An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens

An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).     

Development and evaluation of a human T-cell leukemia virus type I serologic confirmatory assay incorporating a recombinant envelope polypeptide.

A recombinant protein derived from the gp21 region of the human T-cell leukemia virus type I (HTLV-I) env gene was synthesized in Escherichia coli and purified by reversed-phase high-performance liquid chromatography. The purified protein was free of contaminating bacterial proteins and retained reactivity with human HTLV-I- and HTLV-II-positive sera and a gp21 monoclonal antibody. An immunoblot procedure using the recombinant polypeptide in conjunction with native viral proteins was more sensitive than the conventional immunoblot and radioimmunoprecipitation confirmatory assays for detection of antibodies to HTLV-I and HTLV-II env-encoded gene products. The recombinant protein was equally reactive with sera from polymerase chain reaction-confirmed HTLV-I or HTLV-II infections. Furthermore, on the basis of the differential reactivities of gp21-positive sera with the HTLV-I p19 and p24 gag-encoded proteins, an algorithm was proposed to distinguish exposure to HTLV-I from exposure to HTLV-II. These results establish the utility of a modified immunoblot assay incorporating a recombinant envelope polypeptide as an alternative to existing HTLV-I-confirmatory assays.     

Intracellular localization of recombinant vaccinia virus produced HIV antigens and their use for confirmation of HIV seropositivity

Recombinant vaccinia strains vC5 and vE234L expressing the rp50 and rgp160/rgp120 recombinant proteins were used in immunoblot and immunofluorescence assays. No false reactions were found, although 30 sera giving false reactivity with gag or env encoded proteins in a commercial immunoblot assay were included into the test panel. We recommend the recombinant protein-based assay for confirmation and discrimination of HIV seropositivity.     

Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population

Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens. The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6%. Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only [GRO]) (0.4 to 0.5% of donors). Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot. Most GRO sera reacted with p15/p17 bands on HIV immunoblot. Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E. coli-expressed p55gag. This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera. More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.     

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.     

Comparison of Western immunoblot antigens and interpretive criteria for detection of antibody to human T-lymphotropic virus types I and II.

Twenty human T-lymphotropic virus type I (HTLV-I) antibody-positive sera from Japan, Hawaii, and the Marshall Islands and 15 HTLV type II (HTLV-II) antibody-positive sera from intravenous drug users in the United States were tested by immunoblotting with two recombinant HTLV-I proteins and three commercial kits to determine whether there were any differences in reactions between HTLV-I- and HTLV-II-positive sera by the Western immunoblot method and, also, to evaluate the ability of these reagents to detect HTLV-I- and HTLV-II-seropositive individuals by using the recommended Western blot interpretation. These sera were first extensively characterized by immunofluorescence, enzyme immunoassay, radioimmunoprecipitation assay, and Western blot using HTLV-I and HTLV-II viral lysates and an envelope (env) recombinant protein. Although both HTLV-I- and HTLV-II antibody-positive sera reacted with the env protein gp68, reactions with the gp46 env antigens appeared to be specific for HTLV-I. It was found that the use of either p19 or p24 core bands plus an env reaction instead of only the p24 plus env reaction (as presently recommended) increased the number of positive interpretations for HTLV-I but had no effect on the number of HTLV-II-positive interpretations.     

HIV 外膜糖蛋白 gp120 和 gp41 在昆虫细胞中表达及其免疫学特性的评价

将编码完整ｇｐ１２０和完整ｇｐ４１的基因分别克隆到杆状病毒转移质粒中。使用重组转移质粒与野生杆状病毒（ＡｃＮＰＶ）ＤＮＡ共转染Ｓｆ９昆虫细胞，经挑选获得分别带有编码ｇｐ１２０和ｇｐ４１基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中分别表达了ＨＩＶ外膜糖蛋白ｇｐ１２０和ｇｐ４１。其重组蛋白的分子量分别为１２０×１０３和４１×１０３。此重组糖蛋白在免疫荧光、免疫印染和酶联免疫实验中都能被ＨＩＶ阳性血清所识别。动物免疫实验表明此重组糖蛋白能诱导很强的特异性抗体产生。     

Serologic characterization of human immunodeficiency virus infection by Western blot and radioimmunoprecipitation assays

The Western blot and radioimmunoprecipitation assay (RIPA) appear to be the most specific tests available for the detection of antibodies directed against human immunodeficiency virus (HIV). An investigation of 678 HIV-seropositive samples from 518 patients by the Western blot assay indicates that the majority of patients who have been exposed to HIV exhibit antibodies directed against glycoprotein (gp) 41. The HIV-seropositive samples were categorized into four groups according to their Western blot antibody reactive patterns. Group 1 had evidence of reactivities directed against protein (p) 24 and gp41; group 2 had reactivity to gp41 and no reactivity to p24; group 3 had reactivity to p24 and p55 and no reactivity to gp41; and group 4 had an isolated p24 reactivity. The RIPA revealed antibody reactivities directed against HIV envelope proteins (gp 160, gp 120, and gp41) in 91 of 95 samples that were tested. No HIV antibody reactivities were detected by RIPA in four samples from group 4. Specimens exhibiting an isolated antibody reactivity directed against p24 by Western blot analysis should have further evaluation before being labeled as indicative of HIV exposure. A western blot study on a subsequent sequential sample or another confirmatory assay, such as the RIPA, should be performed to identify antibodies directed against HIV envelope components.     

Immunoblot assay for serodiagnosis of Helicobacter pylori infections.

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.     

Sensitivity and specificity of a recombinant transmembrane glycoprotein (rgp21)-spiked western immunoblot for serological confirmation of human T-cell lymphotropic virus type I and type II infections.

Serum specimens (n = 2,712) obtained from individuals residing in diverse geographic regions and categorized as seropositive (n = 122), seroindeterminate (n = 523), or seronegative (n = 2,067) for human T-cell lymphotropic virus (HTLV) infection in accordance with U.S. Public Health Service guidelines were retested by recombinant transmembrane protein (rgp21)-spiked Western immunoblotting. Of the 122 HTLV-positive specimens, those from 85 of 85 (100%) U.S. blood donors, 2 of 2 (100%) Brazilians, 1 of 2 (50%) Indonesians, 14 of 14 (100%) Solomon Islanders, and 18 of 19 (95%) Papua New Guineans reacted with rgp21, yielding an overall sensitivity of 98% (120 of 122). Specimens from individuals whose infections were confirmed to be HTLV type I or HTLV type II by the polymerase chain reaction assay reacted equally well with rgp21. Of the 523 HTLV-indeterminate specimens, those from 21 of 379 (5.5%) U.S. blood donors, 3 of 6 (50%) Brazilians, 10 of 23 (44%) Ugandans, 8 of 49 (16%) Indonesians, 4 of 36 (11%) Solomon Islanders, and 5 of 30 (17%) Papua New Guineans reacted with rgp21. None of these 51 specimens reacted with native gp46 and/or gp61/68 in a radioimmunoprecipitation assay, suggesting a false-positive reaction (9.75%). Of the 2,067 HTLV-negative specimens, 12 reacted with rgp21, yielding a false-positivity rate of 0.6%. These data indicate that while detection of rgp21 is highly sensitive, it can yield false-positive results. Thus, specimens exhibiting reactivity with rgp21 in the absence of reactivity with native gp46 and/or gp61/68 by Western blot should be tested further by a radioimmunoprecipitation assay to verify HTLV type I or type II infection.     

Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies.

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested.     

A sensitive radioimmunoprecipitation assay for human immunodeficiency virus (HIV)

A sensitive and efficient radioimmunoprecipitation procedure is described which provides an alternative to Western blotting assays for characterizing antibodies directed against human immunodeficiency viruses (HIV-1). Reaction of solubilized preparations of HTLV-III with 125I-labeled Bolton-Hunter reagent leads to the efficient labeling of all of the major virus-specific proteins, including gp120, gp41, RT (p66/p51), p24, and p17. These labeled proteins are readily immunoprecipitated by immune human sera, by specific sera derived from hyperimmunized animals, and by monoclonal antibodies. This procedure, referred to as BH-RIP, provides a simple assay for characterizing and titering antibodies against HIV which is equivalent in specificity, and more sensitive and efficient than the Western blotting method. In addition, viral proteins labeled in this way are suitable for biochemical studies. In one such application, the number of high-mannose and complex oligosaccharide side chains of gp120 and gp41 were determined by examining the sensitivities of the two viral glycoproteins labeled by this procedure to the glycosidases Endo H and PNGase F.     

Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation.

A current concept of the serological response to human immunodeficiency virus (HIV) infection in humans is that antibodies to core antigens (p55, p24, and p15) are detectable earlier during initial stages of antibody production than antibodies against envelope antigens (gp160, gp120, and gp41). Comparative studies of Western blot (immunoblot), radioimmunoprecipitation assay (RIPA), and enzyme-linked immunosorbent assay (ELISA) during initial antibody production are limited to case reports and have not resolved the issue. Thirty of the 37 participants who are part of a prospective study had at least one specimen that was negative for anti-gp41 but had one or more other bands on Western blot. Twenty-seven of these 30 specimens were reactive for anti-gp120/160 in the RIPA. Of the same 30 specimens, kits from Bionetics identified 2 (7%), ElectroNucleonics 4 (13%), Abbott 13 (43%), Du Pont 25 (83%), and Genetic Systems 25 (83%). All participants had evidence of serological progression by Western blot, including a gp41 band, on subsequent visits; the ELISA kits of all manufacturers identified these later specimens with greater accuracy. These data show that the RIPA detects anti-envelope antibodies that may be not detectable by Western blot and that the production of anti-envelope antibodies approximately parallels the production of anti-core antibodies. The false-negative results by ELISA would permit transmission of HIV by blood transfusion from donors in early stages of infection. The sensitivity of licensed ELISA kits should be improved to identify antibody as soon as possible after infection.     

Multicenter evaluation of the novel ABN Western blot (immunoblot) system in comparison with an enzyme-linked immunosorbent assay and a different Western blot.

A new, modular Western blot (immunoblot) system for human immunodeficiency virus (HIV) antibodies (ABN WesPage; Wellcome) was compared with enzyme immunoassays (Wellcome, Behringwerke, and Abbott) and with a U.S. Food and Drug Administration (FDA)-licensed Western blot (DuPont) in a multicenter study. A total of 649 serum samples from HIV patients at different stages of the disease, as well as from high-risk patients, from patients with conditions unrelated to AIDS, and from healthy blood donors, were used in the evaluation along with nine seroconversion panels. For evaluation of Western blot reactivity, both Centers for Disease Control (CDC) and FDA criteria were used. With the DuPont Western blot as the reference assay, the overall sensitivity and specificity of the ABN WesPage were 100 and 99.1%, respectively, when indeterminate results were not taken into account and when both tests were interpreted in accordance with CDC criteria. The DuPont Western blot detected significantly more antibodies to pol and gag gene products than the ABN WesPage. The ABN WesPage showed a higher positive rate of detection of viral envelope band gp160. When both Western blots were interpreted in accordance with CDC criteria, the ABN WesPage and the DuPont Western blot yielded 9.3 and 10.4% indeterminate results, respectively. When the DuPont Western blot was interpreted in accordance with the manufacturer's instructions (FDA criteria), 25.7% of the samples tested were regarded as indeterminate. The choice of interpretation criteria is of paramount importance for the evaluation of HIV Western blot patterns.     

Enzyme-linked immunoassay for human immunodeficiency virus type 1 envelope glycoprotein 120.

An enzyme-linked immunosorbent assay (ELISA) that can measure picogram quantities of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) in cell culture medium or body fluids has been developed. Recombinant, soluble CD4 immobilized in microtiter trays was used to capture gp120, which was then detected with polyclonal sheep antibody to gp120 followed by biotinylated rabbit anti-sheep immunoglobulin G and an avidin-alkaline phosphatase indicator system. With a reference recombinant gp120, the assay showed a linear relationship between optical density and concentrations ranging from 60 to 6,000 pg/100-microliters well; precision of the assay varied with the concentrations and ranged from +/- 40% with amounts smaller than 200 pg to +/- 10% with amounts larger than 200 pg. In a group of coded samples containing 60 pg (approximately 10(7) molecules) of reference gp120, the assay correctly identified the samples as containing gp120 99% of the time, with no false-positive results recorded for blank samples. Recombinant gp120 prepared in another cell culture system demonstrated a binding coefficient 13-fold lower than that of reference gp120. Mixing standard amounts of reference gp120 with increasing concentrations of human sera reduced assay sensitivity, although the linear relationship between gp120 concentration and optical density remained. With this assay we were able to detect gp120 in HIV-1 suspensions prepared from cultured lymphoblastoid cells and in the sera of HIV-1-infected patients. This ELISA for gp120 should be useful for studying the biological role of gp120 in HIV infection.     

Antibody-dependent cellular cytotoxicity (ADCC) is directed against immunodominant epitopes of the envelope proteins of human immunodeficiency virus 1 (HIV-1).

In this study, epitopes of HIV envelope proteins that are involved in ADCC were identified. Peripheral blood mononuclear cells (PBMC) were obtained from adults with asymptomatic HIV infection or early symptoms of AIDS. These PBMC, which were reported to be "armed" in vivo with HIV-specific antibodies, were used as effector cells in 51Cr release assays. Target cells consisted of CD4 lymphocytes from healthy seronegative donors, coated with the IIIB strain of HIV-1 or with one of seven synthetic peptides. Cytotoxicity was detected against CD4 lymphocytes coated with HIV-1 IIIB or with the peptides env aa 507-518, corresponding to the carboxy-terminus of gp120, and env aa 597-611, corresponding to the region of the cysteine loop of gp41. The magnitude of target cell lysis was directly related to the quantity of peptide used. In contrast, target cells coated with the peptide gag aa 129-135, corresponding to the p17/p24 cleavage region of the gag precursor, were not killed. The same immunodominant regions which were involved in ADCC were recognized in enzyme-linked immunoabsorbent assays (ELISA) by the majority of 107 sera from HIV-infected adults. We conclude that the immunodominant epitopes located at the carboxy-terminus of gp120 and the cysteine loop of gp41 serve as recognition structure for antibodies, capable of mediating ADCC against HIV-infected cells.     

Characterization of antibody reactivity to human T-cell lymphotropic virus types I and II using immunoblot and radioimmunoprecipitation assays.

We have characterized the immunoreactivity to human T-cell lymphotropic virus type I (HTLV-I) among 26,983 persons of various seroprevalence groups by using enzyme immunoassay, immunoblot (IB), and radioimmunoprecipitation assays (RIPA) in accordance with Public Health Service recommended guidelines for the interpretation of serologic test results for HTLV-I infection. IB-indeterminate serum specimens (n = 178) were reactive to HTLV-I gag proteins, and no serum contained only env reactivity. Overall, RIPA resolved 40% of IB-indeterminate serum samples; however, the probability that RIPA would confirm IB-indeterminate samples depended on the seroprevalence of the population tested. HTLV-I gag p19-only reactivity on IB was not a reliable marker of HTLV-I infection, while gag p24 reactivity on IB was clearly associated with positive seroreactive specimens. IB and RIPA tests did not clearly distinguish between HTLV-I and HTLV-II seroreactivities. These data emphasize that patterns of immunoreactivity to HTLV-I antigens are dependent upon the seroprevalence of the risk groups tested. In addition, RIPA detected antibodies to env proteins present in low titer in a substantial number of IB gag-only reactive sera and resolved the HTLV-I antibody status of these sera.     

Comparison of the sensitivity of various anti-HIV tests in early seroconversion sera.

Paired sera from 4 patients with proven HIV infection whose initial specimens obtained 14-51 days earlier were indeterminate were simultaneously retested with 7 screening anti-HIV test kits and the immunoblot assay. The study aimed to evaluate the sensitivity of various new and old anti-HIV screening tests. The test kits evaluated were 4 ELISA test kits from Wellcome (Wellcozyme), Organon (Vironostika anti-HTLV-III), Pasteur (Rapid Elavia) and Diagnostic Biotechnology (DB, HIV-1 ELISA), 2 rapid tests based on microfiltration enzyme immunoassay procedure from Rapport (SUDS) and Disease Detection International (SeroCard), and 1 particle agglutination (PA) test (Serodia-HIV). Immunoblot strips from Diagnostic Biotechnology (HIV-1 Western blot) were used to confirm the HIV infection in these serum specimens. Out of the 4 initial serum specimens tested, all were positive by PA, 2 by SUDS, Wellcome and Pasteur, 1 by SeroCard and DB, and none by Organon. When tested by immunoblot, 1 was negative (i.e., completely without any bands) whereas 3 were indeterminate (i.e., 1 with very weak band for p18, 1 with weak band for p24, 1 with very weak band for gp160. All repeat specimens obtained 14-51 days later (mean 32.5 +/- 16 days) were positive by all screening tests as well as immunoblot. Therefore, with these 4 early seroconversion sera, the sensitivity of the PA was 100%, that of SUDS, Wellcome and pasteur was 50%, of that SeroCard and DB was 25%, and Organon, 0%. None of these sera was considered positive by immunoblot.     

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu³⁺ chelate or with nanoparticles doped with the Eu³⁺ chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu³⁺ chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.