Veterinary Microbiology, 31 ( 1992 ) 147-160 Elsevier Science Publishers B.V., Amsterdam
147
A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants G. Libeau, A. Diallo, D. Calvez and P.C. Lef6vre lnstitut d'Elevage et de MOdecine Vktkrinaire des Pays Tropicaux/CIRAD - Service de Pathologie infectieuse, 10 rue Pierre Curie, 94704 Maisons-Alfort Cedex, France (Accepted 16 October 1991 )
ABSTRACT Libeau, G., Diallo, A., Calvez, D. and Lef~vre, P.C., 1992. A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants. Vet. Microbiol., 31 : 147-160. A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleoprotein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.
INTRODUCTION
Epidemiological surveys of small ruminants for antibodies to rinderpest virus (RPV) require a rapid serological test, as live attenuated rinderpest vaccine is now being administered to control peste-des-petits-ruminants (PPR) in Africa. Small ruminants being animals of the Artiodactyla group (Provost, 1982), are also susceptible to rinderpest virus and are hence infected from time to time with bovine rinderpest. A small ruminant "adapted" strain is known in India (Rao et al., 1974) where there is evidence for transmissible strains of rinderpest to circulate within these species. 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
148
G. LIBEAUET AL.
The diagnosis of rinderpest in cattle and the assessment of the i m m u n e status of herds before and after vaccination campaigns are made using a rinderpest ELISA kit (Anderson et al., 1982 ), an indirect ELISA test distributed by the International Atomic Energy Agency (IAEA), Vienna. Since this test is unable to specify the identity of the detected antibodies, it can only be applied to epidemiological surveys in cattle. In this report, we describe the use of monoclonal antibodies (mAbs) for improving the sensitivity and the specificity of rinderpest control tests in cattle as well as in small ruminants. Three mAbs were suitable for a competitive ELISA (C-ELISA). Their characteristics have been detailed in a preliminary paper (Libeau and Lef6vre, 1990). The performance of C-ELISA was compared to that of the virus neutralisation test (VNT) on a panel of bovine and small ruminant sera obtained from animals with a known vaccination status. MATERIALS AND METHODS
Monoclonal antibodies Thirteen mAbs against the Old Kabete strain of the rinderpest vaccine virus (RBOK) were produced and characterized as described earlier (Libeau and Lef6vre, 1990). It appeared that these mAbs were immunoglobulins directed against the nucleoprotein (NP) of the virus. Indirect ELISA and immunofluorescence (IIF) on infected cell cultures showed that 6 mAbs of the 13 available recognized only the rinderpest (RP) strains. Three mAbs (VE41, IVB2-4 and 51-5-6) were deemed suitable for use in C-ELISA. VE4-1 and IVB2-4 recognized nine field strains that had been isolated from outbreaks and three vaccine strains, while 51-5-6 recognized only vaccine strains and TABLEI Characteristics of three monoclonal antibodies Mab IgG isotype
VE4-1 2b 5 1 - 5 - 6 2b IVB2-4 1
Detection o f activity on infected cells Site a
N N N
C-ELISA b
ELISA
IIF
log titre
%IH
RPV vac.
RPV
PPR
RPV vac.
RPV
8 7 7
90 55 80
+' + +
+ India +
75-1 -
+ + +
+ India +
PPR
aBinding site was concluded to be nucleoprotein from characteristic immunofluorescence staining and immunoprecipitation. bCompetition with polyclonal bovine R B O K antiserum; % IH at a serum titre of 2. CMAbs reacted with all strains.
COMPETITIVE ELISA FOR SPECIFIC DETECTION OF RINDERPEST ANTIBODIES
149
the Indian RP strain. None of the three mAbs detected any of the wild P P R strains (Table 1 ). Viral strains
The cell culture-attenuated vaccine strain (Plowright, 1962) RBOK used after 98 passages in bovine kidney cells ( B K / 9 8 ) and two passages in Vero cells (Vero/2) and the P P R 75-2 strain (Sheep Kidney (SK) cells/l, BK/1, Vero/24), were grown in Vero cells with Eagle's M i n i m u m Essential Medium (EMEM) supplemented with 2% foetal calf serum. The RBOK strain was used in VNT and for antigen preparation, and the P P R strain in VNT. The PPR 75-1 (SK/1, BK/1, Vero/55) culture-attenuated vaccine strain and the heat-stable clone (BK/98, Vero/6) of the RBOK strain were used to produce post vaccination sera (see test sera). RPV Saudi (BK/2, Vero/7 ) and P P R Meilig (Lamb Kidney cells/2, B K / 4, Vero/1 ) were used in challenge experiments to test the efficacy of the homologous virus used as a vaccine. Serum samples Control negative sera. Sera (n = 85 ) collected from French cattle, which had never been vaccinated or infected with RPV, were used to establish the cutoff value for C-ELISA (see Fig. 1 ). R P sera. Prechallenge sera (n = 40) from 11 prevaccinated, 2 vaccinated and
4 in-contact control animals were collected from young zebu cattle during a vaccine potency test with a heat-stable vaccine in Chad. Vaccinated animals received 50 doses each. Blood samples were collected on days 0, 3, 8, 14, 19 and 26. Post challenge sera ( n = 18) were collected from the two vaccinated and the four control animals of the same experiment. Cattle were challenged 26 d after vaccination with the Saudi rinderpest strain at 102 50% tissue culture infective doses (TCIDso) and 103 TCIDso (Nos. 123 and 181 ). Blood samples were collected 29, 31 and 33 days after vaccination. These 58 sera were used to determine titre correlations and regression coefficients between C-ELISA using mAb VE4-1, 51-5-6 and IVB2-4, and VNT (see Table 3). Vaccinated animals Nos. 123 and 167, and control animals Nos. 181, 165 and 152 from the vaccination experiment were chosen to measure test sensitivity. Their temperature data were recorded daily (see Fig. 2 ). PPR sera. Reference sera (n = 8 ) were prepared in two goats using the atten-
uated vaccine strain of P P R 75-1 virus (Diallo et al., 1989). Two sera were collected after challenge with the Meilig strain and two were from contact
150
G. LIBEAU ET AL.
animals; these sera were used to establish specificity of the tests (see Table 2 and Table 4).
Other sera. Sera (n = 41 ) from sheep and goats vaccinated with the RBOK vaccine strain were collected in Niger. Records from the year before vaccination indicated that 34 animals had exhibited the pneumoenteritis syndrome of PPR. Sera (n = 34) were collected from cattle in Burkina Faso, from a ranch where animals had been subjected to an annual rinderpest vaccination. All sera were screened in a VNT against both PPR and RP viruses, and were used to improve the m e t h o d in practical conditions (see Table 4 ).
Neutralisation test Neutralising antibodies against RPV and PPRV were detected by a VNT (Rioche, 1969 ). Results were obtained within 10 to 12 d. All sera were tested in twofold dilutions in duplicate. Neutralising antibody titres were expressed as the logz of the reciprocal of the serum dilution preventing at least 50% of cytopathic effect of the virus in either or in both wells, the sera being considered positive if log2 titres were >~3.
Antigen preparation. The RP antigen was prepared as described earlier (Libeau and Calvez, 1988). Briefly, supernatants from lysed cells were pooled and clarified and the virus concentrated on a Pellicon cassette system ( 100 kDa cut-off filter). Concentrated viral suspension was centrifuged on a discontinuous sucrose gradient from 20 to 60% prepared in a TNE buffer (0.010 M Tris-HC1, 0.150 M NaC1, 1 m M EDTA, pH 7.4) at 100 000 g for 90 min. The virus band was pelleted ( 100 000 g, 90 rain) then resuspended in the TNE buffer and stored at - 70 °C. The working dilution of this preparation in 0.01M phosphate buffered saline (pH 7.4-PBS) was 1/ 100. This corresponded to a protein concentration of approximately 10 #g/ml.
Competitive ELISA (C-ELISA) Experimental design. Following antigen adsorption and plate washing, the sera were diluted directly in the wells. The mAb was then added and the mixture left to react with the antigen-coated plates. After a suitable incubation time, u n b o u n d antibodies were removed by washing. Possible binding of the mAb was detected by adding a mouse-specific conjugate and its substrate. Absence of chromogenic reaction indicated the presence of circulating antibodies whose specificity was defined by the mAb in competition. When the tested sera did not interfere with the adherence of the mAb, the wells were coloured.
COMPETITIVE ELISA FOR SPECIFIC DETECTION OF RINDERPEST ANTIBODIES
151
Test procedure. An aliquot (50/tl) of antigen diluted in PBS was added to each well of an ELISA plate (Nunc I m m u n o I, Denmark). The plates were incubated for 1 h at 37 °C on an orbital shaker (Vari-shaker, Dynatech). After three washes in PBS 1/5, the sera were diluted in blocking buffer (5% dried skimmed milk (Marvel), 0. I% Tween 20, in PBS ) and 50/~1 of the dilutions were added directly to each well. Sera in duplicate were either tested in twofold serial dilutions or at 1/4 dilution. Immediately thereafter 50/tl of the mAb was added to the test serum at a predetermined dilution of ascites in blocking buffer. The mAb dilution to be used in competitive assay was defined by its titration curve in indirect ELISA and corresponded to 80% of the m a x i m u m plateau height of optical density (OD). Ascites fluids were used at initial dilutions for VE4-1 of 1/ 1600, IVB2-4 of 1/600 and 51-5-6 of 1/ 1600. After incubation at 37°C for 1 h and washing, the mouse monoclonal antibody was detected with 50/zl of a 1-2000 dilution of horse-radish peroxidaselabelled rabbit anti-mouse IgG ( H + L ) (Nordic Immunological Lab.) in blocking buffer, with 2% normal serum of the species under test (bovine, ovine and caprine). After 1 h of incubation at 37 ° C, a substrate solution containing 0.4 m g / m l of phenylenediamine in 0.1 M citric acid-0.2 M NaHPO4 buffer (pH 5.5) and 0.015/zl/ml of 10% H2Oz was put on the plates. Reaction was stopped after 10 min with 1 N H2504. OD492 was read using blocking buffer, substrate plus stop solution as a blank. Expression of percent inhibitions and ELISA titres Using the following formula, the percent of inhibition (%IH) of a serum was calculated from its mean OD value: % I H = 100× (1-OD s a m p l e / O D control). OD of s a m p l e = O D in the presence of inhibitor; OD of control = OD in the absence of inhibitor. For diagnosis we expressed the %IH as an ELISA titre: it was the reciprocal of the last serum dilution (log2) giving a %IH equal to or higher than the cut-off value obtained with the particular mAb on a negative population (Fig. 1 ).
Statistical analys& The standard deviation (#) was calculated for the mean X %IH value, which represents the background level present in negative sera. The correlation between C-ELISA and VNT tests was calculated by linear regression to express the correlation coefficient r.
RESULTS
Cut-off values For each mAb, a cut-off value was established in the C-ELISA at 1/4 dilution with 85 reference sera which had a negative neutralising activity. Because of the distribution of the %IH (Fig. 1 ), the cut-off values were set at X+ 3SD
152
G. LIBEAU ET AL. IVB2-4
VE4-1
;5.
:t
~20.
\ \ \
IS
t tO-
~300
700
~0(~ 5.00 inh~itiorl
11,00
17.00
0
-13.00
\ \ \ -7.00
\ -I.~ 5,00 1~~hibifion
11,00
-I.002,00 5.00 8.OO 11.0Ol4,0017.00~.~23,0O27.0O
Fig. 1. A comparisonof the distribution of% IH valuesobtained in C-ELISAwith mAb VE4-1 (£=2.75, #=5.66), 51-5-6 (£=1.84, 6=4.3), and IVB2-4 (£=15.92) from a negative population. for VE4-1 and 51-5-6, but at 2x for IVB2-4 (risks of false negative values). Using these results, sera with %IH above 19.25 for VE4-1, 32 for IVB2-4 and 13.9 for 51-5-6 were considered as positive. ELISA titres agreed with VNT titres and these results were statistically significant (see below).
Specificity Results in Table 2 show to what extent VE4-1, IVB2-4 and 51-5-6 bind when they are in competition with sera taken from serial bleeds of goats vaccinated with PPR 75-1 attenuated strain. Normal goat sera were included as controls. There was little or no inhibition by any of the sera when using IVB24 mAb as compared with the level obtained with a hyperimmune serum (positive control) against RPV. However, these sera competed with mAb VE4-1 and 51-5-6 even if they were defined as an RPV specific group by IIF and ELISA on cells infected by RP strains of wide geographical origin. The CELISA using IVB2-4 appeared highly specific in detecting antibodies to the RP group, while with VE4-1 and 51-5-4 the test also detected antibodies to PPR. Sera from non-vaccinated goats failed to compete against any of the three mAbs.
Detection of rinderpest antibodies in cattle with mAbs VE4-1, 51-5-6 and IVB2-4. Correlation between VNT and C-ELISA. An examination of the 58 bovine sera in the C-ELISA using mAbs VE4-1, 51-5-6, IVB2-4, and in VNT against the RBOK strain showed (Table 3 ) a highly significant correlation between the two tests (VE4-1: correlation coefficient r=0.86; 51-5-6: r=0.84; IB2-4: r=0.85). Sensitivity. The sensitivity of the C-ELISA using VE4-1, 51-5-6 and IVB2-4 was established by comparing %IH at 1/4 dilution to VNT titres on serial bleeds of calves vaccinated with the thermostable rinderpest vaccine (Nos.
COMPETITIVE ELISAFOR SPECIFICDETECTION OF RINDERPESTANTIBODIES
153
TABLE2
Demonstration of the specificity of C-ELISA with reference to VNT according to seroconversion time in goats vaccinated with PPR 75-1 and then challenged with virulent PPR. Animal/Day
Seroconversion C-ELISA titres
VNT titres
VE4-1
IVB2-4
51-5-6
PPR 75-1
RBOK
147
A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants G. Libeau, A. Diallo, D. Calvez and P.C. Lef6vre lnstitut d'Elevage et de MOdecine Vktkrinaire des Pays Tropicaux/CIRAD - Service de Pathologie infectieuse, 10 rue Pierre Curie, 94704 Maisons-Alfort Cedex, France (Accepted 16 October 1991 )
ABSTRACT Libeau, G., Diallo, A., Calvez, D. and Lef~vre, P.C., 1992. A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants. Vet. Microbiol., 31 : 147-160. A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleoprotein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.
INTRODUCTION
Epidemiological surveys of small ruminants for antibodies to rinderpest virus (RPV) require a rapid serological test, as live attenuated rinderpest vaccine is now being administered to control peste-des-petits-ruminants (PPR) in Africa. Small ruminants being animals of the Artiodactyla group (Provost, 1982), are also susceptible to rinderpest virus and are hence infected from time to time with bovine rinderpest. A small ruminant "adapted" strain is known in India (Rao et al., 1974) where there is evidence for transmissible strains of rinderpest to circulate within these species. 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
148
G. LIBEAUET AL.
The diagnosis of rinderpest in cattle and the assessment of the i m m u n e status of herds before and after vaccination campaigns are made using a rinderpest ELISA kit (Anderson et al., 1982 ), an indirect ELISA test distributed by the International Atomic Energy Agency (IAEA), Vienna. Since this test is unable to specify the identity of the detected antibodies, it can only be applied to epidemiological surveys in cattle. In this report, we describe the use of monoclonal antibodies (mAbs) for improving the sensitivity and the specificity of rinderpest control tests in cattle as well as in small ruminants. Three mAbs were suitable for a competitive ELISA (C-ELISA). Their characteristics have been detailed in a preliminary paper (Libeau and Lef6vre, 1990). The performance of C-ELISA was compared to that of the virus neutralisation test (VNT) on a panel of bovine and small ruminant sera obtained from animals with a known vaccination status. MATERIALS AND METHODS
Monoclonal antibodies Thirteen mAbs against the Old Kabete strain of the rinderpest vaccine virus (RBOK) were produced and characterized as described earlier (Libeau and Lef6vre, 1990). It appeared that these mAbs were immunoglobulins directed against the nucleoprotein (NP) of the virus. Indirect ELISA and immunofluorescence (IIF) on infected cell cultures showed that 6 mAbs of the 13 available recognized only the rinderpest (RP) strains. Three mAbs (VE41, IVB2-4 and 51-5-6) were deemed suitable for use in C-ELISA. VE4-1 and IVB2-4 recognized nine field strains that had been isolated from outbreaks and three vaccine strains, while 51-5-6 recognized only vaccine strains and TABLEI Characteristics of three monoclonal antibodies Mab IgG isotype
VE4-1 2b 5 1 - 5 - 6 2b IVB2-4 1
Detection o f activity on infected cells Site a
N N N
C-ELISA b
ELISA
IIF
log titre
%IH
RPV vac.
RPV
PPR
RPV vac.
RPV
8 7 7
90 55 80
+' + +
+ India +
75-1 -
+ + +
+ India +
PPR
aBinding site was concluded to be nucleoprotein from characteristic immunofluorescence staining and immunoprecipitation. bCompetition with polyclonal bovine R B O K antiserum; % IH at a serum titre of 2. CMAbs reacted with all strains.
COMPETITIVE ELISA FOR SPECIFIC DETECTION OF RINDERPEST ANTIBODIES
149
the Indian RP strain. None of the three mAbs detected any of the wild P P R strains (Table 1 ). Viral strains
The cell culture-attenuated vaccine strain (Plowright, 1962) RBOK used after 98 passages in bovine kidney cells ( B K / 9 8 ) and two passages in Vero cells (Vero/2) and the P P R 75-2 strain (Sheep Kidney (SK) cells/l, BK/1, Vero/24), were grown in Vero cells with Eagle's M i n i m u m Essential Medium (EMEM) supplemented with 2% foetal calf serum. The RBOK strain was used in VNT and for antigen preparation, and the P P R strain in VNT. The PPR 75-1 (SK/1, BK/1, Vero/55) culture-attenuated vaccine strain and the heat-stable clone (BK/98, Vero/6) of the RBOK strain were used to produce post vaccination sera (see test sera). RPV Saudi (BK/2, Vero/7 ) and P P R Meilig (Lamb Kidney cells/2, B K / 4, Vero/1 ) were used in challenge experiments to test the efficacy of the homologous virus used as a vaccine. Serum samples Control negative sera. Sera (n = 85 ) collected from French cattle, which had never been vaccinated or infected with RPV, were used to establish the cutoff value for C-ELISA (see Fig. 1 ). R P sera. Prechallenge sera (n = 40) from 11 prevaccinated, 2 vaccinated and
4 in-contact control animals were collected from young zebu cattle during a vaccine potency test with a heat-stable vaccine in Chad. Vaccinated animals received 50 doses each. Blood samples were collected on days 0, 3, 8, 14, 19 and 26. Post challenge sera ( n = 18) were collected from the two vaccinated and the four control animals of the same experiment. Cattle were challenged 26 d after vaccination with the Saudi rinderpest strain at 102 50% tissue culture infective doses (TCIDso) and 103 TCIDso (Nos. 123 and 181 ). Blood samples were collected 29, 31 and 33 days after vaccination. These 58 sera were used to determine titre correlations and regression coefficients between C-ELISA using mAb VE4-1, 51-5-6 and IVB2-4, and VNT (see Table 3). Vaccinated animals Nos. 123 and 167, and control animals Nos. 181, 165 and 152 from the vaccination experiment were chosen to measure test sensitivity. Their temperature data were recorded daily (see Fig. 2 ). PPR sera. Reference sera (n = 8 ) were prepared in two goats using the atten-
uated vaccine strain of P P R 75-1 virus (Diallo et al., 1989). Two sera were collected after challenge with the Meilig strain and two were from contact
150
G. LIBEAU ET AL.
animals; these sera were used to establish specificity of the tests (see Table 2 and Table 4).
Other sera. Sera (n = 41 ) from sheep and goats vaccinated with the RBOK vaccine strain were collected in Niger. Records from the year before vaccination indicated that 34 animals had exhibited the pneumoenteritis syndrome of PPR. Sera (n = 34) were collected from cattle in Burkina Faso, from a ranch where animals had been subjected to an annual rinderpest vaccination. All sera were screened in a VNT against both PPR and RP viruses, and were used to improve the m e t h o d in practical conditions (see Table 4 ).
Neutralisation test Neutralising antibodies against RPV and PPRV were detected by a VNT (Rioche, 1969 ). Results were obtained within 10 to 12 d. All sera were tested in twofold dilutions in duplicate. Neutralising antibody titres were expressed as the logz of the reciprocal of the serum dilution preventing at least 50% of cytopathic effect of the virus in either or in both wells, the sera being considered positive if log2 titres were >~3.
Antigen preparation. The RP antigen was prepared as described earlier (Libeau and Calvez, 1988). Briefly, supernatants from lysed cells were pooled and clarified and the virus concentrated on a Pellicon cassette system ( 100 kDa cut-off filter). Concentrated viral suspension was centrifuged on a discontinuous sucrose gradient from 20 to 60% prepared in a TNE buffer (0.010 M Tris-HC1, 0.150 M NaC1, 1 m M EDTA, pH 7.4) at 100 000 g for 90 min. The virus band was pelleted ( 100 000 g, 90 rain) then resuspended in the TNE buffer and stored at - 70 °C. The working dilution of this preparation in 0.01M phosphate buffered saline (pH 7.4-PBS) was 1/ 100. This corresponded to a protein concentration of approximately 10 #g/ml.
Competitive ELISA (C-ELISA) Experimental design. Following antigen adsorption and plate washing, the sera were diluted directly in the wells. The mAb was then added and the mixture left to react with the antigen-coated plates. After a suitable incubation time, u n b o u n d antibodies were removed by washing. Possible binding of the mAb was detected by adding a mouse-specific conjugate and its substrate. Absence of chromogenic reaction indicated the presence of circulating antibodies whose specificity was defined by the mAb in competition. When the tested sera did not interfere with the adherence of the mAb, the wells were coloured.
COMPETITIVE ELISA FOR SPECIFIC DETECTION OF RINDERPEST ANTIBODIES
151
Test procedure. An aliquot (50/tl) of antigen diluted in PBS was added to each well of an ELISA plate (Nunc I m m u n o I, Denmark). The plates were incubated for 1 h at 37 °C on an orbital shaker (Vari-shaker, Dynatech). After three washes in PBS 1/5, the sera were diluted in blocking buffer (5% dried skimmed milk (Marvel), 0. I% Tween 20, in PBS ) and 50/~1 of the dilutions were added directly to each well. Sera in duplicate were either tested in twofold serial dilutions or at 1/4 dilution. Immediately thereafter 50/tl of the mAb was added to the test serum at a predetermined dilution of ascites in blocking buffer. The mAb dilution to be used in competitive assay was defined by its titration curve in indirect ELISA and corresponded to 80% of the m a x i m u m plateau height of optical density (OD). Ascites fluids were used at initial dilutions for VE4-1 of 1/ 1600, IVB2-4 of 1/600 and 51-5-6 of 1/ 1600. After incubation at 37°C for 1 h and washing, the mouse monoclonal antibody was detected with 50/zl of a 1-2000 dilution of horse-radish peroxidaselabelled rabbit anti-mouse IgG ( H + L ) (Nordic Immunological Lab.) in blocking buffer, with 2% normal serum of the species under test (bovine, ovine and caprine). After 1 h of incubation at 37 ° C, a substrate solution containing 0.4 m g / m l of phenylenediamine in 0.1 M citric acid-0.2 M NaHPO4 buffer (pH 5.5) and 0.015/zl/ml of 10% H2Oz was put on the plates. Reaction was stopped after 10 min with 1 N H2504. OD492 was read using blocking buffer, substrate plus stop solution as a blank. Expression of percent inhibitions and ELISA titres Using the following formula, the percent of inhibition (%IH) of a serum was calculated from its mean OD value: % I H = 100× (1-OD s a m p l e / O D control). OD of s a m p l e = O D in the presence of inhibitor; OD of control = OD in the absence of inhibitor. For diagnosis we expressed the %IH as an ELISA titre: it was the reciprocal of the last serum dilution (log2) giving a %IH equal to or higher than the cut-off value obtained with the particular mAb on a negative population (Fig. 1 ).
Statistical analys& The standard deviation (#) was calculated for the mean X %IH value, which represents the background level present in negative sera. The correlation between C-ELISA and VNT tests was calculated by linear regression to express the correlation coefficient r.
RESULTS
Cut-off values For each mAb, a cut-off value was established in the C-ELISA at 1/4 dilution with 85 reference sera which had a negative neutralising activity. Because of the distribution of the %IH (Fig. 1 ), the cut-off values were set at X+ 3SD
152
G. LIBEAU ET AL. IVB2-4
VE4-1
;5.
:t
~20.
\ \ \
IS
t tO-
~300
700
~0(~ 5.00 inh~itiorl
11,00
17.00
0
-13.00
\ \ \ -7.00
\ -I.~ 5,00 1~~hibifion
11,00
-I.002,00 5.00 8.OO 11.0Ol4,0017.00~.~23,0O27.0O
Fig. 1. A comparisonof the distribution of% IH valuesobtained in C-ELISAwith mAb VE4-1 (£=2.75, #=5.66), 51-5-6 (£=1.84, 6=4.3), and IVB2-4 (£=15.92) from a negative population. for VE4-1 and 51-5-6, but at 2x for IVB2-4 (risks of false negative values). Using these results, sera with %IH above 19.25 for VE4-1, 32 for IVB2-4 and 13.9 for 51-5-6 were considered as positive. ELISA titres agreed with VNT titres and these results were statistically significant (see below).
Specificity Results in Table 2 show to what extent VE4-1, IVB2-4 and 51-5-6 bind when they are in competition with sera taken from serial bleeds of goats vaccinated with PPR 75-1 attenuated strain. Normal goat sera were included as controls. There was little or no inhibition by any of the sera when using IVB24 mAb as compared with the level obtained with a hyperimmune serum (positive control) against RPV. However, these sera competed with mAb VE4-1 and 51-5-6 even if they were defined as an RPV specific group by IIF and ELISA on cells infected by RP strains of wide geographical origin. The CELISA using IVB2-4 appeared highly specific in detecting antibodies to the RP group, while with VE4-1 and 51-5-4 the test also detected antibodies to PPR. Sera from non-vaccinated goats failed to compete against any of the three mAbs.
Detection of rinderpest antibodies in cattle with mAbs VE4-1, 51-5-6 and IVB2-4. Correlation between VNT and C-ELISA. An examination of the 58 bovine sera in the C-ELISA using mAbs VE4-1, 51-5-6, IVB2-4, and in VNT against the RBOK strain showed (Table 3 ) a highly significant correlation between the two tests (VE4-1: correlation coefficient r=0.86; 51-5-6: r=0.84; IB2-4: r=0.85). Sensitivity. The sensitivity of the C-ELISA using VE4-1, 51-5-6 and IVB2-4 was established by comparing %IH at 1/4 dilution to VNT titres on serial bleeds of calves vaccinated with the thermostable rinderpest vaccine (Nos.
COMPETITIVE ELISAFOR SPECIFICDETECTION OF RINDERPESTANTIBODIES
153
TABLE2
Demonstration of the specificity of C-ELISA with reference to VNT according to seroconversion time in goats vaccinated with PPR 75-1 and then challenged with virulent PPR. Animal/Day
Seroconversion C-ELISA titres
VNT titres
VE4-1
IVB2-4
51-5-6
PPR 75-1
RBOK
