556
APPLICATIONS
[64]
Acknowledgments This work was performed by the author while at Infectious Diseases Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health. The continuous support, at the time, by Drs. John L. Sever and DavidL. Madden is greatly appreciated.
[64] S e n s i t i v e I m m u n o a s s a y for H u m a n I m m u n o d e f i c i e n c y Viral C o r e P r o t e i n s By KENNETH
R. HUSKINS, K E V I N J. REAGAN, JOHN A . WEHRLY, and JEANNE E. NEUMANN
The use of avidin-biotin technology for immunochemical research applications has been gaining steady acceptance. In particular, the methodology has been used for immunoaffinity chromatography, 1,2 for assessment of antigen epitope specificities, 3 and for infectious disease immunoassaysd -7 Nearly all of the immunoassay applications have utilized enzyme-coupled signal generation to correlate with the specific analyte, although recently Hart and Taaffe 8 reported a chemiluminescent reporter system for immunoassay which utilizes acridinium ester-labeled streptavidin. The development of sensitive assays is a critical need in improving the detection, diagnosis, and treatment of disease. It is particularly important in diseases with such devastating ramifications as acquired immunodeficiency syndrome (AIDS). The currently used blood-screening tests are capable of detecting antibodies to human immunodeficiency virus (HIV), the causative viral agent of AIDS. However, these serological tests cannot determine whether or not the specimen actually contains viral proi T. V- Updyke and G. L. Nicolson, J. Immunol. Methods. 73, 83 (1984). 2 D. R. Gretch, M. Surer, and M. F. Stinski, Anal. Biochem. 163, 270 (1987). 3 C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. lmmunol. Methods 68,269 (1984). 4 J.-L. Guesdon, T. Ternynck, and S. Avrameas, J. Histoehem. Cytochem. 27, 1131 (1979). 5 K. Adler-Storthz, C. Kendall, R. C. Kennedy, R. D. Henkel, and G. R. Dreesman, J. Clin. Microbiol. 18, 1329 (1983). 6 L. S. Nerurkar, M. Namba, G. Brashears, A. J. Jacob, Y. J. Lee, and J. L. Sever, J. Clin. Mierobiol. 20, 109 (1984). 7 C. M. Nielsen, K. Hansen, H. M. K. Andersen, J. Gerstoft, and B. F. Vestergaard, J. Virol. Methods 16, 195 (1987). 8 R. C. Hart and L. R. Taaffe, J. Imrnunol. Methods 101, 91 (1987).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[64]
HIV COREPROTEINS
557
TABLE I Du PONT HIV p24 CORE ANTIGEN ELISA Step Antigen capture
Antigen detection Signal amplification
Features 200/.d Triton ® X-100-treated sample MicroweUs coated with rabbit anti-p24 IgG, incubation at room temperature overnight (standard assay) or at 37° for 2 hr (short assay) Biotinylated second rabbit anti-p24 IgG, incubation at 37° for 2 hr Streptavidin-HRP conjugate, incubation at 37° for 30 min o-Phenylenediamine/H202 substrates, 30-rain end-point absorbance (492 nm)
teins. For this reason, we have developed a sensitive, specific, and rapid immunoassay to measure HIV antigen. In this chapter we describe the assay format and detail its performance in various clinical settings. HIV p24 protein is immunologically distinct from the core protein of most other retroviruses. The preparation of monospecific antisera with high specificity and affinity for this viral protein and the use of a streptavidin-biotin amplification system have allowed Du Pont to develop an extremely sensitive ELISA for HIV p24 antigen. The Du Pont HIV p24 Core ELISA test achieves immunological detection of p24 released after sample lysis with 0.5% Triton ®X-100 under conditions gentle enough to retain reactivity. Highly specific rabbit polyclonal antisera provide the capture and detector immunoglobin reagents. Inactivated viral lysate (calibrated against immunoaffinity purified p24) serves as the standard. A biotinstreptavidin-horseradish peroxidase (HRP) couple provides the probe in a microtiter plate sandwich ELISA format. A description of the test format is outlined in Table I. Using this protocol, the amount of assay color generated is directly proportional to the quantity of HIV p24 captured from the specimen. The sensitivity of the p24 ELISA is 30 pg/ml; using a 200-/zl sample, the assay detection limit is 6 pg of p24. When the assay is used to detect viral antigen in supernatants of amplified cultures of peripheral blood lymphocytes, direct comparisons with the reverse transcriptase assay have shown an excellent correlation with additional advantages of increased sensitivity (up to 1000-fold) and reproducibility. A major reason for the excellent sensitivity of the p24 ELISA is the use of the biotin-streptavidin-HRP couple for signal detection. As shown in Table II, a 14-fold increase in specific signal was observed for the
558
APPLICATIONS
[64]
TABLE II COMPARISON OF RABBIT ANTI-p24 CONJUGATES IN ANTIGEN ASSAY SENSITIVITY p24 Antigen in sample (ng/ml)
Biotinylation b
Direct HRP ~
10 5 1.0 0.5 0.25
37.17 32.30 11.03 7.00 4.34
2.76 2.29 1.24 1.10 1.04
Signal-to-noise ratio ~
Conjugates were diluted in PBS-50% normal rabbit serum containing 0.01% thimerasol. b Biotinylated antibodies were subsequently reacted with streptavidin-HRP conjugates and o-phenylenediamine substrate. c HRP-antibody conjugate.
biotinylated rabbit anti-p24 in comparison to the direct antibody-enzyme conjugate. Other attempts at chemical conjugations involving alkaline phosphatase also failed to achieve the sensitivity of the biotin-streptavidin system. It was concluded that the highest analytical sensitivity for HIV p24 core protein required signal amplification using the indirect biotin-avidin methodology. When biotinylated antibodies are employed as immunoassay reagents, assessment of the isoelectic point is critical to the optimization of these reagents as ELISA components.9 The specificity of a positive color response generated in the HIV p24 ELISA is confirmed by specific inhibition of signal by preincubating the sample with human antibody to HIV p24. This procedure forces putative antigen in the sample into immune complexes that are not captured by the anti-p24 coated microplate wells. Then, on retesting in the p24 ELISA, reduction of the signal by at least 50% constitutes evidence of a true positive result. The p24 ELISA also detects free viral antigen without culture amplification from serum, plasma, cell lysates, and cerebrospinal fluid of individuals at high risk for developing AIDS. The significance of the presence of antigen in such human samples is the subject of ongoing research that is directed towards establishing diagnostic or prognostic links to clinical disease staging or patient management. 9 j. j. Wadsley and R. M. Watt, J. lmmunol. Methods 103, 1 (1987).
[64]
HIV COREPROTEINS
559
3.0
~" 2.0 ,0
tO c O"
CUTOFF
1.0
.01
.03
.05
.07 .09 Absorbance (492/620)
>.20
FIG. 1. HIV p24 core antigen EL1SA reactivity in a normal donor population In = 1049; 14 initial reactives (1.3%), 12 repeat reactives (1.1%)]. None of the reactive samples were true positives based on the confirmatory assay methodology (see text). The assay cutoff sensitivity is 0.3 ng/ml of HIV p24.
Our experience with large numbers of seronegative serum and plasma samples obtained from healthy normal donors is shown in Fig. 1. These data were collected using the standard (overnight) format and illustrate a tight absorbance distribution for the large majority of negative samples. An estimate of 1.1% (12 of 1049) for the assay false-positive rate was obtained from the repeat reactive samples that subsequently did not confinn. Extremely hemolysed, lipemic, and icteric samples may be assayed with no interference bias, although clear, nonhemolysed specimens are preferable whenever possible. A low percentage of rheumatoid factor and systemic lupus erythematosus plasma and serum specimens have given false-reactive results. Table III summarizes the results from a preclinical evaluation of the HIV p24 ELISA as a method for detecting serum antigen in individuals either known to be infected or at high risk for infection with HIV. In this limited, coded sample study, the prevalence of antigenemia in patients with AIDS or AIDS-related complex (ARC) was seen to be greater than 40%. Note that nearly all of these individuals were HIV-antibody seropositive (as determined by the Du Pont HIV Antibody ELISA). In other studies of late-stage AIDS patients with markedly depressed HIV antibody titers, the presence of antigen was found in greater than 70% of such
560
APPLICATIONS
[65]
TABLE III HIV ANTIGENEMIAIN AIDS, ARC, AND HIGH-RISKINDIVIDUALS
Disease/risk
Number
HIV Western blot positive
Confirmed p24 positive
AIDS ARC High-risk symptomatics High-risk asymptomatics
29 24 131 117
29 23 101 42
12 (41%) 11 (46%) 15 (11%) 2 (1.7%)
specimens. These results suggest that detection of circulating antigen in peripheral blood by immunoassay is more likely in seropositive individuals with advanced disease where reduced antibody titer likely limits the masking of antigen by endogenous immune complex formation. In conclusion, the use of the biotin-streptavidin-horseradish peroxidase couple for signal detection has contributed to the excellent sensitivity of the Du Pont HIV p24 core antigen ELISA. This assay has demonstrated clinical utility in a variety of settings and provides physicians with another tool in the overall effort for diagnosis and treatment of AIDS.
[65] Gene Probes B y MEIR WILCHEK a n d EDWARD A. BAYER
Owing to its potential for replacement of radioactive DNA probes, the use of the avidin-biotin system as DNA probes in nucleic acid research is very broad, as can be seen from Chapter [3]. Nonetheless, the number of new methods for introducing biotin into DNA is quite limited, and many of these are included in this volume (Chapters [66]-[72]). Most of the previous studies have used the procedure introduced by David Ward and co-workers in the early 1980s.~ Using this approach, a biotinyl derivative of dUTP is incorporated into a suitable DNA probe via nick translation. Following hybridization of the biotinylated probe with the target DNA, an avidin-associated probe is used for localization, isolation, and so on. The major difference in most of these studies is the target DNA, i.e., the organism involved, and for this reason we have not included such 1 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
APPLICATIONS
[64]
Acknowledgments This work was performed by the author while at Infectious Diseases Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health. The continuous support, at the time, by Drs. John L. Sever and DavidL. Madden is greatly appreciated.
[64] S e n s i t i v e I m m u n o a s s a y for H u m a n I m m u n o d e f i c i e n c y Viral C o r e P r o t e i n s By KENNETH
R. HUSKINS, K E V I N J. REAGAN, JOHN A . WEHRLY, and JEANNE E. NEUMANN
The use of avidin-biotin technology for immunochemical research applications has been gaining steady acceptance. In particular, the methodology has been used for immunoaffinity chromatography, 1,2 for assessment of antigen epitope specificities, 3 and for infectious disease immunoassaysd -7 Nearly all of the immunoassay applications have utilized enzyme-coupled signal generation to correlate with the specific analyte, although recently Hart and Taaffe 8 reported a chemiluminescent reporter system for immunoassay which utilizes acridinium ester-labeled streptavidin. The development of sensitive assays is a critical need in improving the detection, diagnosis, and treatment of disease. It is particularly important in diseases with such devastating ramifications as acquired immunodeficiency syndrome (AIDS). The currently used blood-screening tests are capable of detecting antibodies to human immunodeficiency virus (HIV), the causative viral agent of AIDS. However, these serological tests cannot determine whether or not the specimen actually contains viral proi T. V- Updyke and G. L. Nicolson, J. Immunol. Methods. 73, 83 (1984). 2 D. R. Gretch, M. Surer, and M. F. Stinski, Anal. Biochem. 163, 270 (1987). 3 C. Wagener, U. Fenger, B. R. Clark, and J. E. Shively, J. lmmunol. Methods 68,269 (1984). 4 J.-L. Guesdon, T. Ternynck, and S. Avrameas, J. Histoehem. Cytochem. 27, 1131 (1979). 5 K. Adler-Storthz, C. Kendall, R. C. Kennedy, R. D. Henkel, and G. R. Dreesman, J. Clin. Microbiol. 18, 1329 (1983). 6 L. S. Nerurkar, M. Namba, G. Brashears, A. J. Jacob, Y. J. Lee, and J. L. Sever, J. Clin. Mierobiol. 20, 109 (1984). 7 C. M. Nielsen, K. Hansen, H. M. K. Andersen, J. Gerstoft, and B. F. Vestergaard, J. Virol. Methods 16, 195 (1987). 8 R. C. Hart and L. R. Taaffe, J. Imrnunol. Methods 101, 91 (1987).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[64]
HIV COREPROTEINS
557
TABLE I Du PONT HIV p24 CORE ANTIGEN ELISA Step Antigen capture
Antigen detection Signal amplification
Features 200/.d Triton ® X-100-treated sample MicroweUs coated with rabbit anti-p24 IgG, incubation at room temperature overnight (standard assay) or at 37° for 2 hr (short assay) Biotinylated second rabbit anti-p24 IgG, incubation at 37° for 2 hr Streptavidin-HRP conjugate, incubation at 37° for 30 min o-Phenylenediamine/H202 substrates, 30-rain end-point absorbance (492 nm)
teins. For this reason, we have developed a sensitive, specific, and rapid immunoassay to measure HIV antigen. In this chapter we describe the assay format and detail its performance in various clinical settings. HIV p24 protein is immunologically distinct from the core protein of most other retroviruses. The preparation of monospecific antisera with high specificity and affinity for this viral protein and the use of a streptavidin-biotin amplification system have allowed Du Pont to develop an extremely sensitive ELISA for HIV p24 antigen. The Du Pont HIV p24 Core ELISA test achieves immunological detection of p24 released after sample lysis with 0.5% Triton ®X-100 under conditions gentle enough to retain reactivity. Highly specific rabbit polyclonal antisera provide the capture and detector immunoglobin reagents. Inactivated viral lysate (calibrated against immunoaffinity purified p24) serves as the standard. A biotinstreptavidin-horseradish peroxidase (HRP) couple provides the probe in a microtiter plate sandwich ELISA format. A description of the test format is outlined in Table I. Using this protocol, the amount of assay color generated is directly proportional to the quantity of HIV p24 captured from the specimen. The sensitivity of the p24 ELISA is 30 pg/ml; using a 200-/zl sample, the assay detection limit is 6 pg of p24. When the assay is used to detect viral antigen in supernatants of amplified cultures of peripheral blood lymphocytes, direct comparisons with the reverse transcriptase assay have shown an excellent correlation with additional advantages of increased sensitivity (up to 1000-fold) and reproducibility. A major reason for the excellent sensitivity of the p24 ELISA is the use of the biotin-streptavidin-HRP couple for signal detection. As shown in Table II, a 14-fold increase in specific signal was observed for the
558
APPLICATIONS
[64]
TABLE II COMPARISON OF RABBIT ANTI-p24 CONJUGATES IN ANTIGEN ASSAY SENSITIVITY p24 Antigen in sample (ng/ml)
Biotinylation b
Direct HRP ~
10 5 1.0 0.5 0.25
37.17 32.30 11.03 7.00 4.34
2.76 2.29 1.24 1.10 1.04
Signal-to-noise ratio ~
Conjugates were diluted in PBS-50% normal rabbit serum containing 0.01% thimerasol. b Biotinylated antibodies were subsequently reacted with streptavidin-HRP conjugates and o-phenylenediamine substrate. c HRP-antibody conjugate.
biotinylated rabbit anti-p24 in comparison to the direct antibody-enzyme conjugate. Other attempts at chemical conjugations involving alkaline phosphatase also failed to achieve the sensitivity of the biotin-streptavidin system. It was concluded that the highest analytical sensitivity for HIV p24 core protein required signal amplification using the indirect biotin-avidin methodology. When biotinylated antibodies are employed as immunoassay reagents, assessment of the isoelectic point is critical to the optimization of these reagents as ELISA components.9 The specificity of a positive color response generated in the HIV p24 ELISA is confirmed by specific inhibition of signal by preincubating the sample with human antibody to HIV p24. This procedure forces putative antigen in the sample into immune complexes that are not captured by the anti-p24 coated microplate wells. Then, on retesting in the p24 ELISA, reduction of the signal by at least 50% constitutes evidence of a true positive result. The p24 ELISA also detects free viral antigen without culture amplification from serum, plasma, cell lysates, and cerebrospinal fluid of individuals at high risk for developing AIDS. The significance of the presence of antigen in such human samples is the subject of ongoing research that is directed towards establishing diagnostic or prognostic links to clinical disease staging or patient management. 9 j. j. Wadsley and R. M. Watt, J. lmmunol. Methods 103, 1 (1987).
[64]
HIV COREPROTEINS
559
3.0
~" 2.0 ,0
tO c O"
CUTOFF
1.0
.01
.03
.05
.07 .09 Absorbance (492/620)
>.20
FIG. 1. HIV p24 core antigen EL1SA reactivity in a normal donor population In = 1049; 14 initial reactives (1.3%), 12 repeat reactives (1.1%)]. None of the reactive samples were true positives based on the confirmatory assay methodology (see text). The assay cutoff sensitivity is 0.3 ng/ml of HIV p24.
Our experience with large numbers of seronegative serum and plasma samples obtained from healthy normal donors is shown in Fig. 1. These data were collected using the standard (overnight) format and illustrate a tight absorbance distribution for the large majority of negative samples. An estimate of 1.1% (12 of 1049) for the assay false-positive rate was obtained from the repeat reactive samples that subsequently did not confinn. Extremely hemolysed, lipemic, and icteric samples may be assayed with no interference bias, although clear, nonhemolysed specimens are preferable whenever possible. A low percentage of rheumatoid factor and systemic lupus erythematosus plasma and serum specimens have given false-reactive results. Table III summarizes the results from a preclinical evaluation of the HIV p24 ELISA as a method for detecting serum antigen in individuals either known to be infected or at high risk for infection with HIV. In this limited, coded sample study, the prevalence of antigenemia in patients with AIDS or AIDS-related complex (ARC) was seen to be greater than 40%. Note that nearly all of these individuals were HIV-antibody seropositive (as determined by the Du Pont HIV Antibody ELISA). In other studies of late-stage AIDS patients with markedly depressed HIV antibody titers, the presence of antigen was found in greater than 70% of such
560
APPLICATIONS
[65]
TABLE III HIV ANTIGENEMIAIN AIDS, ARC, AND HIGH-RISKINDIVIDUALS
Disease/risk
Number
HIV Western blot positive
Confirmed p24 positive
AIDS ARC High-risk symptomatics High-risk asymptomatics
29 24 131 117
29 23 101 42
12 (41%) 11 (46%) 15 (11%) 2 (1.7%)
specimens. These results suggest that detection of circulating antigen in peripheral blood by immunoassay is more likely in seropositive individuals with advanced disease where reduced antibody titer likely limits the masking of antigen by endogenous immune complex formation. In conclusion, the use of the biotin-streptavidin-horseradish peroxidase couple for signal detection has contributed to the excellent sensitivity of the Du Pont HIV p24 core antigen ELISA. This assay has demonstrated clinical utility in a variety of settings and provides physicians with another tool in the overall effort for diagnosis and treatment of AIDS.
[65] Gene Probes B y MEIR WILCHEK a n d EDWARD A. BAYER
Owing to its potential for replacement of radioactive DNA probes, the use of the avidin-biotin system as DNA probes in nucleic acid research is very broad, as can be seen from Chapter [3]. Nonetheless, the number of new methods for introducing biotin into DNA is quite limited, and many of these are included in this volume (Chapters [66]-[72]). Most of the previous studies have used the procedure introduced by David Ward and co-workers in the early 1980s.~ Using this approach, a biotinyl derivative of dUTP is incorporated into a suitable DNA probe via nick translation. Following hybridization of the biotinylated probe with the target DNA, an avidin-associated probe is used for localization, isolation, and so on. The major difference in most of these studies is the target DNA, i.e., the organism involved, and for this reason we have not included such 1 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
