Journal of Virological Methods, 28 (1990) 41-58
47
Elsevier VIRMET 00998
An enzyme-linked immunosorbent assay for detection of bovine leukaemia virus p24 antibody in cattle John B. Molloy’, Peter J. Walker’, F. Chris Baldock’, Barry J. Rodwell’ and Jeff A. Cowley’ ‘Queensland Department of Primary Indusm’es, Animal Research Institute, Yeerongpilly, Australia and 2CSIR0 Divbion of Tropical Animal Production, Indooroopilly, Alrrmzlia
(Accepted 30 November 1989)
Summary An ELISA for detecting antibody to the bovine leukaemia virus (BLV) core protein p24 is described. The test uses p24 antigen purified from concentrated cell culture supemate by lectin-affinity chromatography and gel tiltration. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection relative to the gp51 agar gel immunodiffusion test, were 98.1 and %.7%, respectively. In the event of widespread use of gp51 based vaccines, the p24-ELISA should differentiate effectively between naturally infected and vaccinated animals. Bovine leukemia virus; Antibody, ~24; Serological test; ELISA
Enzootic bovine leukaemia (EBL) is an infectious lymphoproliferative disease of cattle with worldwide distribution (Ferrer, 1980). The infectious agent, bovine leukaemia virus (BLV), is an exogenous retrovirus (Miller et al., 1969) which infects B-lymphocytes and establishes a persistent infection by integration of proviral DNA into the host chromosomes (Kettmann et al., 1979). The majority of infected cattle show no clinical signs of disease, but up to 30% develop persistent lymphocytosis with a small proportion progressing to lymphosarcoma (Ferrer, 1980; Correspondence to: J.B. Molloy, Queensland Department of Primary Industries, Animal Research Institute, 665 Fairfield Road, Yeerongpilly, 4105, Australia.
0166-0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
48
Burny et al., 1978). Cattle infected with BLV produce antiviral antibodies throughout the term of the lifelong infection (Portetelle et al., 1983). Future strategies for the control of EBL may utilize vaccination with subunit preparations of the major viral surface glycoprotein (gp51), which has been shown to induce protective antibodies (Onuma et al., 1984; Kono et al., 1986). With extensive use of a gp51 vaccine, the widely accepted agar gel immunodiffusion (AGID) test for gp51 antibody (Miller and Van der Maaten, 1976) would become unsuitable for the serological diagnosis of BLV infection. Several sensitive serological tests for antibody to the major viral core protein (~24) have been reported (Gauthier et al., 1982; Walker et al., 1987; De Boer et al., 1987). Of these, radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) techniques are suitable for large scale testing, but require either high quality, radiolabelled antigen or monoclonal antibodies, both of which are expensive to prepare and not readily available. In this paper, we describe an ELISA based on BLV p24 antigen purified from tissue culture supernate. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection were determined relative to the gp51-AGID test.
Materials and Methods Preparation of ACID
antigen
Antigen containing both gp51 and p24 was prepared from the culture supernate of foetal lamb kidney (FLK) cells persistently infected with an Australian isolate of BLV (Chung, 1980; Chung et al., 1984), concentrated lOO-fold using hollow fibre ultrafiltration as described previously (Walker et al., 1987). This antigen was used for AGID tests. Preparation of ELZSA antigen
ELISA antigen was prepared from AGID antigen using lectin-affinity chromatography and gel filtration. Con A-Sepharose chromatography, as described by Miller and Van der Maaten (1976) and modified by Walker et al. (1987), was used to remove gp51 from the AGID antigen. The p24 in the Con A non-binding fraction was concentrated about 15-fold by ultrafiltration (Amicon YMIO membrane) and further purified by gel filtration chromatography on a 750 cm x 2.6 cm Sephadex G-75 Superfine (Pharmacia) column. A 1 ml volume was loaded onto a column previously equilibrated with 20 mM Tris-HCl (pH 7.2), 150 mM NaCl (flow rate 10 ml/h). Fractions containing p24 were identified by SDS-PAGE and immunoblotting with immune sheep serum, pooled and stored at -20°C until required.
49
Virus purification BLV was purified from the culture supernate of persistently infected FLK cells by modifications of methods described previously (Deshayes et al., 1977; Walker et al., 1987). Briefly, culture supemate was clarified by centrifugation at 4000 X g for 45 min and dialysed against half-strength PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HP04, 1.1 mM KH2P0,, pH 7.2) for 16 h at 4°C. The clarified supemate was then dialysed against solid polyethyleneglycol 6000 for 6 h to achieve a 20-fold concentration and again for 16 h against half-strength PBS. This material was applied to a discontinuous gradient of 15% and 40% (w/w) sucrose in NTE buffer (100 mM NaCl, 1 mM EDTA, 10 mM TRIS-HCl pH 7.4) and centrifuged at 100000 x g for 90 min at 4°C in a Beckman SW28 rotor. The band at the interface was harvested and applied to an identical gradient to achieve a two-fold concentration. This preparation was applied to a continuous 15% to 50% (w/w) linear sucrose gradient and centrifuged at 100000 x g for 16 h at 4°C (Beckman SW28 rotor). A visible band at a buoyant density of 1.155 g/ml was harvested and the purified virus pelleted through 15% (w/w) sucrose at 100000 x g for 90 min at 4°C (Beckman SW28 rotor), resuspended in 15% w/w sucrose/NTE (protein concentration = 2 to 3 mg/ml) and stored at -70°C until required.
ACID test AGID tests were conducted using the method of Miller and Van der Maaten (1976) in 0.9% Agarose A (Pharmacia) containing 8.5% NaCl. SDS-PAGE and immunoblom’ng Discontinuous SDS-polyacrylamide gels (Laemmli, 1970) were prepared as described previously (Walker et al., 1987). Immunoblotting tests for BLV antibody were conducted using 60 pg of purified virus applied to a single 75 mm wide slot of a 0.75 mm thickness vertical mini-gel (BioRad). The immunodetection procedure was as described previously (Walker et al., 1987), except that 5% non-dairy coffee whitener (Carnation) was substituted for skim milk powder or gelatin for screening bovine sera and that biotinylated goat anti-bovine IgG (KPL) or biotinylated donkey anti-sheep IgG (Amersham) were used at dilutions of l/50 and l/200, respectively. Single-well ELISA A 100 u.1volume of the partially purified BLV p24 antigen (protein concentration = 0.36 mg/ml), diluted l/50 in 50 mM carbonate buffer (pH 9.6) was adsorbed onto each well of 96-well polyvinyl microtitre plates (Costar) for 16 h at 4°C. Plates were then washed (1 x 5 min) with 0.1% Tween-20 in PBS. Sera diluted l/5 in 1% gelatin, 0.1% Tween-20 in PBS were added (100 u.1per well) and incubated at 37°C for 2 h. Plates were washed (3 X 5 min) with 0.1% Tween-20
50
in PBS, 100 ~1 of goat anti-bovine IgG peroxidase conjugate (KPL) diluted l/l 300 in 1% gelatin, 0.1% Tween-20 in PBS added and incubated at 37°C for 1 h. Washing was repeated, 100 l.~lof peroxidase substrate ABTS (KPL) added, and after 20 min at room temperature, the differential absorbance (4141492 nm) was measured using a dual-beam microtitre plate reader (Titertek Multiscan MC). A standard BLV positive serum was included in triplicate on each plate and each test serum was assayed in triplicate on different plates to account for effects due to well and plate variability. ELISA scores were calculated as a percentage of the standard positive serum and averaged over the three assays. Dual-well ELISA
The procedure for the dual-well ELISA was as described for the single-well ELISA except that either antigen or carbonate buffer only was added to alternate wells of 96-well microtitre plates. Sera were incubated in pairs of wells with and without antigen and the difference in absorbance calculated. Test sera were assayed in duplicate on different plates and ELISA scores calculated as a percentage of the standard positive serum.
A group of 510 bovine sera, taken from 34 herds which had been free of BLV antibody in at least three consecutive AGID tests over a period of 18 months, was used to estimate the specificity of the single-well p24-ELISA. A group of 230 bovine sera from 15 herd submissions for diagnostic testing was tested in blind trials in both the single-well and dual-well p24-ELISA. These were selected from routine diagnostic submissions on the basis of AGID test results, and contained 72 strong positive, 35 weak positive, 115 clear negative and 8 equivocal sera. Data management
and analysis
Data were stored and manipulated using a microcomputer spreadsheet package (SuperCalc 3, Version 2.1, Computer Associates International Inc., San Jose, CA). Statistical analyses were performed using a microcomputer statistics package (Statgraphics, Version 1.2, STSC Inc., Rockville, MD) and specifically written programmes for fitting mixture model curves (R. Young, personal communication) . Single-well ELISA scores were normalised by a log-transformation for fitting mixture model curves. The mean and variance of the log-transformed single-well ELISA scores were estimated from the 510 sera from BLV-free herds. These parameter estimates were then used in a non-linear technique to fit mixture model curves to the 230 log-transformed single-well ELISA scores from BLV-infected herds and the mean and variance of these scores were estimated. The resultant mixture of two normal distributions appeared to fit the bimodal data from BLV-
51
infected herds well, and the amount of overlap of the two populations could be assessed. Agreement between the dual-well ELISA and the AGID test was calculated both as a percent agreement, and a kappa statistic, which measures the level of agreement beyond chance alone (Fleiss, 1981). For this purpose all results were classified as either positive or negative. For the AGID test, strong and weak positive sera were grouped as positive, while clear negative and equivocal sera were classified as negative. For the dual-well ELISA, suspicious results were included with the negatives. Point estimates for sensitivity and specificity of the dual-well ELISA, relative to the AGID test, were calculated as described by Baldock (1988). Exact binomial confidence intervals for sensitivities and specificities were calculated by the method of Rothman and Boyce (1982).
Results
Characterization of the ELISA antigen Antigenic components of crude AGID, Con-A non-binding and p24-ELISA antigens were compared with purified virus in immunoblots using either BLV positive bovine serum or anti-bovine IgG conjugate only (Fig. 1). When diluted in 1% gelatin, the anti-bovine IgG conjugate detected bovine immunoglobulin components of the growth medium in both the AGID and Con-A non-binding antigens, as observed previously (Walker et al., 1987), but not in the p24-ELISA antigen. The use of 0.5% skim milk powder as conjugate diluent in immunoblots with BLV-
12
3,4
5
6
7
6
9P51 IgG H
Fig. 1. Protein immunoblots of BLV antigen preparations. Immunodetections were performed with BLV positive (lanes 1,>5) and negative (lane 2) bovine serum, using serum and anti-bovine IgG conjugate diluted in 0.5% skim milk powder, or with conjugate only diluted in 1% gelatin (lanes 6-8). Antigen preparations compared were purified virus (lanes 1 and 2) AGID antigen (lanes 3 and 6) Con-A sepharose non-binding antigen (lanes 4 and 7) and p24 ELISA antigen (lanes 5 and 8). IgCi-H and IgG-L represent immunoglobulin heavy and light chains, respectively.
52
positive serum suppressed these reactions so that p24 was clearly detected in all three antigen preparations. In addition, reactivity occurred in the region of gp51 in the AGID antigen. This was reduced significantly in the Con-A non-binding and p24-ELISA antigens. As similar reactivity occurred with these two antigens in immunoblots with negative bovine serum (data not shown), it was assumed to be nonspecific. Crude BLV antigens prepared from culture supernates have been found to give poor reactions in immunoblots with gp51 (Walker et al., 1987). For this reason, AGID tests were performed with BLV-positive serum to confirm that the p24ELISA antigen was free of gp51 (data not shown). Serum dilution A set of 8 bovine sera which gave negative, weak positive or strong positive reactions in immunoblots with p24 were compared in the single-well ELISA over a series of dilutions from undiluted to l/50 (Fig. 2). Good differentiation of positive and negative sera was maintained up to a dilution of l/10, although very low dilutions tended to cause erratic results. At higher dilutions the distinction between weak positive and negative sera was poor. A serum dilution of l/5 was selected for use in the ELISA. Comparison of single-well p24-ELISA and gp51-AGID test The distribution of ELISA scores for 230 sera from BLV-infected herds and 510 sera from BLV-free herds is shown in Fig. 3. The frequency distribution of log-
Serum
dilution
Fig. 2. Influence of serum dilution on differentiation of three AGID negative (a), three weak positive (a) and hvo strong positive (A) sera in the single-well p24-ELBA.
53
-=----I 3
25
Fig. 3. Distribution of scores in the single-well p24-ELISA of (a) 510 sera from BLV-free herds and (b) 230 sera from BLV-infected herds which gave (1) strong positive, (2) weak positive, (3) equivocal and (4) clear negative results in AGID tests.
transformed ELISA scores for the sera from BLV-free herds was unimodal and normal. In contrast, the corresponding distribution for the sera from BLV-infected herds was bimodal, indicating two overlapping normal distributions. Fitting of mixture model curves indicated that there was excessive overlap of scores for AGID positive and AGID negative sera from infected herds. The good fit of the normal curve parameter estimates from the sera from BLV-free herds to the first part of the bimodal distribution indicated that this was due to a lack of specificity in the single-well ELISA, rather than superior sensitivity allowing the detection of infected cattle in the AGID negative group. To.coniirm this interpretation, the 19 AGID negative sera with ELISA scores of 850%, from both infected and uninfected herds, were tested for p24 antibody in immunoblots (data not shown). All, except one serum from an infected herd, were negative. That serum had an ELISA score of 56.1% and gave a weak positive result. In an attempt to establish the cause of poor specificity in the single-well ELISA, the immunoblot reactions were compared with the ELISA antigen of 16 sera from BLV-free herds, of which 11 gave high and 5 gave low ELISA scores. No consistent non-specific reactions were observed in high score sera (data not shown). However, by modifying the ELISA format it was possible to demonstrate that most non-specific reactions were due to the direct binding of serum components to the plate, irrespective of the presence of antigen or whether a step was included to block vacant binding sites prior to the addition of serum (data not shown).
54
OIIIL---I 0
2570.-
75
100
125
150
ELlSA Score Fig. 4. Distribution of scores in the dual-well p24-ELISA of 230 sera from BLV-infected herds which gave (1) strong positive, (2) weak positive, (3) equivocal and (4) clear negative results in AGID tests.
Comparison
of dual-well p24-ELISA
and gp.51-AGID
test
In the dual-well ELISA, the contribution of non-specific binding was measured for each serum in a well containing no antigen and the absorbance subtracted from that obtained in an antigen-coated well. The group of 230 sera from BLV-infected herds was again tested in a blind trial using this modified, dual-well format ELISA. The distribution of ELISA scores is shown in Fig. 4. Scores of 25%)
uncertain (20-25%) negative (
47
Elsevier VIRMET 00998
An enzyme-linked immunosorbent assay for detection of bovine leukaemia virus p24 antibody in cattle John B. Molloy’, Peter J. Walker’, F. Chris Baldock’, Barry J. Rodwell’ and Jeff A. Cowley’ ‘Queensland Department of Primary Indusm’es, Animal Research Institute, Yeerongpilly, Australia and 2CSIR0 Divbion of Tropical Animal Production, Indooroopilly, Alrrmzlia
(Accepted 30 November 1989)
Summary An ELISA for detecting antibody to the bovine leukaemia virus (BLV) core protein p24 is described. The test uses p24 antigen purified from concentrated cell culture supemate by lectin-affinity chromatography and gel tiltration. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection relative to the gp51 agar gel immunodiffusion test, were 98.1 and %.7%, respectively. In the event of widespread use of gp51 based vaccines, the p24-ELISA should differentiate effectively between naturally infected and vaccinated animals. Bovine leukemia virus; Antibody, ~24; Serological test; ELISA
Enzootic bovine leukaemia (EBL) is an infectious lymphoproliferative disease of cattle with worldwide distribution (Ferrer, 1980). The infectious agent, bovine leukaemia virus (BLV), is an exogenous retrovirus (Miller et al., 1969) which infects B-lymphocytes and establishes a persistent infection by integration of proviral DNA into the host chromosomes (Kettmann et al., 1979). The majority of infected cattle show no clinical signs of disease, but up to 30% develop persistent lymphocytosis with a small proportion progressing to lymphosarcoma (Ferrer, 1980; Correspondence to: J.B. Molloy, Queensland Department of Primary Industries, Animal Research Institute, 665 Fairfield Road, Yeerongpilly, 4105, Australia.
0166-0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
48
Burny et al., 1978). Cattle infected with BLV produce antiviral antibodies throughout the term of the lifelong infection (Portetelle et al., 1983). Future strategies for the control of EBL may utilize vaccination with subunit preparations of the major viral surface glycoprotein (gp51), which has been shown to induce protective antibodies (Onuma et al., 1984; Kono et al., 1986). With extensive use of a gp51 vaccine, the widely accepted agar gel immunodiffusion (AGID) test for gp51 antibody (Miller and Van der Maaten, 1976) would become unsuitable for the serological diagnosis of BLV infection. Several sensitive serological tests for antibody to the major viral core protein (~24) have been reported (Gauthier et al., 1982; Walker et al., 1987; De Boer et al., 1987). Of these, radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) techniques are suitable for large scale testing, but require either high quality, radiolabelled antigen or monoclonal antibodies, both of which are expensive to prepare and not readily available. In this paper, we describe an ELISA based on BLV p24 antigen purified from tissue culture supernate. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection were determined relative to the gp51-AGID test.
Materials and Methods Preparation of ACID
antigen
Antigen containing both gp51 and p24 was prepared from the culture supernate of foetal lamb kidney (FLK) cells persistently infected with an Australian isolate of BLV (Chung, 1980; Chung et al., 1984), concentrated lOO-fold using hollow fibre ultrafiltration as described previously (Walker et al., 1987). This antigen was used for AGID tests. Preparation of ELZSA antigen
ELISA antigen was prepared from AGID antigen using lectin-affinity chromatography and gel filtration. Con A-Sepharose chromatography, as described by Miller and Van der Maaten (1976) and modified by Walker et al. (1987), was used to remove gp51 from the AGID antigen. The p24 in the Con A non-binding fraction was concentrated about 15-fold by ultrafiltration (Amicon YMIO membrane) and further purified by gel filtration chromatography on a 750 cm x 2.6 cm Sephadex G-75 Superfine (Pharmacia) column. A 1 ml volume was loaded onto a column previously equilibrated with 20 mM Tris-HCl (pH 7.2), 150 mM NaCl (flow rate 10 ml/h). Fractions containing p24 were identified by SDS-PAGE and immunoblotting with immune sheep serum, pooled and stored at -20°C until required.
49
Virus purification BLV was purified from the culture supernate of persistently infected FLK cells by modifications of methods described previously (Deshayes et al., 1977; Walker et al., 1987). Briefly, culture supemate was clarified by centrifugation at 4000 X g for 45 min and dialysed against half-strength PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HP04, 1.1 mM KH2P0,, pH 7.2) for 16 h at 4°C. The clarified supemate was then dialysed against solid polyethyleneglycol 6000 for 6 h to achieve a 20-fold concentration and again for 16 h against half-strength PBS. This material was applied to a discontinuous gradient of 15% and 40% (w/w) sucrose in NTE buffer (100 mM NaCl, 1 mM EDTA, 10 mM TRIS-HCl pH 7.4) and centrifuged at 100000 x g for 90 min at 4°C in a Beckman SW28 rotor. The band at the interface was harvested and applied to an identical gradient to achieve a two-fold concentration. This preparation was applied to a continuous 15% to 50% (w/w) linear sucrose gradient and centrifuged at 100000 x g for 16 h at 4°C (Beckman SW28 rotor). A visible band at a buoyant density of 1.155 g/ml was harvested and the purified virus pelleted through 15% (w/w) sucrose at 100000 x g for 90 min at 4°C (Beckman SW28 rotor), resuspended in 15% w/w sucrose/NTE (protein concentration = 2 to 3 mg/ml) and stored at -70°C until required.
ACID test AGID tests were conducted using the method of Miller and Van der Maaten (1976) in 0.9% Agarose A (Pharmacia) containing 8.5% NaCl. SDS-PAGE and immunoblom’ng Discontinuous SDS-polyacrylamide gels (Laemmli, 1970) were prepared as described previously (Walker et al., 1987). Immunoblotting tests for BLV antibody were conducted using 60 pg of purified virus applied to a single 75 mm wide slot of a 0.75 mm thickness vertical mini-gel (BioRad). The immunodetection procedure was as described previously (Walker et al., 1987), except that 5% non-dairy coffee whitener (Carnation) was substituted for skim milk powder or gelatin for screening bovine sera and that biotinylated goat anti-bovine IgG (KPL) or biotinylated donkey anti-sheep IgG (Amersham) were used at dilutions of l/50 and l/200, respectively. Single-well ELISA A 100 u.1volume of the partially purified BLV p24 antigen (protein concentration = 0.36 mg/ml), diluted l/50 in 50 mM carbonate buffer (pH 9.6) was adsorbed onto each well of 96-well polyvinyl microtitre plates (Costar) for 16 h at 4°C. Plates were then washed (1 x 5 min) with 0.1% Tween-20 in PBS. Sera diluted l/5 in 1% gelatin, 0.1% Tween-20 in PBS were added (100 u.1per well) and incubated at 37°C for 2 h. Plates were washed (3 X 5 min) with 0.1% Tween-20
50
in PBS, 100 ~1 of goat anti-bovine IgG peroxidase conjugate (KPL) diluted l/l 300 in 1% gelatin, 0.1% Tween-20 in PBS added and incubated at 37°C for 1 h. Washing was repeated, 100 l.~lof peroxidase substrate ABTS (KPL) added, and after 20 min at room temperature, the differential absorbance (4141492 nm) was measured using a dual-beam microtitre plate reader (Titertek Multiscan MC). A standard BLV positive serum was included in triplicate on each plate and each test serum was assayed in triplicate on different plates to account for effects due to well and plate variability. ELISA scores were calculated as a percentage of the standard positive serum and averaged over the three assays. Dual-well ELISA
The procedure for the dual-well ELISA was as described for the single-well ELISA except that either antigen or carbonate buffer only was added to alternate wells of 96-well microtitre plates. Sera were incubated in pairs of wells with and without antigen and the difference in absorbance calculated. Test sera were assayed in duplicate on different plates and ELISA scores calculated as a percentage of the standard positive serum.
A group of 510 bovine sera, taken from 34 herds which had been free of BLV antibody in at least three consecutive AGID tests over a period of 18 months, was used to estimate the specificity of the single-well p24-ELISA. A group of 230 bovine sera from 15 herd submissions for diagnostic testing was tested in blind trials in both the single-well and dual-well p24-ELISA. These were selected from routine diagnostic submissions on the basis of AGID test results, and contained 72 strong positive, 35 weak positive, 115 clear negative and 8 equivocal sera. Data management
and analysis
Data were stored and manipulated using a microcomputer spreadsheet package (SuperCalc 3, Version 2.1, Computer Associates International Inc., San Jose, CA). Statistical analyses were performed using a microcomputer statistics package (Statgraphics, Version 1.2, STSC Inc., Rockville, MD) and specifically written programmes for fitting mixture model curves (R. Young, personal communication) . Single-well ELISA scores were normalised by a log-transformation for fitting mixture model curves. The mean and variance of the log-transformed single-well ELISA scores were estimated from the 510 sera from BLV-free herds. These parameter estimates were then used in a non-linear technique to fit mixture model curves to the 230 log-transformed single-well ELISA scores from BLV-infected herds and the mean and variance of these scores were estimated. The resultant mixture of two normal distributions appeared to fit the bimodal data from BLV-
51
infected herds well, and the amount of overlap of the two populations could be assessed. Agreement between the dual-well ELISA and the AGID test was calculated both as a percent agreement, and a kappa statistic, which measures the level of agreement beyond chance alone (Fleiss, 1981). For this purpose all results were classified as either positive or negative. For the AGID test, strong and weak positive sera were grouped as positive, while clear negative and equivocal sera were classified as negative. For the dual-well ELISA, suspicious results were included with the negatives. Point estimates for sensitivity and specificity of the dual-well ELISA, relative to the AGID test, were calculated as described by Baldock (1988). Exact binomial confidence intervals for sensitivities and specificities were calculated by the method of Rothman and Boyce (1982).
Results
Characterization of the ELISA antigen Antigenic components of crude AGID, Con-A non-binding and p24-ELISA antigens were compared with purified virus in immunoblots using either BLV positive bovine serum or anti-bovine IgG conjugate only (Fig. 1). When diluted in 1% gelatin, the anti-bovine IgG conjugate detected bovine immunoglobulin components of the growth medium in both the AGID and Con-A non-binding antigens, as observed previously (Walker et al., 1987), but not in the p24-ELISA antigen. The use of 0.5% skim milk powder as conjugate diluent in immunoblots with BLV-
12
3,4
5
6
7
6
9P51 IgG H
Fig. 1. Protein immunoblots of BLV antigen preparations. Immunodetections were performed with BLV positive (lanes 1,>5) and negative (lane 2) bovine serum, using serum and anti-bovine IgG conjugate diluted in 0.5% skim milk powder, or with conjugate only diluted in 1% gelatin (lanes 6-8). Antigen preparations compared were purified virus (lanes 1 and 2) AGID antigen (lanes 3 and 6) Con-A sepharose non-binding antigen (lanes 4 and 7) and p24 ELISA antigen (lanes 5 and 8). IgCi-H and IgG-L represent immunoglobulin heavy and light chains, respectively.
52
positive serum suppressed these reactions so that p24 was clearly detected in all three antigen preparations. In addition, reactivity occurred in the region of gp51 in the AGID antigen. This was reduced significantly in the Con-A non-binding and p24-ELISA antigens. As similar reactivity occurred with these two antigens in immunoblots with negative bovine serum (data not shown), it was assumed to be nonspecific. Crude BLV antigens prepared from culture supernates have been found to give poor reactions in immunoblots with gp51 (Walker et al., 1987). For this reason, AGID tests were performed with BLV-positive serum to confirm that the p24ELISA antigen was free of gp51 (data not shown). Serum dilution A set of 8 bovine sera which gave negative, weak positive or strong positive reactions in immunoblots with p24 were compared in the single-well ELISA over a series of dilutions from undiluted to l/50 (Fig. 2). Good differentiation of positive and negative sera was maintained up to a dilution of l/10, although very low dilutions tended to cause erratic results. At higher dilutions the distinction between weak positive and negative sera was poor. A serum dilution of l/5 was selected for use in the ELISA. Comparison of single-well p24-ELISA and gp51-AGID test The distribution of ELISA scores for 230 sera from BLV-infected herds and 510 sera from BLV-free herds is shown in Fig. 3. The frequency distribution of log-
Serum
dilution
Fig. 2. Influence of serum dilution on differentiation of three AGID negative (a), three weak positive (a) and hvo strong positive (A) sera in the single-well p24-ELBA.
53
-=----I 3
25
Fig. 3. Distribution of scores in the single-well p24-ELISA of (a) 510 sera from BLV-free herds and (b) 230 sera from BLV-infected herds which gave (1) strong positive, (2) weak positive, (3) equivocal and (4) clear negative results in AGID tests.
transformed ELISA scores for the sera from BLV-free herds was unimodal and normal. In contrast, the corresponding distribution for the sera from BLV-infected herds was bimodal, indicating two overlapping normal distributions. Fitting of mixture model curves indicated that there was excessive overlap of scores for AGID positive and AGID negative sera from infected herds. The good fit of the normal curve parameter estimates from the sera from BLV-free herds to the first part of the bimodal distribution indicated that this was due to a lack of specificity in the single-well ELISA, rather than superior sensitivity allowing the detection of infected cattle in the AGID negative group. To.coniirm this interpretation, the 19 AGID negative sera with ELISA scores of 850%, from both infected and uninfected herds, were tested for p24 antibody in immunoblots (data not shown). All, except one serum from an infected herd, were negative. That serum had an ELISA score of 56.1% and gave a weak positive result. In an attempt to establish the cause of poor specificity in the single-well ELISA, the immunoblot reactions were compared with the ELISA antigen of 16 sera from BLV-free herds, of which 11 gave high and 5 gave low ELISA scores. No consistent non-specific reactions were observed in high score sera (data not shown). However, by modifying the ELISA format it was possible to demonstrate that most non-specific reactions were due to the direct binding of serum components to the plate, irrespective of the presence of antigen or whether a step was included to block vacant binding sites prior to the addition of serum (data not shown).
54
OIIIL---I 0
2570.-
75
100
125
150
ELlSA Score Fig. 4. Distribution of scores in the dual-well p24-ELISA of 230 sera from BLV-infected herds which gave (1) strong positive, (2) weak positive, (3) equivocal and (4) clear negative results in AGID tests.
Comparison
of dual-well p24-ELISA
and gp.51-AGID
test
In the dual-well ELISA, the contribution of non-specific binding was measured for each serum in a well containing no antigen and the absorbance subtracted from that obtained in an antigen-coated well. The group of 230 sera from BLV-infected herds was again tested in a blind trial using this modified, dual-well format ELISA. The distribution of ELISA scores is shown in Fig. 4. Scores of 25%)
uncertain (20-25%) negative (
