study effectively degraded the 146S Those given in brackets were typed as epithelium suspensions particles as this was monitored by sucrose density ELISA

g

1~

232

P. L. Roeder, P. M. Le Blanc Smith

gradient analysis of some preparations (data not shown). Strong homotypic ELISA reactions were obtained, although less than those of an equivalent mass of 146S antigen, yet cross reactions were minimal, being similar to those of 146S before degradation, and clear typings resulted. One explanation of the discrepancy between our results and those of Have et al (1984) obtained in similar assay systems could be found in our use of guinea pig detection sera raised against purified inactivated 146S antigen as opposed to hyperimmune guinea pig sera. The latter might recognise antigenic determinants common to 12S subunits of different serotypes whereas the former might recognise only the serotype-specific determinants shared with 146S particles; such relationships have been indicated by Meloen and Briare (1980) and McCullough and Butcher (1982). It is conceivable that the double-inoculation rabbit antisera, used for trapping, capture and orientate the 12S subunits for serotype-specific detection by the single-inoculation (and thus probably more specific) guinea pig antisera. However, Ouldridge et al (1982) used an'ti-146Sguinea pig sera which did not detect 12S to any appreciable extent. Whatever the explanation the serotype-specific detection of degraded virus antigen is clearly a desirable attribute for this diagnostic assay favouring the detection and typing of the virus content of samples subjected to adverse conditions in transit even possibly where the infectious virus content has been reduced to undetectable levels. This was almost certainly the case for the two pig samples from the Philippines which clearly typed as serotype C, a likely result for that region, although confirmation by virus isolation and CF was not possible. The attributes of high sensitivity, at least 125 times the minimum detection limit of I J.lg ml! quoted for 146S by CF (Garland et aI1977), and precise specificity reflected in the efficiency and rapidity of diagnosis achieved by use of the test described here suggests that it merits adoption for routine use. Its use in conjunction with similar ELISA tests for the viruses of swine vesicular disease, vesicular stomatitis and

vesicular exanthema should facilitate the rapid differentiation of the vesicular diseases of livestock allowing the rapid implementation of control measures. Acknowledgements We gratefully acknowledge the help afforded by the director and many staff of the Animal Virus Research Institute in the work described here. Dr J. R. Crowther and Mr C. Hamblin generously gave us access to their considerable knowledge and experience. Advice given by Dr A. I. Donaldson and Mr N. P. Ferris was invaluable as was their practical involvement in supplying material and data from the WRL. Miss C. N. Hebert of the Central Veterinary Laboratory, Weybridge kindly performed the statistical analysis. References ABU ELZEIN, E. M. E. & CROWTHER, J. R. (1979) Journal of Hygiene 83,127-134 BAHNEMANN, H. G. (1975) Archives of Virology 47, 47-56 CASEY, H. L. (1965) Public Health Monograph number 74. Washington, DC, United States Government Printing Office CROWTHER, J. R. & ABU ELZEIN, E. M. E. (1979) Journal of Hygiene 83,513-519 FERRIS, N. P. & DONALDSON, A. I. (1984) Revue Scientifique et Technique de l'Office International des Epizooties 3,563-574 GARLAND, A. J. M., MOWAT, G. N. & FLETTON, B. (1977) Developments in Biological Standardization 35, 323-33~ HAMBLIN, c., ARMSTRONG, R. M. & HEDGER, R. S. (1984) Veterinary Microbiology 9, 435-443 HAVE, P., LEI, J. C. & SCHJERNING-THIESEN, K. (1984) Acta Veterinaria Scandinavica 25, 280-296 JOHNSTONE, A. & THORPE, R. (1982) Immunochemistry in Practice. Oxford, Blackwell Scientific Publications. pp 44-47 McCULLOUGH, K. C. & BUTCHER, R. (1982) Archives of Virology 74, 1-9 MELOEN, R. H. & BRIARE, J. (1980) Journal ofGeneral Virology 51, 107-116 OULDRIDGE, E., BARNETT, P. & RWEYEMAMU, M. M. (1982) Current Topics in Veterinary Medicine and Animal Science 22,142-151

Received July 16, 1986 Accepted April 27, 1987