Journal of Virological Methods. 30
VIRh4ET
( 1990) 109-114
01071
An ELISA-inhibition immunodeficiency
assay for antibody to human virus core antigen (~24)
Jacob Povolotsky, Jonathan W.M. Gold, Patricia Darminin, Nancy Chein, Penny Baron and Donald Armstrong The Infectious Diseuse Service ond Speciul Microbiology Luhorarory. Memorial Sloan-Kettering Cancer Center, New York. Nen* York, U.S.A. (Accepted
26
June
1990)
Summary An ELISA-inhibition assay based on commercially available HIV- 1 p24 antigen tests was devclopcd for detecting p24 antibodies. The test is specific and simple. p24 antibody was detectable in all p24 antigen negative Western blot positive sera, but was detectable infrequently in antigen positive sera. Sera from patients with indeterminate HIV-I reactions (individuals without evidence of HIV-I infection who nevertheless had p24 antibody) were p24 antigen negative and p24 antibody negative in the ELISA-inhibition reaction. HIV- 1 p24 antigen; HIV-I p24 antibody
Introduction The humoral immune response to HIV-l includes antibodies to the gag-gene protein (~24 core antigen) of the virus. Titers of p24 antibody are thought to decline in patients with AIDS and in HIV-1 infected patients who are at risk for progression of immune deficiency. (Allain et al., 1987; Andrieu et al., 1988; Frosner et al., 1987; Schiavini et al.. 1989; Sei et al., 1989; Webcr et al., 1987). Antibody reactive with gag or gag precursor is the most commonly found antibody in the sera of individuals with so-called indeterminate HIV-l antibody tests. Such individuals have not been shown to have infection with any known retrovirus. Establishing that such apparently sero-reactive people do not in fact have HIV-l infection is a fairly common and trying clinical problem. Correspond&e fo: J.W.M. Gold. The Infectious Disease Service and Special Microbiology Memorial Sloan-Kettering Cancer Center. 1275 York Ave.. New York, NY 10021, U.S.A.
0168-85 lO/9O/SO.l.S0@ 1990 Elscvier Science Publisher> B.V. (Biomedical
Division)
Laboratory.
110
Studies of HIV-l p24 antibodies have been performed using Western blot analysis or competition ELISA employing recombinant HIV-l core protein. (Cao et al., 1987; De Wolf et al., 1988; Goudsmit et al., 1987; Groopman et al., 1986; Mayer et al., 1987; Pedersen et al., 1987). We describe a test for detection of HIV-l p24 antibodies by ELISA-inhibition using commercially available HIV-l p24 core antigen kits.
Materials
and Methods
Serum specimens One hundred and fifty-three sera from 140 HIV-l infected individuals positive by ELISA and Western blot (104 people with asymptomatic HIV-l infection and 36 people with AIDS) were studied. Antibody to HIV-l was detected by ELISA (DuPont HIV-l ELISA, DuPont, Wilmington, DE 19898) and confirmed by Western blot (DuPont). HIV-l p24 antigen was detected by ELISA (Antibody to human immunodeficiency virus type 1 (HIV-l), Abbott Laboratories, Abbott Park, IL 60064).
ELBA inhibition assay for detection of HIV-l p-24 antibody Purified viral HIV- 1 p24 antigen (provided in the DuPont HIV p24 core antigen ELISA kits) and HIV-l p24 recombinant antigen (Micro Gen Sys, Inc., West Haven, CT 065 16) derived from an HIV- 1 gene fragment which codes for gag polyprotein including all of the amino acids of p24 were used. Both preparations of p24 antigen were diluted in PBS to contain 200 pg/ml (working solution). 200 ~1 of test serum was mixed with 200 ,~l of the antigen working solution. After thorough mixing, the mixture was incubated for 1 h at 37OC and 200 /-11 were added to a well of the p24 core antigen ELISA microtiter plate (HIV p24 Core Antigen Elisa, DuPont, Wilmington, DE 19898). If the Abbott kit was used the polystyrene bead coated with p24 antibodies was added to the mixture. The antigen control was prepared by mixing 500 ~1 of the working solution of p24 antigen with 500 ~1 of PBS. After thorough mixing the mixture was incubated for 1 h at 37°C and 200 ~1 were added to 3 wells. The negative control consisted of 200 ~1 of PBS which was added to 3 wells. Specimens and controls were then run in the p24 ELISA kits according to the manufacturer’s instructions. p24 antibodies present in a sample bind the p24 antigen and inhibit its detection, resulting in a reduction in OD compared to control antigen. The percent inhibition of the sample was calculated as: (A - S) x 100 (A - c> where A = average absorbance
value of 3 wells with antigen; S = absorbance
111
value of the mixture of antigen and a sample; and C = absorbance value of the negative control. We considered a sample positive for p24 antibody if the percent inhibition was 2 50%. This was based on the manufacturer’s (DuPont) recommendation for conformation of a p24 antigen test using a known positive antiserum. The p24 antibody titer was considered, to be the highest dilution of a serum which gave 50% inhibition.
Results p24 antibodies were detected by ELISA-inhibition in 84 (54.9%) of the 153 samples of HIV-l infected individuals (Table 1). In p24 antigen negative sera antibody was found in 79 of 91 samples (86.8%). In p24 antigen positive sera antibody was detected in only 5 of 62 samples (8.1%). In sera with 25-100 pg/ml of p24 antigen antibody could be detected in 4 of 27 (14.8%) samples. In sera with more than 100 pg/ml of p24 antigen, antibody was detected in only 1 of 35 samples (2.9%). Thus the ability to detect antibody by ELISA-inhibition appeared to correlate inversely with the presence of p24 antigen. Comparison of detection of anti-p24 with ELISA-inhibition with kits from different manufacturers showed that antibody was detected in 9.7% and 6.4% of antigen positive sera by, the DuPont and Abbott kits respectively. In p24 antigen negative sera antibody was detectable in 78.4% and 92.6% by each kit respectively. The median % inhibition for all positive sera tested was 74.4% with the DuPont kits and 88.3% with the Abbott kits. Titers of p24 antibodies in antigen negative sera ranged from 1:2 to 1:20 000. Titers of 1:2 were found in 10.3%, I:10 in 20.7%, 1:lOO in 34.5%, 1:lOOOand higher in 34.5%. Sera TABLE 1 p24 antibody and p24 antigen patterns in HIV-l infected individuals Kits and samples
No. p24 of antibody sera positive
Level of p24 antigen (pg/ml) 25-50 No
51-100 No. Antibody(+)
An&dy(+)
>I00 No. Antibody(+)
Dupont Sera with ~24 antigen Sera without p24 antigen Total
31 37 68
3 (9.7%) 29 (78.4%) 32 (47.1%)
7
1
9
1
15
I
Abbott Sera with ~24 antigen Sera without p24 antigen Total
31 54 85
2 (6.4%) 50 (92.6%) 52 (61.2%)
4
1
7
1
20
-
62 5 (8.1%) 91 79 (86.8%) 153 84 (54.9%)
11
Sera with p24 antigen Sera without ~24 antigen Total
2 (18.2%:6
2 35 (12.5%)
:.9%)
112
TABLE 2 Titers of HIV-1 p24 antibody in HIV-l infected individuals Sera
Number
Titers of ~24’ antibodies
Sera with p24 antigen Sera without p24 antigen Total
5 53 58
I:2 2 (40.0%) 4 (7.5%) 6 (10.3%)
I:10 3 (60.0%) 9 (17.0%) 12 (20.7%)
I:100 _ 20 (37.8%) 20 (34.5%)
>l:lOOO _ 20 (37.8%) 20 (34.5%)
with p24 antigen never had p24 antibody titers greater than 1: 10 (Table 2). All 42 p24 antigen negative, ELBA-inhibition positive sera were positive for p24 antibodies in Western blot analysis (Table 3). Twenty-four of 35 (68.5%) p24 antigen positive samples which were negative for p24 antibodies by ELISA inhibitionwere p24 antibody positive by Western blotting. Thus, in the absence of p24 serum antigen, p24 antibody is detectable at the same rate by both ELBA and Western blot, whereas in the presence of p24 antigen, the Western blot is more sensitive for detecting p24 antibody. None of 27 sera from people not infected with HIV-l were positive for antibody to p24 by ELBA-inhibition. None of 36 sera from patients with indeterminate HIV-l antibody tests who had p24 antibody by Western blot were positive by ELISA-inhibition. This suggests that the ELISA inhibition test may be useful for distinguishing individuals whose p24 antibody reaction is not related to HIV-l infection from those with HIV-l infection. p24 antigen from DuPont kits was compared to that of the Recombinant HIV-l p24 antigen (Micro Gene Sys, Inc.) in the ELISA inhibition assay by titration of 13 seropositive samples. The overall agreement was 100% in detection of p24 antibodies.
TABLE 3 Comparison of inhibition test and Western blot test for detection of p24 antibody Sera
Number
Western blot Positive
Positive inhibition test Sera of HIV-I infected persons without p24 antigen Negative inhibition test Sera of HIV-I infected persons with p24 antigen Negative inhibition test Sera of HIV-I uninfected people
Indeterminate
42
42 (100%)
-
35
24 (68%)
II (31.5%)
27
-
Negative
-
27 (100%)
113
Discussion An ELISA inhibition test is described for detection of HIV-l p24 antibodies using p24 core antigen ELISA. The frequency of finding p24 antibodies was 86.8% in sera without p24 antigen and 8.1% in sera with detectable p24 antigen. These frequencies agree with others found in p24 antibody studies which used ELISA competition methods. All patients who were positive by ELISA inhibition were also positive by Western blot analysis. Positive readings were not detected in samples which were negative in Western blot. The inhibition test showed an inverse correlation between HIV-l p24 antigenemia and levels of anti-p24 antibodies. Patients with indeterminate HIV-l antibody test results who were p24 antibody positive by Western blot were ELISA inhibition negative for p24 antibody. While the origin of such indeterminate reactions is not yet understood, such patients do not appear to be infected with HIV-l and a negative ELISAinhibition assay may provide additional information distinguishing them from HIV- 1 infected patients.
References Allain, J.-P., Laurian, Y., Paul, D.A., Verroust, F., Leuther, M., Gazengel C., Senn D., Larrieu M.-J. and Bosser C. (1987) Long-term evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. N. Engl. J. Med. 317, 1114-1121. Andrieu, J.-M., Eme, D., Venet, A., Audroin, C., Tourani, J.M., Stem, M., Israel-Biet, D., Beldjord, K., Driss, F. and Even, P. (1988) Serum HIV antigen and anti-P24-antibodies in 200 HIV seropositive patients: correlation with CD4 and CD8 lymphocyte subsets. Clin. Exp. Immunol. 73. l-5. Cao, Y., Valentine, F., Hojvat, S., Allain, J.P., Rubinstein, P., Mirabile, M., Czelusniak, S., Leuther, M., Baker, L. and Friedman-Kien, A.E. (1987) Detection of HIV antigen and specific antibodies to HIV core and envelope proteins in sera of patients with HIV infection. Blood 70, 575-578. De Wolf, F., Lange, J.M.A., Houweling, J.T.M., Coutinho, R.A., Schellekens, P.T., Van der Noordaa, J. and Goudsmit, J. (1988) Numbers of CD4+ cells and the levels of core antigens of and antibodies to the human immunodeficiency virus as predictors of AIDS among seropositive homosexual men. J. Infect. Dis. 158, 615-622. Frosner, G.G., Erfle, V., Mellert, W. and Hehlmann, R. (1987) Diagnostic significance of quantitative determination of HIV antibody specific for envelope and core proteins. Lancet i. 159-160. Goudsmit, J., Lange, J.M.A., Paul, D.A. and Dawson, G.J. (1987) Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J. Infect. Dis. 155, 558-560. Groopman, J.E., Chen, F.W., Hope, J.A., Andrews, J.M., Swift, R.L., Benton, C.V., Sullivan, J.L., Volberding. P.A., Sites, D.P., Landesman, S., Gold, J., Baker, L., Craven, D. and Boches, F.S. (1986) Serological characterization of HTLV-III infection in AIDS and related disorders. J. Infect. Dis. 153, 736-742. Mayer, K.H., Falk, L.A., Paul, D.A., Dawson, G.J., Stoddard, A.M., McCusker. J.. Saltzman, S.P., Moon. M.W., Ferriani, R. and Groopman, J.E. (1987) Correlation of enzyme-linked immunosorbent assays for serum human immunodeficiency virus antigen and antibodies to recombinant viral proteins with subsequent clinical outcomes in a cohort of asymptomatic homosexual men. Am. J. Med. 83, 208-2 12. Pedersen, C., Nielsen, C.M.. Vestergaard, B.F., Gerstoft, J., Krogsgaard, K. and Nielsen, J.O. (1987) Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection. Br. Med. J. 295, 567-569. Schiavini, D.G., Puel, J.M.L.. Averous, S.A. and Bazex. J.A. (1989) Quantitative Western immunoblot-
114
ting analysis in survey of human immunodeficiency virus-seropositive patients. J. Clin. Microbial. 21. 2062-2066. Sei, Y., Tsang, P.H., Chu, F.N., Wallace, I., Roboz, J.P.. Sarin. P.S. and Bekesi, J.G. (1989) Inverse relationship between HIV-I. p24 antigenemia, anti-p24 antibody and neutralizing antibody response in all stages of HIV-l infection. Immunol. Lett. 20, 223-230. Weber, J.N., Weiss, R.A., Roberts, C., Weller, I., Tedder, R.S., Clapham, P.R., Parker, D., Duncan, J., Came, C., Pinching, A.J. and Cheingsong-Popov, R. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralising sera and association of anti-GAG antibody with prognosis. Lancet i, 119-122.
VIRh4ET
( 1990) 109-114
01071
An ELISA-inhibition immunodeficiency
assay for antibody to human virus core antigen (~24)
Jacob Povolotsky, Jonathan W.M. Gold, Patricia Darminin, Nancy Chein, Penny Baron and Donald Armstrong The Infectious Diseuse Service ond Speciul Microbiology Luhorarory. Memorial Sloan-Kettering Cancer Center, New York. Nen* York, U.S.A. (Accepted
26
June
1990)
Summary An ELISA-inhibition assay based on commercially available HIV- 1 p24 antigen tests was devclopcd for detecting p24 antibodies. The test is specific and simple. p24 antibody was detectable in all p24 antigen negative Western blot positive sera, but was detectable infrequently in antigen positive sera. Sera from patients with indeterminate HIV-I reactions (individuals without evidence of HIV-I infection who nevertheless had p24 antibody) were p24 antigen negative and p24 antibody negative in the ELISA-inhibition reaction. HIV- 1 p24 antigen; HIV-I p24 antibody
Introduction The humoral immune response to HIV-l includes antibodies to the gag-gene protein (~24 core antigen) of the virus. Titers of p24 antibody are thought to decline in patients with AIDS and in HIV-1 infected patients who are at risk for progression of immune deficiency. (Allain et al., 1987; Andrieu et al., 1988; Frosner et al., 1987; Schiavini et al.. 1989; Sei et al., 1989; Webcr et al., 1987). Antibody reactive with gag or gag precursor is the most commonly found antibody in the sera of individuals with so-called indeterminate HIV-l antibody tests. Such individuals have not been shown to have infection with any known retrovirus. Establishing that such apparently sero-reactive people do not in fact have HIV-l infection is a fairly common and trying clinical problem. Correspond&e fo: J.W.M. Gold. The Infectious Disease Service and Special Microbiology Memorial Sloan-Kettering Cancer Center. 1275 York Ave.. New York, NY 10021, U.S.A.
0168-85 lO/9O/SO.l.S0@ 1990 Elscvier Science Publisher> B.V. (Biomedical
Division)
Laboratory.
110
Studies of HIV-l p24 antibodies have been performed using Western blot analysis or competition ELISA employing recombinant HIV-l core protein. (Cao et al., 1987; De Wolf et al., 1988; Goudsmit et al., 1987; Groopman et al., 1986; Mayer et al., 1987; Pedersen et al., 1987). We describe a test for detection of HIV-l p24 antibodies by ELISA-inhibition using commercially available HIV-l p24 core antigen kits.
Materials
and Methods
Serum specimens One hundred and fifty-three sera from 140 HIV-l infected individuals positive by ELISA and Western blot (104 people with asymptomatic HIV-l infection and 36 people with AIDS) were studied. Antibody to HIV-l was detected by ELISA (DuPont HIV-l ELISA, DuPont, Wilmington, DE 19898) and confirmed by Western blot (DuPont). HIV-l p24 antigen was detected by ELISA (Antibody to human immunodeficiency virus type 1 (HIV-l), Abbott Laboratories, Abbott Park, IL 60064).
ELBA inhibition assay for detection of HIV-l p-24 antibody Purified viral HIV- 1 p24 antigen (provided in the DuPont HIV p24 core antigen ELISA kits) and HIV-l p24 recombinant antigen (Micro Gen Sys, Inc., West Haven, CT 065 16) derived from an HIV- 1 gene fragment which codes for gag polyprotein including all of the amino acids of p24 were used. Both preparations of p24 antigen were diluted in PBS to contain 200 pg/ml (working solution). 200 ~1 of test serum was mixed with 200 ,~l of the antigen working solution. After thorough mixing, the mixture was incubated for 1 h at 37OC and 200 /-11 were added to a well of the p24 core antigen ELISA microtiter plate (HIV p24 Core Antigen Elisa, DuPont, Wilmington, DE 19898). If the Abbott kit was used the polystyrene bead coated with p24 antibodies was added to the mixture. The antigen control was prepared by mixing 500 ~1 of the working solution of p24 antigen with 500 ~1 of PBS. After thorough mixing the mixture was incubated for 1 h at 37°C and 200 ~1 were added to 3 wells. The negative control consisted of 200 ~1 of PBS which was added to 3 wells. Specimens and controls were then run in the p24 ELISA kits according to the manufacturer’s instructions. p24 antibodies present in a sample bind the p24 antigen and inhibit its detection, resulting in a reduction in OD compared to control antigen. The percent inhibition of the sample was calculated as: (A - S) x 100 (A - c> where A = average absorbance
value of 3 wells with antigen; S = absorbance
111
value of the mixture of antigen and a sample; and C = absorbance value of the negative control. We considered a sample positive for p24 antibody if the percent inhibition was 2 50%. This was based on the manufacturer’s (DuPont) recommendation for conformation of a p24 antigen test using a known positive antiserum. The p24 antibody titer was considered, to be the highest dilution of a serum which gave 50% inhibition.
Results p24 antibodies were detected by ELISA-inhibition in 84 (54.9%) of the 153 samples of HIV-l infected individuals (Table 1). In p24 antigen negative sera antibody was found in 79 of 91 samples (86.8%). In p24 antigen positive sera antibody was detected in only 5 of 62 samples (8.1%). In sera with 25-100 pg/ml of p24 antigen antibody could be detected in 4 of 27 (14.8%) samples. In sera with more than 100 pg/ml of p24 antigen, antibody was detected in only 1 of 35 samples (2.9%). Thus the ability to detect antibody by ELISA-inhibition appeared to correlate inversely with the presence of p24 antigen. Comparison of detection of anti-p24 with ELISA-inhibition with kits from different manufacturers showed that antibody was detected in 9.7% and 6.4% of antigen positive sera by, the DuPont and Abbott kits respectively. In p24 antigen negative sera antibody was detectable in 78.4% and 92.6% by each kit respectively. The median % inhibition for all positive sera tested was 74.4% with the DuPont kits and 88.3% with the Abbott kits. Titers of p24 antibodies in antigen negative sera ranged from 1:2 to 1:20 000. Titers of 1:2 were found in 10.3%, I:10 in 20.7%, 1:lOO in 34.5%, 1:lOOOand higher in 34.5%. Sera TABLE 1 p24 antibody and p24 antigen patterns in HIV-l infected individuals Kits and samples
No. p24 of antibody sera positive
Level of p24 antigen (pg/ml) 25-50 No
51-100 No. Antibody(+)
An&dy(+)
>I00 No. Antibody(+)
Dupont Sera with ~24 antigen Sera without p24 antigen Total
31 37 68
3 (9.7%) 29 (78.4%) 32 (47.1%)
7
1
9
1
15
I
Abbott Sera with ~24 antigen Sera without p24 antigen Total
31 54 85
2 (6.4%) 50 (92.6%) 52 (61.2%)
4
1
7
1
20
-
62 5 (8.1%) 91 79 (86.8%) 153 84 (54.9%)
11
Sera with p24 antigen Sera without ~24 antigen Total
2 (18.2%:6
2 35 (12.5%)
:.9%)
112
TABLE 2 Titers of HIV-1 p24 antibody in HIV-l infected individuals Sera
Number
Titers of ~24’ antibodies
Sera with p24 antigen Sera without p24 antigen Total
5 53 58
I:2 2 (40.0%) 4 (7.5%) 6 (10.3%)
I:10 3 (60.0%) 9 (17.0%) 12 (20.7%)
I:100 _ 20 (37.8%) 20 (34.5%)
>l:lOOO _ 20 (37.8%) 20 (34.5%)
with p24 antigen never had p24 antibody titers greater than 1: 10 (Table 2). All 42 p24 antigen negative, ELBA-inhibition positive sera were positive for p24 antibodies in Western blot analysis (Table 3). Twenty-four of 35 (68.5%) p24 antigen positive samples which were negative for p24 antibodies by ELISA inhibitionwere p24 antibody positive by Western blotting. Thus, in the absence of p24 serum antigen, p24 antibody is detectable at the same rate by both ELBA and Western blot, whereas in the presence of p24 antigen, the Western blot is more sensitive for detecting p24 antibody. None of 27 sera from people not infected with HIV-l were positive for antibody to p24 by ELBA-inhibition. None of 36 sera from patients with indeterminate HIV-l antibody tests who had p24 antibody by Western blot were positive by ELISA-inhibition. This suggests that the ELISA inhibition test may be useful for distinguishing individuals whose p24 antibody reaction is not related to HIV-l infection from those with HIV-l infection. p24 antigen from DuPont kits was compared to that of the Recombinant HIV-l p24 antigen (Micro Gene Sys, Inc.) in the ELISA inhibition assay by titration of 13 seropositive samples. The overall agreement was 100% in detection of p24 antibodies.
TABLE 3 Comparison of inhibition test and Western blot test for detection of p24 antibody Sera
Number
Western blot Positive
Positive inhibition test Sera of HIV-I infected persons without p24 antigen Negative inhibition test Sera of HIV-I infected persons with p24 antigen Negative inhibition test Sera of HIV-I uninfected people
Indeterminate
42
42 (100%)
-
35
24 (68%)
II (31.5%)
27
-
Negative
-
27 (100%)
113
Discussion An ELISA inhibition test is described for detection of HIV-l p24 antibodies using p24 core antigen ELISA. The frequency of finding p24 antibodies was 86.8% in sera without p24 antigen and 8.1% in sera with detectable p24 antigen. These frequencies agree with others found in p24 antibody studies which used ELISA competition methods. All patients who were positive by ELISA inhibition were also positive by Western blot analysis. Positive readings were not detected in samples which were negative in Western blot. The inhibition test showed an inverse correlation between HIV-l p24 antigenemia and levels of anti-p24 antibodies. Patients with indeterminate HIV-l antibody test results who were p24 antibody positive by Western blot were ELISA inhibition negative for p24 antibody. While the origin of such indeterminate reactions is not yet understood, such patients do not appear to be infected with HIV-l and a negative ELISAinhibition assay may provide additional information distinguishing them from HIV- 1 infected patients.
References Allain, J.-P., Laurian, Y., Paul, D.A., Verroust, F., Leuther, M., Gazengel C., Senn D., Larrieu M.-J. and Bosser C. (1987) Long-term evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. N. Engl. J. Med. 317, 1114-1121. Andrieu, J.-M., Eme, D., Venet, A., Audroin, C., Tourani, J.M., Stem, M., Israel-Biet, D., Beldjord, K., Driss, F. and Even, P. (1988) Serum HIV antigen and anti-P24-antibodies in 200 HIV seropositive patients: correlation with CD4 and CD8 lymphocyte subsets. Clin. Exp. Immunol. 73. l-5. Cao, Y., Valentine, F., Hojvat, S., Allain, J.P., Rubinstein, P., Mirabile, M., Czelusniak, S., Leuther, M., Baker, L. and Friedman-Kien, A.E. (1987) Detection of HIV antigen and specific antibodies to HIV core and envelope proteins in sera of patients with HIV infection. Blood 70, 575-578. De Wolf, F., Lange, J.M.A., Houweling, J.T.M., Coutinho, R.A., Schellekens, P.T., Van der Noordaa, J. and Goudsmit, J. (1988) Numbers of CD4+ cells and the levels of core antigens of and antibodies to the human immunodeficiency virus as predictors of AIDS among seropositive homosexual men. J. Infect. Dis. 158, 615-622. Frosner, G.G., Erfle, V., Mellert, W. and Hehlmann, R. (1987) Diagnostic significance of quantitative determination of HIV antibody specific for envelope and core proteins. Lancet i. 159-160. Goudsmit, J., Lange, J.M.A., Paul, D.A. and Dawson, G.J. (1987) Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J. Infect. Dis. 155, 558-560. Groopman, J.E., Chen, F.W., Hope, J.A., Andrews, J.M., Swift, R.L., Benton, C.V., Sullivan, J.L., Volberding. P.A., Sites, D.P., Landesman, S., Gold, J., Baker, L., Craven, D. and Boches, F.S. (1986) Serological characterization of HTLV-III infection in AIDS and related disorders. J. Infect. Dis. 153, 736-742. Mayer, K.H., Falk, L.A., Paul, D.A., Dawson, G.J., Stoddard, A.M., McCusker. J.. Saltzman, S.P., Moon. M.W., Ferriani, R. and Groopman, J.E. (1987) Correlation of enzyme-linked immunosorbent assays for serum human immunodeficiency virus antigen and antibodies to recombinant viral proteins with subsequent clinical outcomes in a cohort of asymptomatic homosexual men. Am. J. Med. 83, 208-2 12. Pedersen, C., Nielsen, C.M.. Vestergaard, B.F., Gerstoft, J., Krogsgaard, K. and Nielsen, J.O. (1987) Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection. Br. Med. J. 295, 567-569. Schiavini, D.G., Puel, J.M.L.. Averous, S.A. and Bazex. J.A. (1989) Quantitative Western immunoblot-
114
ting analysis in survey of human immunodeficiency virus-seropositive patients. J. Clin. Microbial. 21. 2062-2066. Sei, Y., Tsang, P.H., Chu, F.N., Wallace, I., Roboz, J.P.. Sarin. P.S. and Bekesi, J.G. (1989) Inverse relationship between HIV-I. p24 antigenemia, anti-p24 antibody and neutralizing antibody response in all stages of HIV-l infection. Immunol. Lett. 20, 223-230. Weber, J.N., Weiss, R.A., Roberts, C., Weller, I., Tedder, R.S., Clapham, P.R., Parker, D., Duncan, J., Came, C., Pinching, A.J. and Cheingsong-Popov, R. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralising sera and association of anti-GAG antibody with prognosis. Lancet i, 119-122.
