Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 May;35(5):1080–1089. doi: 10.1128/jcm.35.5.1080-1089.1997

Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay.

R J Awad 1, G E Corrigan 1, D H Ekstrand 1, R Thorstensson 1, C F Källander 1, J S Gronowitz 1
PMCID: PMC232707  PMID: 9114385

Abstract

Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms of primary infection. In addition, HIV-1 RTb-Ab was detected in the same recently infected individuals in most cases within 1 month and in some cases as early as 10 to 12 days after the onset of symptoms. A cross-reactivity study involving HIV-1 and HIV-2 RTb-Abs and their homologous RT showed HIV-1 RTb-Ab to be highly type specific. None of 10 serum samples from HIV-1-infected individuals showed cross-reacting RTb-Ab toward HIV-2 RT, whereas 4 of 10 serum samples from HIV-2-infected patients showed cross-reactivity toward HIV-1 RT; however, the cross-reactivity toward HIV-1 RT was 3,000 times lower than that toward its homologous RT. Future uses for the assay with reference to the recent World Health Organization proposal for other methods instead of Western blotting (immunoblotting) for confirming HIV-1 infection and for methods for the diagnosis of infection as follow-up in vaccine trials are also discussed.

Full Text

The Full Text of this article is available as a PDF (302.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Advani M., Imagawa D. T., Lee M. H., Sano K., Morales F., Mitsuyasu R. T., Detels R. Cross-sectional study of reverse transcriptase-inhibiting antibody as a marker of acquired immune deficiency syndrome. J Clin Microbiol. 1989 Jul;27(7):1453–1455. doi: 10.1128/jcm.27.7.1453-1455.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chatterjee R., Rinaldo C. R., Jr, Gupta P. Immunogenicity of human immunodeficiency virus (HIV) reverse transcriptase: detection of high levels of antibodies to HIV reverse transcriptase in sera of homosexual men. J Clin Immunol. 1987 May;7(3):218–224. doi: 10.1007/BF00915727. [DOI] [PubMed] [Google Scholar]
  3. De Leys R., Vanderborght B., Vanden Haesevelde M., Heyndrickx L., van Geel A., Wauters C., Bernaerts R., Saman E., Nijs P., Willems B. Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin. J Virol. 1990 Mar;64(3):1207–1216. doi: 10.1128/jvi.64.3.1207-1216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeVico A. L., Veronese F. D., Lee S. L., Gallo R. C., Sarngadharan M. G. High prevalence of serum antibodies to reverse transcriptase in HIV-1-infected individuals. AIDS Res Hum Retroviruses. 1988 Feb;4(1):17–22. doi: 10.1089/aid.1988.4.17. [DOI] [PubMed] [Google Scholar]
  5. Ekstrand D. H., Awad R. J., Källander C. F., Gronowitz J. S. A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation. Biotechnol Appl Biochem. 1996 Apr;23(Pt 2):95–105. [PubMed] [Google Scholar]
  6. Grimison B., Laurence J. Immunodominant epitope regions of HIV-1 reverse transcriptase: correlations with HIV-1+ serum IgG inhibitory to polymerase activity and with disease progression. J Acquir Immune Defic Syndr Hum Retrovirol. 1995 May 1;9(1):58–68. [PubMed] [Google Scholar]
  7. Gronowitz J. S., Neumüller M., Lennerstrand J., Bhikhabhai R., Unge T., Weltman H., Källander C. F. Carrier bound templates for single tube reverse transcriptase assays and for combined purification and activity analyses, with special reference to HIV. Biotechnol Appl Biochem. 1991 Feb;13(1):127–142. [PubMed] [Google Scholar]
  8. Gürtler L. G., Hauser P. H., Eberle J., von Brunn A., Knapp S., Zekeng L., Tsague J. M., Kaptue L. A new subtype of human immunodeficiency virus type 1 (MVP-5180) from Cameroon. J Virol. 1994 Mar;68(3):1581–1585. doi: 10.1128/jvi.68.3.1581-1585.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gürtler L. G., Zekeng L., Simon F., Eberle J., Tsague J. M., Kaptue L., Brust S., Knapp S. Reactivity of five anti-HIV-1 subtype O specimens with six different anti-HIV screening ELISAs and three immunoblots. J Virol Methods. 1995 Feb;51(2-3):177–183. doi: 10.1016/0166-0934(94)00102-m. [DOI] [PubMed] [Google Scholar]
  10. Hu D. J., Dondero T. J., Rayfield M. A., George J. R., Schochetman G., Jaffe H. W., Luo C. C., Kalish M. L., Weniger B. G., Pau C. P. The emerging genetic diversity of HIV. The importance of global surveillance for diagnostics, research, and prevention. JAMA. 1996 Jan 17;275(3):210–216. [PubMed] [Google Scholar]
  11. Kornfeld H., Riedel N., Viglianti G. A., Hirsch V., Mullins J. I. Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses. Nature. 1987 Apr 9;326(6113):610–613. doi: 10.1038/326610a0. [DOI] [PubMed] [Google Scholar]
  12. Laurence J., Saunders A., Kulkosky J. Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. Science. 1987 Mar 20;235(4795):1501–1504. doi: 10.1126/science.2435004. [DOI] [PubMed] [Google Scholar]
  13. Lelie P. N., van der Poel C. L., Reesink H. W., Huisman H. G., Boucher C. A., Goudsmit J. Efficacy of the latest generation of antibody assays for (early) detection of HIV 1 and HIV 2 infection. Vox Sang. 1989;56(1):59–61. doi: 10.1111/j.1423-0410.1989.tb03051.x. [DOI] [PubMed] [Google Scholar]
  14. Lennerstrand J., Rytting A. S., Orvell C., Gronowitz J. S., Kallander C. F. A method for combined immunoaffinity purification and assay of HIV-1 reverse transcriptase activity useful for crude samples. Anal Biochem. 1996 Mar 15;235(2):141–152. doi: 10.1006/abio.1996.0106. [DOI] [PubMed] [Google Scholar]
  15. Loussert-Ajaka I., Ly T. D., Chaix M. L., Ingrand D., Saragosti S., Couroucé A. M., Brun-Vézinet F., Simon F. HIV-1/HIV-2 seronegativity in HIV-1 subtype O infected patients. Lancet. 1994 Jun 4;343(8910):1393–1394. doi: 10.1016/s0140-6736(94)92524-0. [DOI] [PubMed] [Google Scholar]
  16. Loveday C., Tedder R. S. Enzyme-linked immunosorbent assays for the measurement of human immunodeficiency virus, type 1 reverse transcriptase antigen and antibodies. J Virol Methods. 1993 Feb;41(2):181–192. doi: 10.1016/0166-0934(93)90125-b. [DOI] [PubMed] [Google Scholar]
  17. Martin D. J., Blackburn N. K., O'Connell K. F., Brant E. T., Goetsch E. A. Evaluation of the World Health Organisation antibody-testing strategy for the individual patient diagnosis of HIV infection (strategy III). S Afr Med J. 1995 Sep;85(9):877–880. [PubMed] [Google Scholar]
  18. Neumüller M., Karlsson A., Lennerstrand J., Källander C. F., Holmberg V., Långström-Persson U., Thorstensson R., Sandström E., Gronowitz J. S. HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables. J Med Virol. 1991 May;34(1):55–63. doi: 10.1002/jmv.1890340110. [DOI] [PubMed] [Google Scholar]
  19. Neumüller M., Karlsson A., Lennerstrand J., Källander C. F., Sandström E., Holmberg V., Gronowitz J. S. HIV-1 reverse transcriptase inhibiting antibody titer in serum: relation to disease progression and to core-antibody levels. J Med Virol. 1992 Apr;36(4):283–291. doi: 10.1002/jmv.1890360410. [DOI] [PubMed] [Google Scholar]
  20. Race E. M., Ramsey K. M., Lucia H. L., Cloyd M. W. Human immunodeficiency virus infection elicits early antibody not detected by standard tests: implications for diagnostics and viral immunology. Virology. 1991 Oct;184(2):716–722. doi: 10.1016/0042-6822(91)90441-d. [DOI] [PubMed] [Google Scholar]
  21. Sano K., Lee M. H., Morales F., Nishanian P., Fahey J., Detels R., Imagawa D. T. Antibody that inhibits human immunodeficiency virus reverse transcriptase and association with inability to isolate virus. J Clin Microbiol. 1987 Dec;25(12):2415–2417. doi: 10.1128/jcm.25.12.2415-2417.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schable C., Zekeng L., Pau C. P., Hu D., Kaptue L., Gurtler L., Dondero T., Tsague J. M., Schochetman G., Jaffe H. Sensitivity of United States HIV antibody tests for detection of HIV-1 group O infections. Lancet. 1994 Nov 12;344(8933):1333–1334. doi: 10.1016/s0140-6736(94)90695-5. [DOI] [PubMed] [Google Scholar]
  23. Urabe T., Sano K., Nakano T., Odawara F., Lee M. H., Otake T., Okubo S., Hayami M., Misaki H., Baba M. Differentiation between human immunodeficiency virus type 1 (HIV-1) and HIV-2 isolates by nonradioisotopic reverse transcriptase-typing assay. J Clin Microbiol. 1994 Aug;32(8):1870–1875. doi: 10.1128/jcm.32.8.1870-1875.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vanden Haesevelde M., Decourt J. L., De Leys R. J., Vanderborght B., van der Groen G., van Heuverswijn H., Saman E. Genomic cloning and complete sequence analysis of a highly divergent African human immunodeficiency virus isolate. J Virol. 1994 Mar;68(3):1586–1596. doi: 10.1128/jvi.68.3.1586-1596.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES