J. gen. ViroL (I979), 42, 597-602 Printed in Great Britain

597

Detection and Quantification of Foot and Mouth Disease Virus by Enzyme Labelled Immunosorbent Assay Techniques By J. R. C R O W T H E R

AND E. M. E. A B U - E L Z E I N

Animal Virus Research Institute, Pirbright, Woking, Surrey, U.K. (Accepted 20 September I978 ) SUMMARY

Enzyme labelled immunosorbent assays (ELISA) have been developed to detect and quantify foot and mouth disease (FMD) virus using flexible plastic microtitre plates. The methods were successful for the specific detection of F M D virus and were 5o to Ioo times more sensitive than the complement fixation test. The application of the ELISA techniques to F M D virus typing and subtyping, and to the assay of antigen concentration during manufacture of vaccines is discussed.

INTRODUCTION The micro ELISA test is frequently used for the detection and quantification of viruses; (Voller et al. 1976; Wolters et al. I976, I977; Halbert & Anken, 1977; Miranda et al. 1977; Yolken et al. ~977). The technique had been found to be rapid and can be as sensitive as radioimmunoassay (RIA), and makes it possible to detect small amounts of material without the problems associated with the use of radioactive compounds. The measurement of bovine antibodies to F M D virus was shown to be possible using the micro ELISA test (Abu-E1 Zein & Crowther, I978). In this paper we describe methods used to detect and quantify F M D virus and indicate the applications of the test to problems associated with F M D virus. METHODS Viruses. F M D viruses type O1/UK 186o/1967 and type A/Allier/I96O, were grown in monolayers of BHK 21 cells and purified by sucrose density gradient centrifugation as described by Brown & Cartwright (1963) using 1% SDS instead of deoxycholate. Purified viruses were stored at - 7 0 °C in siliconized glass vials. Swine vesicular disease (SVD) virus U K 27/72 was grown in IBRSz cells and purified using the same method as for F M D virus. Antisera. Antiserum against purified (I4oS) O1/UK/I86O/I967 F M D virus was prepared in guinea pigs. Purified virus was inactivated using acetylethyleneimine (AEI), at a final concentration o"o5 %, by incubation at 26 °C for 3 ° h. One vol. of inactivated virus was emulsified with one vol. of Freund's complete adjuvant (Difco Labs., Detroit, Michigan, U.S.A.) and five guinea pigs each recieved 5o #g of virus, intramuscularly at two sites. Animals were exsanguinated after 28 days and the sera pooled. Goat anti guinea antiserum was obtained commercially (Miles Labs. Ltd, U.K.). The IgG fractions of the antisera were prepared as described previously (Abu-E1-Zein &

oo23-1317/7/oooo-3391 $02.00 (~) I979 SGM

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J. R. CROWTHER AND E. M. E. ABU-EL ZEIN

Crowther, 1978). The IgG concentration was calculated from the absorbance at 278 nm (A~Ts = 1.4). Samples were stored at - 2 0 °C in siliconized glass vials. Conjugation o f enzyme to IgG. Alkaline phosphatase (EC.3.I.3.I., Type VII, Sigma, England) was conjugated to the IgG fraction of the antisera according to the method of Avrameas (1969). The working dilution of each conjugate was determined as described by Voller et al. (1976). Stock conjugates were stored at 4 °C after the addition of 5% ovalbumin (final concentration) and sodium azide (final concentration 0.02 %). Virus detection experiments. Tests were performed using purified virus of known concentration, determined after spectrophotometric analysis at 259 nm of sucrose density gradient material, as described by Bachrach et al. (I964). The approximate particle concentration of purified SVD virus U K 27/72 was determined using the same formula, where this virus was used as a control. The methods described state conditions found to be optimal for the tests based on preliminary studies to determine parameters such as incubation times and temperatures number of washes after each step, addition of different concentrations of Tween 2o a n d / o r ovalbumin. Several important points concerning conditions are raised in the results. Direct test. Viruses were attached to the solid phase (Flexible polyvinyl microplates, U-bottomed wells, Dynatech, England, catalogue n o . 1 - 2 2 0 - 2 4 ) , by incubating the plates with varying dilution of purified virus diluted in sodium carbonate/bicarbonate buffer, 0"05 M, pH 9"6, for 3 h at 37 °C (200 #1 virus dilutions per well). Unattached virus was then removed by flooding and emptying the wells three times with a solution of saline (o"15 M-NaC1) containing a final concentration of o-I % (w/v) Tween 20. Each well then received 200 #1 of a dilution (pre-titrated) of homologous anti-FMD virus IgG conjugate diluted in PBS with 0"05 % (w/v) Tween 20 (PBST), containing 3 % final concentration of ovalbumin (Miles Laboratories, Slough, England). Plates were incubated for I h at 37 °C while being shaken on a rotary shaking machine (Rloo Rotatest, Luckham Ltd, West Sussex, England) at 60 rev/min. Plates were washed three times and 2oo #l Of substrate was added to each well (p-nitrophenyl phosphate, Sigma, England, at I mg/ml in IO %, w/v, diethanolamine buffer, pH 9"8). Plates were left for 30 rain at room temperature, then the colour reaction stopped by the addition of 5o #1 of 3 M-NaOH. Colour readings were made by measuring the absorbance at 405 nm using a Uvichem Spectrophotometer (Hilger and Watts). Indirect test. Dilution series of purified virus were attached to wells as described above. Homologous guinea pig anti-FMD virus (I4oS) IgG was then added to a concentration o f 2-5/~g/ml in PBST containing I ~o ovalbumin (200/d/well) and the plates were incubated at 37 °C for I h with shaking. After washing three times, each well received 2oo #1 of enzyme conjugated goat anti-guinea pig IgG diluted in PBST containing 3 % ovalbumin. Plates were incubated at 37 °C for I h with shaking. Substrate additions and incubation procedures were then made as described in the direct test. Sandwich test. The procedure described by Voller et al. (I976) was followed. Guinea pig anti-FMD virus (I4oS) IgG was added to wells after dilution in carbonate buffer to IO #g/ml (2oo/,1/well). After incubation for 2 h at 37 °C the wells were washed three times and 200 #1 of dilutions of purified homologous virus in PBST containing 1% ovalbumin was added. The plates were incubated for I h at 37 °C with shaking. After washing three times, 2oo/~1 of enzyme-conjugated anti-FMD virus (I4oS) guinea pig IgG were added to each well and the plates were incubated at 37 °C for I h with shaking. Plates were read after addition of substrate as already described. For all the methods described, triplicate samples of each virus dilutions were examined. Controls were included to examine the specificity of the tests and are indicated in the results.

Identification of FMD V by ELISA I

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Fig. I. Comparison of ELISA techniques for the quantification of purified F M D virus type O 1 / U K / 186o/1967. Amounts of different viruses added to wells are shown as: O - - I , F M D O1/UK/I86O/ 1967 virus; I , F M D A/Allier/I96O; O, SDV virus U K 27/72; A, blank wells. Dotted lines indicate 2 x s.d. from the mean absorbance reading of type A/Allier/I96O F M D virus control, the arrowed points show the accepted end-point for each assay. RESULTS

Attachment of purified F M D virus O1/UK/I86O/I967 or antibody to wells was successfully achieved at 37 °C for 3 h although incubation at room temperature overnight gave similar results Successful assays were also made after drying virus on to wells by leaving plates overnight without lids, or drying in a stream of air. Virus antigens contained in different volumes of diluent (5o/~1 to 200 #1) were examined, and all gave successful assays; thus 5° #1 dilution loops could be used to dilute virus samples in the direct and indirect tests. The use of the rotary shaker increased the reaction of antigen and antibody at the reported stages, presumably by increasing the probability of contact between the reactants; equivalent results were obtained after 3 h at 37 °C on stationary plates. The inclusion of both Tween 20 and ovalbumin where stated was essential to minimize non-specific effects. Results for the titration of purified type O 1 / U K / I 8 6 o / I 9 6 7 F M D virus, using the three ELISA techniques outlined, are shown graphically in Fig. I. SVD virus U K 27/72, was used as an antigen control as it had been found to attach to wells in a similar way to F M D viruses and can be titrated using ELISA techniques with antiserum to SVD. The limits for the detection of O1/UK/~86o/I967 F M D viruses were similar using all three methods. These were determined as the last well showing a colour reaction reading above the background control readings with suitable confidence limits. The standard deviation from the mean of each assay point was always very low and the end-points were read as illustrated on the specific titration curves on Fig. I. All three methods were specific in measuring only type O1/UK/I86O/I967 virus using the described antisera; neither SVD virus nor type A F M D virus gave colour development under the same test conditions. Fig. 2 and Table I show the results of titrating a narrow range of virus concentrations using the sandwich-ELISA

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Virus concentration (ng/ml) Fig. 2. Quantification of purified type O 1 / U K / I 8 6 O / I 9 6 7 F M D virus by sandwich ELISA. 0 - - - 0 , Titration of O 1 / U K / I 8 6 o / I 9 6 7 . D a s h e d lines indicate m a x i m u m control a b s o r b a n c e levels for: I - F M D virus type A / A l l i e r / x 9 6 o ; z - S V D virus U K 27/72; 3 - b l a n k wells.

Table I. Titration of FMD virus type 01/UK/I86o/1976 by sandwich ELISA technique* A4o5 r

t

Samples ~ 3

3

Mean

s.d.

I "402 I'202 I'I02

I '397 I'260 I q9I

I "41o I'302 I'I65

I "403 I'255 I'I53

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I'045

0"062

xoo 75 5o 25 SVD (2o0-~---n g / m l ) F M D type A (200 n g / m l ) Blank

o-95o 0"892 o'7IO O'614 0"335 0"485 0"306

i.oo8 0-889 0"794 0"599 o'3oi 0"480 o'34I

0"907 0"729 o-7to 0"630 o'32t 0"45o o-312

0"955 0"854 0.738 O'614 o'319 0"473 o-32o

o'o5I 0"o63 0'048 O'OI6 o'o17 o'oi6 o'o18

Virus concentration (ng/ml)

,

200 I75 I50

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* Regression coefficient of O ~ / U K / ] 8 6 o / I 9 6 7 titration line = o'9815.

technique, in order to assess the relationship of virus dilution to colour development. A linear relationship was obtained over the range of 25 to 200 ng examined. The small standard deviation for each assay point is illustrated in Table I. The line in Fig. 2 was fitted using linear regression statistics; the regression coefficient was 0"9815. Similar plots were obtained for the direct and indirect techniques using equivalent ranges of virus concentrations. Similar assays have been performed to titrate F M D virus types A, C, and SAT t, and all

Identification of F M D V by ELISA

6Ol

confirm the test as being type specific under the conditions described, using purified viruses. Titration curves were reproducible when the same antiviral antibodies and conjugated antisera are used. Tests performed three months apart produced identical titration curves where the reactants had been stored as described. DISCUSSION

The ELISA technique had been successfully applied to the quantification of purified F M D viruses. The three methods described showed similar limits for the detection of virus. Cumulative data indicate a range of 5 to 30 ng/ml F M D virus may be measured distinct from background controls. This level can be compared to the complement fixation (CF) test which had been reported to detect approx. I #g/ml of purified F M D virus (Garland et al. 1977). The methods were specific for type O F M D in this study; SVD and type A F M D virus did not interfere. The specificity and reproducibility of the assays indicate that standard curves relating known virus concentrations to colour development, using defined reagents, can be constructed in order to assay unknown virus concentrations. The three methods utilizing the ELISA technique are being examined for different applications to F M D virus research. The sandwich technique is now being used successfully in this laboratory to assess the mass of I4oS virus in the presence of I2S and tissue culture material, using antisera prepared against inactivated purified F M D viruses. This is useful in the examination of the quantity of antigenic material to be incorporated in vaccines and eventually it is hoped to produce an automatic system for the quantification of I4oS virus throughout the preparative procedures in vaccine manufacture. The indirect technique, where only a single anti-species conjugate is needed, is being used to detect and type F M D viruses form epithelial samples sent to the World Reference Laboratory, Pirbright. Results are being compared to the conventionally used CF test. Preliminary studies indicate that the increased sensitivity of the ELISA technique over the CF test (5o to IOO times), the lack of anti-complementary factors and the high specificity of the test will make it a useful method for the routine diagnosis of F M D virus. The direct test is being used to examine subtype differences between F M D virus strains using competition assays. Studies on the application of ELISA tests to F M D virus research will probably modify the techniques further to deal with specific problems. The versatility of the ELISA when applied to other animal viruses is indicated by the fact that the direct and indirect tests have recently been used to develop assays to quantify viruses as different as African swine fever and SVD in this laboratory. REFERENCES ABU-EL ZEIN, E. M. E. & CROWTHER, J. R. (I978). Enzyme-labelled i m m u n o s o r b e n t assay techniques in foot a n d m o u t h disease virus research. Journal of Hygiene, Cambridge 80, 39I-4OO. AVRAMEAS,S. (I969). Coupling of enzymes to proteins with gluteraldehyde. Immunochemistry 6, 43-52. BACHRACH, H. L., TRAUTMAN, Ft. & BREESE, S. S. (1964). Chemical a n d physical properties o f virtually pure f o o t - a n d - m o u t h disease virus. American Journal of Veterinary Research 25, 333. BROWN, E & CARTWRIGHT,B. (I963). Purification of radioactive foot-and-mouth disease virus. Nature,

London I99, It68-117o.

GARLAND, A. J. M., MOWAT~G. N. & FLETTON, B. (I977)- A n evaluation of s o m e m e t h o d s o f a s s a y o f f o o t a n d m o u t h disease antigen for vaccines. International Symposium on Foot-and-Mouth Disease, Lyon 1976 Development of Biological Standardization, vol. 35, PP. 323-332. Basel: S. Karger. HALBERT, S. P. & ANKEN, M. (1977). Detection o f hepatitis B surface antigen (HBsAg) with use of alkaline phosphatase-labelled antibody to H B s A g . Journal of Infectious Diseases 136, $318-$323. 38

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MIRANDA, O. R., BAILEY, G. D., FRASER, A. S. & TENOSO, H. J. (1977). Solid p h a s e e n z y m e i m m u n o a s s a y for H e r p e s simplex virus. Journal of Infectious Diseases 136, $3o5-$31o. VOLLER, A., BIDWELL, D. & BARTLETT,A. (1976). Microplate e n z y m e i m m u n o a s s a y s for the i m m u n o diagnosis o f virus infections. In Manual of Clinical Immunology, pp. 506-512. Edited by N. R. R o s e & H. F r i e d m a n . W a s h i n g t o n D.C. : A m e r i c a n Society of Microbiology. WOLTERS, G., KUIJPERS, L. P. C., KACAKI, J. & SCHUURS, A. H. W. M. (I976). Solid p h a s e e n z y m e - i m m u n o l o g y for detection o f hepatitis B surface antigen. Journal of Clinical Pathology 29, 873-879. WOLTERS, G., KUIJPERS, L. P. C., KACAKI, J. & SCFIUURS,A. H. W. M. (1977). Enzyme-linked i m m u n o s o r b e n t assay for hepatitis B surface antigen. Journal of Infectious Diseases 136, $3I I-$317. YOLKEN, R. H., KIM, H. W., CLEM, T., WYATT, R. G., CHANOCK, R. M., KALICIA, A. R. & KAPIKIAN, A. Z. (1977). Enzyme-linked i m m u n o s o r b e n t assay (ELISA) for detection of h u m a n reovirus like agent o f infantile gastroenteritis. Lancet 263-266.

(Received 18 July I 9 7 8 )

Detection and quantification of foot and mouth disease virus by enzyme labelled immunosorbent assay techniques.

J. gen. ViroL (I979), 42, 597-602 Printed in Great Britain 597 Detection and Quantification of Foot and Mouth Disease Virus by Enzyme Labelled Immun...
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