Biozogicuzs
(1990)
18, 31!%319
Quantification of Intact 146s Foot-and-mouth Antigen for Vaccine Production by a Double Antibody Sandwich ELBA using Monoclonal Antibodies C. Van Maanen Central
Veterinary
Institute,
and C. Terpstra
Department of Virology, P. 0. Box 365, 8200 AJ Lelystad, The Netherlands
Abstract. A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146s antigen of foot-and-mouth disease virus (FMDV) strain Al 0 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin-which affects VP1 , the most immunogenic structural protein of FMDV-the concentration of 146s antigen as determined by ELISA was reduced by >90%. Therefore, the test detected essentially only those virus particles with intact VPl. When the test was compared with the sucrose density gradient method, concentrations of 146s antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146s antigen in many samples, and also indicates when VP1 of 146s particles has disintegrated by the action of proteases.
introduction The potency of foot-and-mouth disease (FMD) vaccines is strongly correlated to the 146s content of the tissue cultures used to prepare the vaccines.’ The whole virus particle of foot-and-mouth disease virus (FMDV) has a sedimentation coefficient of 146S, and the subunits of the virus have a sedimentation coefficient of 12s. Brown et aZ.2 demonstrated that compared to 146s particles, 12s particles were inefficient immunogens. To quantify 146s antigen, Barteling & Meloen3 developed the sucrose density gradient method, which is now commonly used in FMD research and vaccine production. Although quantitative sucrose density gradient (SDG) analysis is rapid, it requires expensive equipment. It is also not suitable for testing large numbers of samples, and it does not distinguish between intact 146s particles and trypsin-cleaved particles. Trypsin cleaves an important epitope on VPl, the most immunogenic structural protein of foot-and-mouth disease virus (FMDV). 4,5 Most FMDV strains are less immunogenic after treatment with trypsin or other proteases,5,6 and because of this, discrepancies can be found between the expected potency based on the 146s content of vaccine and its real potency in cattle or pigs. In 1984, Ouldridge et ~1.~ introduced an indirect 1045-1056/90/040315+05
$03.00/O
sandwich enzyme-linked immunosorbent assay (ELISA) to specifically quantify the 146s content of vaccine virus harvests. Polyvalent rabbit serum was used to coat the wells of microtitre plates, and polyvalent guinea-pig serum was used for the second phase of the test. The investigators reported that the test did not detect 12s antigen and was therefore suitable for quantifying 146s antigen. When the ELISA was compared with the SDG method, the concentration of 146s antigen quantified in the two tests correlated well. Moreover, they stated that the ELISA preferentially detected the trypsin-sensitive site of 146s. These findings were in contrast to those of other investigators,‘,’ including us, who found that the ELISA detected 12s antigen as well as 146s antigen, and was thus unsuitable for quantifying whole virus. To quantify 146s antigen of FMDV exclusively, we developed a double antibody sandwich (DAS) ELISA with two monoclonal antibodies (MAbs). One was directed against a 146Sspecific site of FMDV A10 Holland; the other was directed against a trypsinsensitive site of this strain, and its epitope contained an essential amino acid residue at VPl, 144.” This paper describes the standardization of the test procedure, its specificity for detecting intact 146s antigen in tissue culture harvests, and the correlation with the sucrose density gradient method. @ 1990 The International
Association
of Biological
Standardization
316
Material
C. Van Maanen
and methods
Antigen
Standard virus. FMDV A10 Holland was propagated in a suspension of bovine tongue epithelium, clarified by filtration, and stored in ampoules of 1 ml at -70°C. The 146s content of the standard virus was determined 36 times by the SDG method, and the mean 146s content was calculated. Subunits (12s) of the virus were prepared by acidifying antigens according to the method of Abu Elzein & Crowther.” Vaccine virus harvests. Thirty harvests from different FMDV A10 Holland virus cultures were frozen at -70°C. The antigens were prepared as described for the standard virus. The 146s content of all samples was determined by the SDG method and by the DAS-ELISA. Trypsin-treated virus. One ml volumes of standard virus were mixed with 100 yl volumes of phosphatebuffered saline (PBS) containing various concentrations of trypsin. Final concentrations of trypsin were 1 mg ml- ‘, 100 pg ml-r, and 10 pg ml-‘. The mixtures were incubated for 15 min at 37°C. Reactions were stopped by adding soybean trypsin inhibitor in concentrations of 1.5 mg ml-‘, 150 pg ml-‘, and 15 pg ml-‘. The 146s content of all mixtures was determined by the SDG method and by the DAS-ELISA. Monoclonal
antibodies
A panel of MAbs directed against FMDV A10 Holland has been described and characterized before.““’ Monoclonal antibody 22.9 is directed against an epitope on VP3, and therefore, treatment of virus with trypsin does not reduce the binding of this MAb in the ELISA. This MAb recognizes only the intact 146s particle, including trypsin-treated virus, and not the 12s subunits of strain A10 Holland. Monoclonal antibody 22.11 is directed against a part of the antigenic site of VPl, amino acids 140-166, which is cleaved by trypsin. Therefore, treatment of virus with trypsin reduces the binding of this MAb in the ELISA. This MAb does recognize 12s subunits of FMDV A10 Holland. Both MAbs were partly purified from ascites fluid by ammonium sulphate precipitation. Monoclonal antibody 22.9 was diluted to a final protein concentration of 4 mg/ml, and MAb 22.11 was conjugated to horseradish peroxidase according to the method of Wilson & Nakane.13 Both MAbs were stored with 50% v/v/ glycerol at -20°C.
and
C. Terpstra
Double antibody
sandwich
(DAS) ELISA
The wells of ELISA plates (Costar 3590) were coated for 20 h at 4°C with 50 ~1 MAb CVI-FMD-22.9, diluted 1:lOOO in PBS. In a dummy microtitre plate with low binding capacity, five virus harvests were diluted two-fold in duplicate in dilution medium (PBS containing 1% Tween 80,5% fetal bovine serum, and 2.4% NaClJ; the standard virus was titrated on each plate. The plates were washed five times with PBS containing 0.05% Tween 80; 50 ~1 volumes of the antigen dilutions were transferred from the dummy plates to the washed ELISA plates. After incubation for 1 h at 37°C on a Luckham rotary shaker, plates were washed as described. The wells were then filled with 50 ,~l of MAb 22.11 conjugated to horseradish peroxidase, diluted 1:5000 in dilution medium. After incubation for 1 h at 37°C on a Luckham rotary shaker, plates were washed as described. A mixture of 0.006% v/v Hz02 and 0.4 mg ml-’ ortho-phenylenediamine in 0.1 M NasHPOd and 0.1 M citric acid buffer (pH 5.0) was used as substrate and chromogen. A volume of 100 ~1 of this mixture was added to each well. After 15 min, colour development was stopped by adding 100 ~11 M HsS04, and the extinction values were measured at 492 nm in an Easy Reader spectrophotometer (SLT, Vienna). For each virus harvest the mean logic antigen titre corresponding to the dilution with an optical density of 1.5 was determined graphically. On each plate the antigen dilution of the standard virus with an optical density of 1.5 was determined, and for each plate a calibration coefficient was calculated for converting the antigen dilution to 146s concentration. Sucrose
density
gradients
The 146s content of virus harvests was determined according to the method of Barteling & Meloen.3 Briefly, 200 ,ul of virus harvest was layered on lo-25% sucrose gradients, and centrifuged at 243 000 g for 40 min. The gradients were scanned by ultraviolet light at 254 nm with a continuous flow cell monitor (LKB Instruments).
Results Specificity
of DAS-ELISA
for 146s particles
To determine the 146s specificity of the test, 146s particles and 12s particles of the standard virus were titrated in the DAS-ELISA in quadruplicate in twofold dilutions. Mean extinction values were determined for each antigen dilution. Whereas hardly any 12s subunits were detected even in the
Quantification
of 146s foot-and-mouth
antigen
317
1
3000 2500
500 I.2
I.8 log,,
antigen
2.4 dilution
3.0
3.6
0
0.6
I.2 log,,
I.8 antigen
2.4
3.0
dilutton
Figure 1. Lack of interference of 12s subunits with 146s
detection: titration curves of 146s particles and 12s subunits in dilution medium, and titration curve of 146s particles in a suspension of 12s subunits. -+-, 146s in D&l; --G--, 12s in DM; -0-, 146s in 12s suspension.
Figure 2. Influence of trypsin treatment
lowest dilution, 146s antigen was still detected in high dilutions (Fig. 1). To determine whether high concentrations of 12s subunits interfered with quantifying 146s antigen, we titrated the standard virus twice in dilution medium, both with and without 12s subunits; both titrations were performed in quadruplicate using two-fold dilutions. The two titrations were almost identical; in both curves an extinction value of 1.5 corresponded to the same antigen dilution (Fig. 1).
ml-’ of trypsin the values were 1.73,162, ml-l, respectively.
Influence of trypsin-treatment on virus quantified in the SDG and ELISA
The influence of trypsin on the binding of 146s antigen in the DAS-ELISA is shown in Fig. 2; compared with the untreated virus, virus that was incubated with trypsin 100 pg ml-’ or more reduced the binding of virus considerably. Apparently the 10 pg ml-l concentration of trypsin was too low to affect VP1 of most virus particles. At an optical density of 1.5 the logic antigen dilution was 1.57 for untreated virus and O-30 for virus treated with 1 mg ml-’ trypsin. In the SDG, the mean 146s content of the untreated virus was 2.04 pg ml-i. As a logi0 antigen dilution of 1.57 in the linear part of the titration curve corresponds to a 146s concentration of 2.04 pg ml-‘, a loglo antigen dilution of O-30 corresponds to a 146s concentration of 0.11 pg ml-‘. Assuming that MAb 22.11 did not recognize VP1 that was destroyed by trypsin, 95% of the immunodominant epitopes on VP1 molecules were cleaved by trypsin. In the SDG, the 146s content of untreated virus was 1.74 pg ml-l; for virus treated with 10 pg ml-‘, 100 pg ml-‘, or 1 mg
of FMDV A10 Holland on optical densities determined in the DAS-ELISA. -A-, trypsin 1 mg/ml; -U--, trypsin 100 @g/ml; -W--, trypsin 10 pg/ml; -0---, control. and 1.96 pg
1465 antigen content of tissue culture virus harvests
Thirty samples of virus harvested from cultures of surviving bovine tongue epithelium were tested in duplicate in the SDG and the DAS-ELISA. Five samples plus the standard virus were titrated per DAS-ELISA microtitre plate; the logic antigen dilution that corresponded to an extinction value of 1.5 was determined for each virus sample (Fig. 3); the logic standard virus titre was 1.71, which is equal to a standard virus titre of 51.3. Because the mean 146s concentration for standard virus is 2.04 pg ml-’ in the SDG, the calibration coefficient for virus samples
I
0
0.6
I.2 log,,
Figure 3. Titration
I.8 ontigen
2.4
3.0
dilution
curves of five different FMDV A10 Holland tissue culture harvests and standard virus, tested in duplicate on the same microtitre plate. The standard virus contains 2-04 ,ug/ml of 146s ( + --- + 1,virus samples 1-5 contain 0.85, 1.45, 1.70, 2.34, and 3-80 pg/ml.
318
C. Van Maanen and C. Terpstra
tested on this particular microtitre plate was O-040. The 146s content for virus samples l-5 was 085, 1.45, 1.70, 2.34, and 380 ,ug ml-’ (Fig. 3). The 146s content of the 30 virus harvests correlated significantly (P < 0.001, Student’s t-test) in the two tests. The correlation coefficient was 087, and the regression coefficient was 1.00 (Fig. 4). Statistical variation within and between tests The variation within and between each run of the SDG test was determined in six test runs each using six samples (total 36) of standard virus. The mean 146s concentration of the samples was 2.04 pg ml-‘, and the standard error was 0.21. The pooled standard error within the test runs was 0.14, significantly 0’ < O-05, F-test) smaller than that between test runs, which was 0.20. Because virus harvests were tested in duplicate in the SDG test, the 36 samples of standard virus were also considered as 18 duplicate samples; the pooled standard error for these was 0.17. The variation within and between tests of standard virus was determined in four tests each using six microtitre plates; the virus was titrated in duplicate on each plate. The mean 146s concentration of the 24 samples of standard virus was 1.80 ,ug ml-‘, and the standard error was 0.23. The pooled standard error of 0.20 within tests did not differ significantly from the pooled standard error of 0.21 between tests. The variation between tests was also determined by titrating 30 virus harvests in three different ELISA tests. Standard virus was also titrated on each microtitre plate, and a calibration coefficient for conversion of loglo antigen titre to 146s concentration was calculated. The pooled standard error was O-18, which did not differ significantly from the pooled standard error for the variation between tests of the standard virus (0.21). 4,
n l
;: 3 i w s
n;/
n
l
*
2
0 3 :
/d
Wm
v 0
I
I I
1 pg/ml
I 2
,
146s
SDG
I 3
1
1 4
Figure 4. Regressionand correlation between 1465 concentrations as determined by SDG and DAS-ELISA in FMDV harvests from tissue culture. r = 0.87, y = x.
Discussion The SDG method has been used for many years in our vaccine production plant to determine the 146s content of FMDV harvested from tissue culture and suspension cultures of bovine tongue epithelium.” Sometimes, however, the 146s content of a vaccine, as determined by SDG analysis, does not indicate its potency. This can be caused by the action of proteases which damage an important immunogenic site on VP1 of FMDV.5,6 ELISAs using reagents that are mainly directed against the immunodominant site on VP1 of 146s particles can indicate whether the VP1 proteins of the virus particles are still intact or not. Therefore, we developed a DAS-ELISA using MAbs specific for 146S, one of which recognized a trypsin-sensitive site on FMDV A10 Holland. Strain A10 Holland 146s particles were specifically detected by the DAS-ELISA, and 146s antigen titres did not change in the presence of 12s subunits (Fig. 1). Apparently 12s particles do not interfere at all with the quantification of 146s in this ELISA. When the epitopes on VP1 of FMDV A10 Holland were cut by trypsin, the extinction values at any given antigen dilution were much lower (Fig. 2). In contrast, the SDG method measured comparable 146s contents for both trypsin-treated virus and untreated virus. The 146s contents of the virus harvests correlated well in both tests. Moreover, the regression line coincided with the line of equivalence. Therefore, for virus harvests produced under the same conditions, 146s contents determined in the DAS-ELISA can be converted without any correction to 146s contents determined in the SDG method. The variation of results is comparable in both tests, whereas many samples can be tested at the same time in the DAS-ELISA. Furthermore the DAS-ELISA has the unique ability to determine whether VP1 epitopes are intact, which is extremely critical for the potency of FMD vaccines. We suggest that this DASELISA is a valuable tool for FMD vaccine producers and examiners. Acknowledgements We thank H. Gaasenbeek and F. van Hemert for skilful technical assistance, and S. J. Barteling for kindly supplying the MAbs directed against FMDV strain A10 Holland and for critical comments. References 1. Pay TWF, Hingley PH, Radlett PJ, Black L, O’Reilly KJ. The correlation of 140s antigen dose with the serum neutralisation antibody response and with
Quantlficatlon
2. 3. 4.
5. 6.
7.
8.
of 146s foot-and-mouth
protection from challenge induced by FMD vaccines. Session of the Research Group of the European Commissionfor the Control of FMD, Lelystad 1983; App. IX: 52-55. Brown F, Newman JFE. In vitro measurement of the potency of inactivated foot-and-mouth disease vaccines. J Hyg Camb 1963; 61: 345-351. Barteling SJ, Meloen RH. A simple method for the quantification of 140s particles of foot and mouth diseasevirus. Arch Ges Virusforsch 1974;45: 362-364. Bachrach HL, Moore DM, McKercher PD, Polatnick J. Immune and antibody responsesto an isolated capsid protein of foot-and-mouth diseasevirus. J Immoll975; 115: 16361641. Wild TF, Brown F. Nature of the inactivating action of trypsin on foot-and-mouth diseasevirus. J Gen Virol 1967; 1: 247-250. Doe1TR, Collen T. Quantitative assessmentof 146s particles of foot-and-mouth diseasevirus in preparations destined for vaccines. J Biol Stand 1982; 10: 69-81. Ouldridge JE, Barnett PV, Hingley PJ, Rweyemamu MM. An indirect enzyme labelled immunosorbent assayfor the detection of foot and mouth diseasevirus immunizing antigen in tissue culture harvests. J Biol Stand 1984; 12: 339-351. Have P, Lei JC, Schjerning-Thiesen K. An enzymelinked immunosorbent assay (ELISA) for the primary
319
antigen
diagnosis of foot-and-mouth disease;characterization and comparison with complement fixation. Acta Vet Stand 1984; 25: 280-296. 9. Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis.ResVet Sci 1987; 43: 225-232. 10. Thomas AAM, Woortmeijer RJ, Puyck W, Barteling SJ. Antigenic sites on foot-and-mouth diseasevirus type AlO. J Virol 1988; 62: 2782-2789. 11. Abu Elzein EME, Crowther JR. The specific detection of foot-and-mouth diseasevirus whole particle antigen (140s) by enzyme labelled immunosorbent assay. J Hyg 1979; 83: 127-134. 12. Barteling SJ, Boerke J, Woortmeijer R, Thomas A. Neutralising monoclonal antibodies are directed toward antigenic sites on VPl, VP2, and VP3. Bull Int Epizoot 1986; 17: 141-151. 13. Wilson MB, Nakane PK. Recent developments in the periodate method of conjugating horseradish peroxidase(HRPO) to antibodies. In: W. Knap, K. Holubar, and G. Wick (Eds), Immunofluorescence and Related Staining Techniques, Amsterdam: Elsevier/North Holland Biomedical Press, 1987: 215-224.
Received for publication 2 April accepted 30 May 1990.
1990:
(1990)
18, 31!%319
Quantification of Intact 146s Foot-and-mouth Antigen for Vaccine Production by a Double Antibody Sandwich ELBA using Monoclonal Antibodies C. Van Maanen Central
Veterinary
Institute,
and C. Terpstra
Department of Virology, P. 0. Box 365, 8200 AJ Lelystad, The Netherlands
Abstract. A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146s antigen of foot-and-mouth disease virus (FMDV) strain Al 0 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin-which affects VP1 , the most immunogenic structural protein of FMDV-the concentration of 146s antigen as determined by ELISA was reduced by >90%. Therefore, the test detected essentially only those virus particles with intact VPl. When the test was compared with the sucrose density gradient method, concentrations of 146s antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146s antigen in many samples, and also indicates when VP1 of 146s particles has disintegrated by the action of proteases.
introduction The potency of foot-and-mouth disease (FMD) vaccines is strongly correlated to the 146s content of the tissue cultures used to prepare the vaccines.’ The whole virus particle of foot-and-mouth disease virus (FMDV) has a sedimentation coefficient of 146S, and the subunits of the virus have a sedimentation coefficient of 12s. Brown et aZ.2 demonstrated that compared to 146s particles, 12s particles were inefficient immunogens. To quantify 146s antigen, Barteling & Meloen3 developed the sucrose density gradient method, which is now commonly used in FMD research and vaccine production. Although quantitative sucrose density gradient (SDG) analysis is rapid, it requires expensive equipment. It is also not suitable for testing large numbers of samples, and it does not distinguish between intact 146s particles and trypsin-cleaved particles. Trypsin cleaves an important epitope on VPl, the most immunogenic structural protein of foot-and-mouth disease virus (FMDV). 4,5 Most FMDV strains are less immunogenic after treatment with trypsin or other proteases,5,6 and because of this, discrepancies can be found between the expected potency based on the 146s content of vaccine and its real potency in cattle or pigs. In 1984, Ouldridge et ~1.~ introduced an indirect 1045-1056/90/040315+05
$03.00/O
sandwich enzyme-linked immunosorbent assay (ELISA) to specifically quantify the 146s content of vaccine virus harvests. Polyvalent rabbit serum was used to coat the wells of microtitre plates, and polyvalent guinea-pig serum was used for the second phase of the test. The investigators reported that the test did not detect 12s antigen and was therefore suitable for quantifying 146s antigen. When the ELISA was compared with the SDG method, the concentration of 146s antigen quantified in the two tests correlated well. Moreover, they stated that the ELISA preferentially detected the trypsin-sensitive site of 146s. These findings were in contrast to those of other investigators,‘,’ including us, who found that the ELISA detected 12s antigen as well as 146s antigen, and was thus unsuitable for quantifying whole virus. To quantify 146s antigen of FMDV exclusively, we developed a double antibody sandwich (DAS) ELISA with two monoclonal antibodies (MAbs). One was directed against a 146Sspecific site of FMDV A10 Holland; the other was directed against a trypsinsensitive site of this strain, and its epitope contained an essential amino acid residue at VPl, 144.” This paper describes the standardization of the test procedure, its specificity for detecting intact 146s antigen in tissue culture harvests, and the correlation with the sucrose density gradient method. @ 1990 The International
Association
of Biological
Standardization
316
Material
C. Van Maanen
and methods
Antigen
Standard virus. FMDV A10 Holland was propagated in a suspension of bovine tongue epithelium, clarified by filtration, and stored in ampoules of 1 ml at -70°C. The 146s content of the standard virus was determined 36 times by the SDG method, and the mean 146s content was calculated. Subunits (12s) of the virus were prepared by acidifying antigens according to the method of Abu Elzein & Crowther.” Vaccine virus harvests. Thirty harvests from different FMDV A10 Holland virus cultures were frozen at -70°C. The antigens were prepared as described for the standard virus. The 146s content of all samples was determined by the SDG method and by the DAS-ELISA. Trypsin-treated virus. One ml volumes of standard virus were mixed with 100 yl volumes of phosphatebuffered saline (PBS) containing various concentrations of trypsin. Final concentrations of trypsin were 1 mg ml- ‘, 100 pg ml-r, and 10 pg ml-‘. The mixtures were incubated for 15 min at 37°C. Reactions were stopped by adding soybean trypsin inhibitor in concentrations of 1.5 mg ml-‘, 150 pg ml-‘, and 15 pg ml-‘. The 146s content of all mixtures was determined by the SDG method and by the DAS-ELISA. Monoclonal
antibodies
A panel of MAbs directed against FMDV A10 Holland has been described and characterized before.““’ Monoclonal antibody 22.9 is directed against an epitope on VP3, and therefore, treatment of virus with trypsin does not reduce the binding of this MAb in the ELISA. This MAb recognizes only the intact 146s particle, including trypsin-treated virus, and not the 12s subunits of strain A10 Holland. Monoclonal antibody 22.11 is directed against a part of the antigenic site of VPl, amino acids 140-166, which is cleaved by trypsin. Therefore, treatment of virus with trypsin reduces the binding of this MAb in the ELISA. This MAb does recognize 12s subunits of FMDV A10 Holland. Both MAbs were partly purified from ascites fluid by ammonium sulphate precipitation. Monoclonal antibody 22.9 was diluted to a final protein concentration of 4 mg/ml, and MAb 22.11 was conjugated to horseradish peroxidase according to the method of Wilson & Nakane.13 Both MAbs were stored with 50% v/v/ glycerol at -20°C.
and
C. Terpstra
Double antibody
sandwich
(DAS) ELISA
The wells of ELISA plates (Costar 3590) were coated for 20 h at 4°C with 50 ~1 MAb CVI-FMD-22.9, diluted 1:lOOO in PBS. In a dummy microtitre plate with low binding capacity, five virus harvests were diluted two-fold in duplicate in dilution medium (PBS containing 1% Tween 80,5% fetal bovine serum, and 2.4% NaClJ; the standard virus was titrated on each plate. The plates were washed five times with PBS containing 0.05% Tween 80; 50 ~1 volumes of the antigen dilutions were transferred from the dummy plates to the washed ELISA plates. After incubation for 1 h at 37°C on a Luckham rotary shaker, plates were washed as described. The wells were then filled with 50 ,~l of MAb 22.11 conjugated to horseradish peroxidase, diluted 1:5000 in dilution medium. After incubation for 1 h at 37°C on a Luckham rotary shaker, plates were washed as described. A mixture of 0.006% v/v Hz02 and 0.4 mg ml-’ ortho-phenylenediamine in 0.1 M NasHPOd and 0.1 M citric acid buffer (pH 5.0) was used as substrate and chromogen. A volume of 100 ~1 of this mixture was added to each well. After 15 min, colour development was stopped by adding 100 ~11 M HsS04, and the extinction values were measured at 492 nm in an Easy Reader spectrophotometer (SLT, Vienna). For each virus harvest the mean logic antigen titre corresponding to the dilution with an optical density of 1.5 was determined graphically. On each plate the antigen dilution of the standard virus with an optical density of 1.5 was determined, and for each plate a calibration coefficient was calculated for converting the antigen dilution to 146s concentration. Sucrose
density
gradients
The 146s content of virus harvests was determined according to the method of Barteling & Meloen.3 Briefly, 200 ,ul of virus harvest was layered on lo-25% sucrose gradients, and centrifuged at 243 000 g for 40 min. The gradients were scanned by ultraviolet light at 254 nm with a continuous flow cell monitor (LKB Instruments).
Results Specificity
of DAS-ELISA
for 146s particles
To determine the 146s specificity of the test, 146s particles and 12s particles of the standard virus were titrated in the DAS-ELISA in quadruplicate in twofold dilutions. Mean extinction values were determined for each antigen dilution. Whereas hardly any 12s subunits were detected even in the
Quantification
of 146s foot-and-mouth
antigen
317
1
3000 2500
500 I.2
I.8 log,,
antigen
2.4 dilution
3.0
3.6
0
0.6
I.2 log,,
I.8 antigen
2.4
3.0
dilutton
Figure 1. Lack of interference of 12s subunits with 146s
detection: titration curves of 146s particles and 12s subunits in dilution medium, and titration curve of 146s particles in a suspension of 12s subunits. -+-, 146s in D&l; --G--, 12s in DM; -0-, 146s in 12s suspension.
Figure 2. Influence of trypsin treatment
lowest dilution, 146s antigen was still detected in high dilutions (Fig. 1). To determine whether high concentrations of 12s subunits interfered with quantifying 146s antigen, we titrated the standard virus twice in dilution medium, both with and without 12s subunits; both titrations were performed in quadruplicate using two-fold dilutions. The two titrations were almost identical; in both curves an extinction value of 1.5 corresponded to the same antigen dilution (Fig. 1).
ml-’ of trypsin the values were 1.73,162, ml-l, respectively.
Influence of trypsin-treatment on virus quantified in the SDG and ELISA
The influence of trypsin on the binding of 146s antigen in the DAS-ELISA is shown in Fig. 2; compared with the untreated virus, virus that was incubated with trypsin 100 pg ml-’ or more reduced the binding of virus considerably. Apparently the 10 pg ml-l concentration of trypsin was too low to affect VP1 of most virus particles. At an optical density of 1.5 the logic antigen dilution was 1.57 for untreated virus and O-30 for virus treated with 1 mg ml-’ trypsin. In the SDG, the mean 146s content of the untreated virus was 2.04 pg ml-i. As a logi0 antigen dilution of 1.57 in the linear part of the titration curve corresponds to a 146s concentration of 2.04 pg ml-‘, a loglo antigen dilution of O-30 corresponds to a 146s concentration of 0.11 pg ml-‘. Assuming that MAb 22.11 did not recognize VP1 that was destroyed by trypsin, 95% of the immunodominant epitopes on VP1 molecules were cleaved by trypsin. In the SDG, the 146s content of untreated virus was 1.74 pg ml-l; for virus treated with 10 pg ml-‘, 100 pg ml-‘, or 1 mg
of FMDV A10 Holland on optical densities determined in the DAS-ELISA. -A-, trypsin 1 mg/ml; -U--, trypsin 100 @g/ml; -W--, trypsin 10 pg/ml; -0---, control. and 1.96 pg
1465 antigen content of tissue culture virus harvests
Thirty samples of virus harvested from cultures of surviving bovine tongue epithelium were tested in duplicate in the SDG and the DAS-ELISA. Five samples plus the standard virus were titrated per DAS-ELISA microtitre plate; the logic antigen dilution that corresponded to an extinction value of 1.5 was determined for each virus sample (Fig. 3); the logic standard virus titre was 1.71, which is equal to a standard virus titre of 51.3. Because the mean 146s concentration for standard virus is 2.04 pg ml-’ in the SDG, the calibration coefficient for virus samples
I
0
0.6
I.2 log,,
Figure 3. Titration
I.8 ontigen
2.4
3.0
dilution
curves of five different FMDV A10 Holland tissue culture harvests and standard virus, tested in duplicate on the same microtitre plate. The standard virus contains 2-04 ,ug/ml of 146s ( + --- + 1,virus samples 1-5 contain 0.85, 1.45, 1.70, 2.34, and 3-80 pg/ml.
318
C. Van Maanen and C. Terpstra
tested on this particular microtitre plate was O-040. The 146s content for virus samples l-5 was 085, 1.45, 1.70, 2.34, and 380 ,ug ml-’ (Fig. 3). The 146s content of the 30 virus harvests correlated significantly (P < 0.001, Student’s t-test) in the two tests. The correlation coefficient was 087, and the regression coefficient was 1.00 (Fig. 4). Statistical variation within and between tests The variation within and between each run of the SDG test was determined in six test runs each using six samples (total 36) of standard virus. The mean 146s concentration of the samples was 2.04 pg ml-‘, and the standard error was 0.21. The pooled standard error within the test runs was 0.14, significantly 0’ < O-05, F-test) smaller than that between test runs, which was 0.20. Because virus harvests were tested in duplicate in the SDG test, the 36 samples of standard virus were also considered as 18 duplicate samples; the pooled standard error for these was 0.17. The variation within and between tests of standard virus was determined in four tests each using six microtitre plates; the virus was titrated in duplicate on each plate. The mean 146s concentration of the 24 samples of standard virus was 1.80 ,ug ml-‘, and the standard error was 0.23. The pooled standard error of 0.20 within tests did not differ significantly from the pooled standard error of 0.21 between tests. The variation between tests was also determined by titrating 30 virus harvests in three different ELISA tests. Standard virus was also titrated on each microtitre plate, and a calibration coefficient for conversion of loglo antigen titre to 146s concentration was calculated. The pooled standard error was O-18, which did not differ significantly from the pooled standard error for the variation between tests of the standard virus (0.21). 4,
n l
;: 3 i w s
n;/
n
l
*
2
0 3 :
/d
Wm
v 0
I
I I
1 pg/ml
I 2
,
146s
SDG
I 3
1
1 4
Figure 4. Regressionand correlation between 1465 concentrations as determined by SDG and DAS-ELISA in FMDV harvests from tissue culture. r = 0.87, y = x.
Discussion The SDG method has been used for many years in our vaccine production plant to determine the 146s content of FMDV harvested from tissue culture and suspension cultures of bovine tongue epithelium.” Sometimes, however, the 146s content of a vaccine, as determined by SDG analysis, does not indicate its potency. This can be caused by the action of proteases which damage an important immunogenic site on VP1 of FMDV.5,6 ELISAs using reagents that are mainly directed against the immunodominant site on VP1 of 146s particles can indicate whether the VP1 proteins of the virus particles are still intact or not. Therefore, we developed a DAS-ELISA using MAbs specific for 146S, one of which recognized a trypsin-sensitive site on FMDV A10 Holland. Strain A10 Holland 146s particles were specifically detected by the DAS-ELISA, and 146s antigen titres did not change in the presence of 12s subunits (Fig. 1). Apparently 12s particles do not interfere at all with the quantification of 146s in this ELISA. When the epitopes on VP1 of FMDV A10 Holland were cut by trypsin, the extinction values at any given antigen dilution were much lower (Fig. 2). In contrast, the SDG method measured comparable 146s contents for both trypsin-treated virus and untreated virus. The 146s contents of the virus harvests correlated well in both tests. Moreover, the regression line coincided with the line of equivalence. Therefore, for virus harvests produced under the same conditions, 146s contents determined in the DAS-ELISA can be converted without any correction to 146s contents determined in the SDG method. The variation of results is comparable in both tests, whereas many samples can be tested at the same time in the DAS-ELISA. Furthermore the DAS-ELISA has the unique ability to determine whether VP1 epitopes are intact, which is extremely critical for the potency of FMD vaccines. We suggest that this DASELISA is a valuable tool for FMD vaccine producers and examiners. Acknowledgements We thank H. Gaasenbeek and F. van Hemert for skilful technical assistance, and S. J. Barteling for kindly supplying the MAbs directed against FMDV strain A10 Holland and for critical comments. References 1. Pay TWF, Hingley PH, Radlett PJ, Black L, O’Reilly KJ. The correlation of 140s antigen dose with the serum neutralisation antibody response and with
Quantlficatlon
2. 3. 4.
5. 6.
7.
8.
of 146s foot-and-mouth
protection from challenge induced by FMD vaccines. Session of the Research Group of the European Commissionfor the Control of FMD, Lelystad 1983; App. IX: 52-55. Brown F, Newman JFE. In vitro measurement of the potency of inactivated foot-and-mouth disease vaccines. J Hyg Camb 1963; 61: 345-351. Barteling SJ, Meloen RH. A simple method for the quantification of 140s particles of foot and mouth diseasevirus. Arch Ges Virusforsch 1974;45: 362-364. Bachrach HL, Moore DM, McKercher PD, Polatnick J. Immune and antibody responsesto an isolated capsid protein of foot-and-mouth diseasevirus. J Immoll975; 115: 16361641. Wild TF, Brown F. Nature of the inactivating action of trypsin on foot-and-mouth diseasevirus. J Gen Virol 1967; 1: 247-250. Doe1TR, Collen T. Quantitative assessmentof 146s particles of foot-and-mouth diseasevirus in preparations destined for vaccines. J Biol Stand 1982; 10: 69-81. Ouldridge JE, Barnett PV, Hingley PJ, Rweyemamu MM. An indirect enzyme labelled immunosorbent assayfor the detection of foot and mouth diseasevirus immunizing antigen in tissue culture harvests. J Biol Stand 1984; 12: 339-351. Have P, Lei JC, Schjerning-Thiesen K. An enzymelinked immunosorbent assay (ELISA) for the primary
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diagnosis of foot-and-mouth disease;characterization and comparison with complement fixation. Acta Vet Stand 1984; 25: 280-296. 9. Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis.ResVet Sci 1987; 43: 225-232. 10. Thomas AAM, Woortmeijer RJ, Puyck W, Barteling SJ. Antigenic sites on foot-and-mouth diseasevirus type AlO. J Virol 1988; 62: 2782-2789. 11. Abu Elzein EME, Crowther JR. The specific detection of foot-and-mouth diseasevirus whole particle antigen (140s) by enzyme labelled immunosorbent assay. J Hyg 1979; 83: 127-134. 12. Barteling SJ, Boerke J, Woortmeijer R, Thomas A. Neutralising monoclonal antibodies are directed toward antigenic sites on VPl, VP2, and VP3. Bull Int Epizoot 1986; 17: 141-151. 13. Wilson MB, Nakane PK. Recent developments in the periodate method of conjugating horseradish peroxidase(HRPO) to antibodies. In: W. Knap, K. Holubar, and G. Wick (Eds), Immunofluorescence and Related Staining Techniques, Amsterdam: Elsevier/North Holland Biomedical Press, 1987: 215-224.
Received for publication 2 April accepted 30 May 1990.
1990:
