Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1990 Sep;28(9):2022–2029. doi: 10.1128/jcm.28.9.2022-2029.1990

New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

H Siitari 1, P Turunen 1, J Schrimsher 1, M Nunn 1
PMCID: PMC268097  PMID: 2121790

Abstract

A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-amino-acid sequence from a highly conserved region of the HIV-1 gp41 envelope protein. This recombinant antigen enabled the detection of antibodies to both gag and env gene products. When this assay was compared with a commercially available recombinant enzyme-linked immunoabsorbent assay (ELISA) by using four quality-control panels, the TR-FIA detected all 20 positive specimens, while the recombinant ELISA detected only 16 of them. This increased sensitivity could be demonstrated directly by the assay of dilution series of HIV-1-positive sera. The analysis of two seroconversion panels by TR-FIA and six ELISAs showed that TR-FIA allowed detection of antibody in infected individuals 16 days earlier than the other assays did. In addition to being highly sensitive, the assay was highly specific; of the 57 samples shown to be repeatedly positive by ELISA but known to be HIV-1 negative by Western immunoblot analysis, only 1 sample reacted positively in this assay. The specificity of the assay was 99.9% when 1.054 random serum specimens were tested.

Full text

PDF
2022

Selected References

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

  1. Aceti A., Titti F., Verani P., Buttò S., Pennica A., Sebastiani A., Rossi G. B. Time-resolved immunofluorescence: a sensitive and specific assay for anti-HIV antibody detection in human sera. J Virol Methods. 1987 Jul;16(4):303–315. doi: 10.1016/0166-0934(87)90015-2. [DOI] [PubMed] [Google Scholar]
  2. Allain J. P., Laurian Y., Paul D. A., Verroust F., Leuther M., Gazengel C., Senn D., Larrieu M. J., Bosser C. Long-term evaluation of HIV antigen and antibodies to p24 and gp41 in patients with hemophilia. Potential clinical importance. N Engl J Med. 1987 Oct 29;317(18):1114–1121. doi: 10.1056/NEJM198710293171804. [DOI] [PubMed] [Google Scholar]
  3. Bedarida G., Crocchiolo P., Cambié G., D'Agostino F., Uberti Foppa C., Berto E., Grisi M. E., Arcidiacono I., Ronsivalle M. G., Pasquali M. HIV-IgM serology. Infection. 1988 Sep-Oct;16(5):301–302. doi: 10.1007/BF01645079. [DOI] [PubMed] [Google Scholar]
  4. Biberfeld G., Bredberg-Rådén U., Böttiger B., Putkonen P. O., Blomberg J., Juto P., Wadell G. Blood donor sera with false-positive Western blot reactions to human immunodeficiency virus. Lancet. 1986 Aug 2;2(8501):289–290. doi: 10.1016/s0140-6736(86)92111-2. [DOI] [PubMed] [Google Scholar]
  5. Boscato L. M., Stuart M. C. Incidence and specificity of interference in two-site immunoassays. Clin Chem. 1986 Aug;32(8):1491–1495. [PubMed] [Google Scholar]
  6. Chiodi F., von Gegerfeldt A., Albert J., Fenyö E. M., Gaines H., von Sydow M., Biberfeld G., Parks E., Norrby E. Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein. J Med Virol. 1987 Sep;23(1):1–9. doi: 10.1002/jmv.1890230102. [DOI] [PubMed] [Google Scholar]
  7. Cooper D. A., Imrie A. A., Penny R. Antibody response to human immunodeficiency virus after primary infection. J Infect Dis. 1987 Jun;155(6):1113–1118. doi: 10.1093/infdis/155.6.1113. [DOI] [PubMed] [Google Scholar]
  8. Courouce A. M. Evaluation of eight ELISA kits for the detection of anti-LAV/HTLV-III antibodies. Lancet. 1986 May 17;1(8490):1152–1153. doi: 10.1016/s0140-6736(86)91863-5. [DOI] [PubMed] [Google Scholar]
  9. Dawson G. J., Heller J. S., Wood C. A., Gutierrez R. A., Webber J. S., Hunt J. C., Hojvat S. A., Senn D., Devare S. G., Decker R. H. Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins. J Infect Dis. 1988 Jan;157(1):149–155. doi: 10.1093/infdis/157.1.149. [DOI] [PubMed] [Google Scholar]
  10. Dowbenko D. J., Bell J. R., Benton C. V., Groopman J. E., Nguyen H., Vetterlein D., Capon D. J., Lasky L. A. Bacterial expression of the acquired immunodeficiency syndrome retrovirus p24 gag protein and its use as a diagnostic reagent. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7748–7752. doi: 10.1073/pnas.82.22.7748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gnann J. W., Jr, Schwimmbeck P. L., Nelson J. A., Truax A. B., Oldstone M. B. Diagnosis of AIDS by using a 12-amino acid peptide representing an immunodominant epitope of the human immunodeficiency virus. J Infect Dis. 1987 Aug;156(2):261–267. doi: 10.1093/infdis/156.2.261. [DOI] [PubMed] [Google Scholar]
  12. Goudsmit J., de Wolf F., Paul D. A., Epstein L. G., Lange J. M., Krone W. J., Speelman H., Wolters E. C., Van der Noordaa J., Oleske J. M. Expression of human immunodeficiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet. 1986 Jul 26;2(8500):177–180. doi: 10.1016/s0140-6736(86)92485-2. [DOI] [PubMed] [Google Scholar]
  13. Griffith B. P., Ferguson D., Mellors J. W. Acute human immunodeficiency virus infection: false-negative ELISA in a patient with viremia and a positive western blot test. J Infect Dis. 1989 Jul;160(1):160–161. doi: 10.1093/infdis/160.1.160. [DOI] [PubMed] [Google Scholar]
  14. Gürtler L. G., Eberle J., Lorbeer B., Deinhardt F. Sensitivity and specificity of commercial ELISA kits for screening anti-LAV/HTLV III. J Virol Methods. 1987 Jan;15(1):11–23. doi: 10.1016/0166-0934(87)90044-9. [DOI] [PubMed] [Google Scholar]
  15. Hemmilä I., Dakubu S., Mukkala V. M., Siitari H., Lövgren T. Europium as a label in time-resolved immunofluorometric assays. Anal Biochem. 1984 Mar;137(2):335–343. doi: 10.1016/0003-2697(84)90095-2. [DOI] [PubMed] [Google Scholar]
  16. Huisman J. G., Winkel I. N., Lelie P. N., Tersmette M., Goudsmit J., Miedema F. Detection of early anti-p24 HIV responses in EIA- and immunoblot-negative individuals. Implications for confirmatory testing. Vox Sang. 1987;53(1):31–36. doi: 10.1111/j.1423-0410.1987.tb04910.x. [DOI] [PubMed] [Google Scholar]
  17. Imrie A., Best S., Penny R., Cooper D. A. False-negative ELISA result for HIV antibody due to an apparent prozone phenomenon. Med J Aust. 1987 Jul 6;147(1):42–42. doi: 10.5694/j.1326-5377.1987.tb133235.x. [DOI] [PubMed] [Google Scholar]
  18. Janvier D., Reviron M., Reviron J. False negative results in a competitive immunoassay for anti-HIV-antibodies due to the presence of rheumatoid factors. Vox Sang. 1989;56(4):237–242. doi: 10.1111/j.1423-0410.1989.tb02035.x. [DOI] [PubMed] [Google Scholar]
  19. Jendis J. B., Tomasik Z., Hunziker U., Nadal D., Seger R., Wetzel J. C., Kind C., Schüpbach J. Evaluation of diagnostic tests for HIV infection in infants born to HIV-infected mothers in Switzerland. AIDS. 1988 Aug;2(4):273–279. doi: 10.1097/00002030-198808000-00006. [DOI] [PubMed] [Google Scholar]
  20. Joller-Jemelka H. I., Joller P. W., Müller F., Schüpbach J., Grob P. J. Anti-HIV IgM antibody analysis during early manifestations of HIV infections. AIDS. 1987 May;1(1):45–47. [PubMed] [Google Scholar]
  21. Kennedy R. C., Henkel R. D., Pauletti D., Allan J. S., Lee T. H., Essex M., Dreesman G. R. Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein. Science. 1986 Mar 28;231(4745):1556–1559. doi: 10.1126/science.3006246. [DOI] [PubMed] [Google Scholar]
  22. Ludlam C. A., Tucker J., Steel C. M., Tedder R. S., Cheingsong-Popov R., Weiss R. A., McClelland D. B., Philp I., Prescott R. J. Human T-lymphotropic virus type III (HTLV-III) infection in seronegative haemophiliacs after transfusion of factor VIII. Lancet. 1985 Aug 3;2(8449):233–236. doi: 10.1016/s0140-6736(85)90288-0. [DOI] [PubMed] [Google Scholar]
  23. Mathiesen T., Chiodi F., Broliden P. A., Albert J., Houghten R. A., Utter G., Wahren B., Norrby E. Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides. Immunology. 1989 May;67(1):1–7. [PMC free article] [PubMed] [Google Scholar]
  24. 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., Groopman J. E. 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. 1987 Aug;83(2):208–212. doi: 10.1016/0002-9343(87)90686-3. [DOI] [PubMed] [Google Scholar]
  25. Meurman O. H., Ziola B. R. IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies. J Clin Pathol. 1978 May;31(5):483–487. doi: 10.1136/jcp.31.5.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Närvänen A., Korkolainen M., Suni J., Korpela J., Kontio S., Partanen P., Vaheri A., Huhtala M. L. Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection. J Med Virol. 1988 Oct;26(2):111–118. doi: 10.1002/jmv.1890260202. [DOI] [PubMed] [Google Scholar]
  27. Parry J. V., Mortimer P. P. Place of IgM antibody testing in HIV serology. Lancet. 1986 Oct 25;2(8513):979–980. doi: 10.1016/s0140-6736(86)90632-x. [DOI] [PubMed] [Google Scholar]
  28. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  29. Reesink H. W., Lelie P. N., Huisman J. G., Schaasberg W., Gonsalves M., Aaij C., Winkel I. N., van der Does J. A., Hekker A. C., Desmyter J. Evaluation of six enzyme immunoassays for antibody against human immunodeficiency virus. Lancet. 1986 Aug 30;2(8505):483–486. doi: 10.1016/s0140-6736(86)90358-2. [DOI] [PubMed] [Google Scholar]
  30. Saah A. J., Farzadegan H., Fox R., Nishanian P., Rinaldo C. R., Jr, Phair J. P., Fahey J. L., Lee T. H., Polk B. F. Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation. J Clin Microbiol. 1987 Sep;25(9):1605–1610. doi: 10.1128/jcm.25.9.1605-1610.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sayers M. H., Beatty P. G., Hansen J. A. HLA antibodies as a cause of false-positive reactions in screening enzyme immunoassays for antibodies to human T-lymphotropic virus type III. Transfusion. 1986 Jan-Feb;26(1):113–115. doi: 10.1046/j.1537-2995.1986.26186124012.x. [DOI] [PubMed] [Google Scholar]
  32. Scorza Smeraldi R., Fabio G., Lazzarin A., Eisera N. B., Moroni M., Zanussi C. HLA-associated susceptibility to acquired immunodeficiency syndrome in Italian patients with human-immunodeficiency-virus infection. Lancet. 1986 Nov 22;2(8517):1187–1189. doi: 10.1016/s0140-6736(86)92197-5. [DOI] [PubMed] [Google Scholar]
  33. Shoeman R. L., Young D., Pottathil R., Victor J., Conroy R. R., Crowl R. M., Coleman T., Heimer E., Lai C. Y., Ganguly K. Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents. Anal Biochem. 1987 Mar;161(2):370–379. doi: 10.1016/0003-2697(87)90465-9. [DOI] [PubMed] [Google Scholar]
  34. Siitari H., Hemmilä I., Soini E., Lövgren T., Koistinen V. Detection of hepatitis B surface antigen using time-resolved fluoroimmunoassay. Nature. 1983 Jan 20;301(5897):258–260. doi: 10.1038/301258a0. [DOI] [PubMed] [Google Scholar]
  35. Steimer K. S., Puma J. P., Power M. D., Powers M. A., George-Nascimento C., Stephans J. C., Levy J. A., Sanchez-Pescador R., Luciw P. A., Barr P. J. Differential antibody responses of individuals infected with AIDS-associated retroviruses surveyed using the viral core antigen p25gag expressed in bacteria. Virology. 1986 Apr 15;150(1):283–290. doi: 10.1016/0042-6822(86)90289-8. [DOI] [PubMed] [Google Scholar]
  36. Stramer S. L., Heller J. S., Coombs R. W., Parry J. V., Ho D. D., Allain J. P. Markers of HIV infection prior to IgG antibody seropositivity. JAMA. 1989 Jul 7;262(1):64–69. [PubMed] [Google Scholar]
  37. Stute R. Comparison in sensitivity of 10 HIV antibody detection tests by serial dilutions of Western blot-confirmed samples. J Virol Methods. 1988 Jul;20(3):269–274. doi: 10.1016/0166-0934(88)90130-9. [DOI] [PubMed] [Google Scholar]
  38. Wang J. J., Steel S., Wisniewolski R., Wang C. Y. Detection of antibodies to human T-lymphotropic virus type III by using a synthetic peptide of 21 amino acid residues corresponding to a highly antigenic segment of gp41 envelope protein. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6159–6163. doi: 10.1073/pnas.83.16.6159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van der Poel C. L., Reesink H. W., Tersmette T., Lelie P. N., Huisman H., Miedema F. Blood donations reactive for HIV in Western blot, but non-infective in culture and recipients of blood. Lancet. 1986 Sep 27;2(8509):752–753. doi: 10.1016/s0140-6736(86)90273-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES