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.     

Indeterminate human immunodeficiency virus Western blot profiles in ethiopians with discordant screening-assay results.

The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB reactivity among Ethiopians are hardly known. Here, we describe the profiles of indeterminate WB reactivity in Ethiopians with discordant screening assays. Between 1996 and 2000, a total of 12,124 specimens were tested for HIV-1 antibodies. Overall, 1,437 (11.9%) were positive for HIV-1 antibody. Ninety-one ( approximately 0.8%) gave equivocal results because of discordant results among the various screening assays and indeterminate WB profiles by the American Red Cross (ARC) criteria. Most (30.4%) of these indeterminate WB results were due to p24 reactivity. However, 12 samples (13.2%) displayed reactivity to p24 and gp41 or to p24 and gp120/160 proteins (positive by Centers for Disease Control and Prevention [CDC] criteria). Only two samples (2.2%) were reactive to both env glycoproteins gp41 and gp120/160 (positive by the World Health Organization [WHO] criteria). Of 31 WB assays initially indeterminate by the ARC criteria and with follow-up samples, 29 (93.5%) became negative when retested subsequently while 2 (6.5%) remained indeterminate for more than a year and were thus considered negative. Using CDC and WHO criteria, 6 (19.4%) and 2 (6.5%), respectively, of these WB assays would have been considered falsely positive. In addition, 17 indeterminate samples were negative when assessed by a nucleic acid-based amplification assay for HIV-1 viremia. In general, there was 97.8% concordance between the ARC and WHO criteria and 85.7% concordance between the ARC and CDC criteria for an indeterminate WB result. The ARC criteria best met the specified objectives for diagnosis in our setting.     

Indeterminate Human Immunodeficiency Virus Western Blot Profiles in Ethiopians with Discordant Screening-Assay Results

The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB reactivity among Ethiopians are hardly known. Here, we describe the profiles of indeterminate WB reactivity in Ethiopians with discordant screening assays. Between 1996 and 2000, a total of 12,124 specimens were tested for HIV-1 antibodies. Overall, 1,437 (11.9%) were positive for HIV-1 antibody. Ninety-one (≈0.8%) gave equivocal results because of discordant results among the various screening assays and indeterminate WB profiles by the American Red Cross (ARC) criteria. Most (30.4%) of these indeterminate WB results were due to p24 reactivity. However, 12 samples (13.2%) displayed reactivity to p24 and gp41 or to p24 and gp120/160 proteins (positive by Centers for Disease Control and Prevention [CDC] criteria). Only two samples (2.2%) were reactive to both env glycoproteins gp41 and gp120/160 (positive by the World Health Organization [WHO] criteria). Of 31 WB assays initially indeterminate by the ARC criteria and with follow-up samples, 29 (93.5%) became negative when retested subsequently while 2 (6.5%) remained indeterminate for more than a year and were thus considered negative. Using CDC and WHO criteria, 6 (19.4%) and 2 (6.5%), respectively, of these WB assays would have been considered falsely positive. In addition, 17 indeterminate samples were negative when assessed by a nucleic acid-based amplification assay for HIV-1 viremia. In general, there was 97.8% concordance between the ARC and WHO criteria and 85.7% concordance between the ARC and CDC criteria for an indeterminate WB result. The ARC criteria best met the specified objectives for diagnosis in our setting.     

Assessment of a New Immunoassay for Serological Confirmation and Discrimination of Human T-Cell Lymphotropic Virus Infections

The present study evaluated a new confirmatory assay for antibodies to human T-cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) proteins performed with serum samples from various commercial sources. The new test is a line immunoassay (LIA) with a nylon membrane sensitized with the most relevant antigens of HTLVs: the envelope gp46 and gp21 as well as the gag p24 and p19 antigens, represented by either recombinant proteins or synthetic peptides. A total of 176 serum or plasma samples were tested, of which 66 were HTLV-1 positive, 72 were HTLV-2 positive, and 38 were HTLV negative; of the 38 HTLV-negative samples 23 were indeterminate by Western blotting (WB). Serially diluted samples (n = 33) from HTLV-1- and HTLV-2-infected patients were also analyzed to determine the sensitivity of the new assay. The new confirmatory assay (INNO-LIA HTLV) performed markedly better than WB assays for those samples reactive by screening. Accurate confirmation of the presence of HTLV-1 and HTLV-2 antibodies and accurate discrimination of HTLV-1 and HTLV-2 antibodies were obtained for all the HTLV-seropositive samples. Due to its enhanced specificity and sensitivity, the new assay not only improves the ability to confirm and discriminate HTLV infections but also eliminates the vast majority of WB-indeterminate and false-positive specimens.     

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).     

Suitability of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus in Ghana, West Africa

In West African countries such as Ghana, efficient human immunodeficiency virus (HIV) testing is a priority in the fight against AIDS. A new immunochromatographic rapid test, Determine HIV-1/2 (Abbott Diagnostics, North Chicago, Ill.), that detects antibodies against HIV type 1 (HIV-1) and/or HIV-2 was evaluated using Ghanaian blood samples. Two hundred four serum and/or plasma specimens were tested. HIV screening was done by a particle agglutination test and confirmed by a Western blot (WB) test as the "gold standard." The results revealed 125 HIV-seropositive AIDS patients, 75 HIV-seronegative healthy individuals, and 4 individuals for whom the HIV-1 result was indeterminate. The results obtained by the Determine HIV-1/2 assay and Diagnostic HIV SPOT (Genelabs), which is currently widely used in many districts in Ghana, were compared with those of the WB test, excluding the four HIV-1-indeterminate samples. The sensitivity of the Determine HIV-1/2 assay was 100%, compared with 98.0% for the HIV SPOT assay. The specificity was 100% for both tests. Determine HIV-1/2 is a single-step assay and was found to be rapid and easy to perform without any special equipment. It was highly sensitive and specific. The kit can be applied without electricity and water supplies, making it suitable for the detection of HIV antibodies especially in the rural areas of Ghana, West Africa.     

Predictive value of two commercial human immunodeficiency virus serological tests in cases with indeterminate Western blot results.

BACKGROUND AND PURPOSE: Serodiagnosis of human immunodeficiency virus (HIV) infection typically requires repeatedly reactive positive screening test followed by Western blot (WB) assay. When WB assay result is indeterminate, the results of follow-up WB assay may remain inconclusive. This study evaluated use of enzyme-linked immunosorbent assay (ELISA) and particle agglutination test (PAT) as sequential screening tests with WB assay for the diagnosis of HIV infection. METHODS: From January 1, 2000 to December 31, 2003, a total of 565 serum samples collected from individuals with a previous positive or borderline positive ELISA test for HIV at regular check-up (281 samples) and a second group of individuals with known risk of HIV exposure or suspected infection based on clinical presentation (284 samples) were tested for HIV infection by ELISA, PAT and WB assay. RESULTS: The result was positive for HIV infection and confirmed by WB assay in 197 samples (22.5%), indeterminate in 127 samples (22.5%) and negative in 241 samples (42.6%). The sensitivity and specificity of ELISA were 100% and 21.6% and of PAT were 99.5% and 95%, respectively. Among the 197 HIV-infected cases, all ELISA and PAT results were concordant with WB assay except 1 (1/197) with negative PAT. Positive ELISA, positive PAT and indeterminate WB assay results were found in 9 of the 284 samples (7 individuals) from at-risk patients. Among these 7 individuals, 5 were later proved to have HIV infection. WB assay in 1 of the 7 individuals remained indeterminate 1 year later, and the remaining case was lost to follow-up. CONCLUSION: We suggest initial ELISA followed by PAT as sequential screening for HIV infection. When both screening tests are concordant but subsequent WB assay is indeterminate, further evaluation (such as nucleic acid amplification test) should be arranged as soon as possible.     

Human immunodeficiency virus antibody testing by enzyme-linked fluorescent and western blot assays using serum, gingival-crevicular transudate, and urine samples

The aim of the present study was to evaluate the possible utilization of saliva and urine as alternative samples to serum for the diagnosis of human immunodeficiency virus (HIV) infection. A total of 302 individuals participated in the study: 187 HIV-infected individuals (106 had Centers for Disease Control and Prevention [CDC] stage II infection, 19 had CDC stage III infection, and 62 had CDC stage IV infection) and 115 noninfected persons (46 of the noninfected persons were blood donors and 69 belonged to a group at high risk of HIV infection). Paired saliva and urine samples were taken from each of the participants in the study. The presence of HIV-specific antibodies was detected by an enzyme-linked fluorescent assay (ELFA), and the result was confirmed by Western blot analysis (WB). The ELFA with saliva gave maximum sensitivity and specificity values, while ELFA had lower sensitivity (95.2%) and specificity (97. 4%) values for detection of HIV antibody in urine samples. WB with all saliva samples fulfilled the World Health Organization criterion for positivity, while only 96.8% of the urine samples were confirmed to be positive by WB. Among the four reactivity patterns found by WB of these alternative samples, the most frequent included bands against three groups of HIV structural proteins (was ENV, POL, and GAG). The reactivity bands most frequently observed were those for the proteins gp160 and gp120. The least common reactivity band was the band for protein p17. The detection of HIV antibodies in saliva samples by means of ELFA with the possibility of later confirmation by WB makes saliva an alternative to serum for possible use in the diagnosis of infection. In contrast, HIV antibody detection in urine samples by the same methodology (ELFA) could be taken into consideration for use in epidemiological studies.     

HIV antibodies in whole saliva detected by ELISA and western blot assays

Paired serum and saliva samples were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) for the presence of human immunodeficiency virus (HIV) antibodies. The study group included 36 individuals known to be HIV seropositive and 14 healthy, seronegative controls. HIV antibodies were detected in all but one of the saliva samples of the seropositive subjects. In this particular patient, seroconversion was documented 1 week earlier by sequential testings. A further saliva sample obtained 2 months later was ELISA positive for salivary HIV antibodies. Antibodies against HIV proteins gp120 and gp160 were detected by Western blot assay in all saliva specimens taken from HIV seropositive subjects (including the ELISA-negative patient who seroconverted. Antibodies against other viral proteins (p65, p55, p51, gp41, p35, p24 p18) were found in saliva haphazardly without any clear-cut correlation with the clinical stage of the disease. Pretreatment of the saliva with protease inhibitor was essential for the diagnostic use of saliva for the detection of HIV antibodies by Western blot assay. Calculation of the ratio of titres in serum to those in saliva showed the highest ratios in symptomless subjects (mean +/- SD; 1844 +/- 1412) and the lowest in patients with acquired immune deficiency syndrome (AIDS) (mean +/- SD; 811 +/- 445). The ratio of serum to saliva by ELISA showed a positive correlation with salivary flow rate, indicating a dilution of salivary HIV antibodies with increasing salivary flow rate. The gingival bleeding index was negatively correlated with the ratio, supporting the concept that salivary HIV antibodies transudate from blood to saliva via gingival fluid.     

Detection of human immunodeficiency virus (HIV) type 1 antibodies by new automated microparticle enzyme immunoassay for HIV types 1 and 2.

We compared an automated microparticle double-antigen sandwich enzyme immunoassay (EIA) for the IMx test system recently developed by Abbott with two established assays (the automated indirect Vidas IgG EIA and the double-antigen sandwich EIA from Murex/Wellcome) devised for the detection of human immunodeficiency virus type 1 (HIV-1) and HIV-2 antibodies. A total of 1,078 consecutive serum samples were tested prospectively with the three assays. In addition, we used retrospectively selected panels of serum samples with discrepant results in two different screening tests and with indeterminate or positive Western immunoblot (WB) results, as well as five commercially available HIV-1 seroconversion panels. The new assay showed excellent discriminatory characteristics for the separation of samples from HIV-1-positive and HIV-1-negative persons according to Centers for Disease Control and Prevention WB criteria. The sensitivities were 98.1, 92.9, and 96.1% for the new test and the two other assays, respectively, and the specificities were 99.7, 97.9, and 98.1%, respectively. With the seroconversion panels this new test was positive several days earlier than the two other assays; i.e., seroconversion was evident at the peak of p24 antigenemia and often several weeks before WB became positive by the most stringent criteria.     

Retrospective study of Western blot profiles in immune sera of natural dengue virus infections

The Western blot (WB) assay was used to determine dengue virus antibodies present in human immune sera arising from recent primary and secondary dengue virus infections in Singapore. Cell lysates of dengue-2 virus-infected C6/36 and Vero cells were used. Antibodies directed against structural proteins of dengue-2 virus including envelope (E, gp60/50), capsid-premembrane (C-PrM, gp35), and premembrane (PrM, gp20) were detected, with antibody against envelope protein being most dominant. Similar WB profiles were detected in both primary and secondary dengue virus infections. The reactivity rate of antibodies to dengue-2 virus proteins was higher in infected Vero cell lysate than in infected C6/36 cell lysate, with the exception of antibodies to nonstructural proteins of NS1 and NS3, which were detected predominantly in infected C6/36 cell lysate. More than 75% of "normal" individuals (with no complaint of recent dengue virus infection) examined had low levels of dengue virus antibodies, but all presented with similar WB profiles as patients with recent dengue virus infections. This finding reflects a high seroprevalence of dengue virus infections and the long lasting nature of E, C-PrM, and PrM antibodies. Results from this study indicate that in natural dengue virus infections, native E, C-PrM, and PrM antigens of dengue virus are immunogenic and elicit long-lasting antibodies.     

Utility of various commercially available human immunodeficiency virus (HIV) antibody diagnostic kits for use in conjunction with efficacy trials of HIV-1 vaccines.

There is a need for human immunodeficiency virus (HIV) screening assays which will distinguish uninfected HIV vaccine recipients from HIV-infected individuals. Commercial screening kits were used to test serum samples from low- and high-risk participants in clinical trials before and after immunization with various recombinant HIV type 1 (HIV-1) envelope glycoprotein 120 (gp120) candidate vaccines. All kits were 100% sensitive in detecting HIV infection. Both Murex Single Use Diagnostic System and United Biomedical, Inc., HIV type 1 or 2 (HIV-1/2) enzyme immunoassay (EIA) kits, which detect antibodies to HIV-1 gp41, were 98 to 100% specific when used to screen baseline or recombinant gp120-vaccinated populations as vaccine-induced antibodies to gp120 were nonreactive in these tests. The Abbott HIVAB HIV-1 EIA (lysate of whole infected cells, reactive with anti-gp120 antibodies) gave high levels of reactivity due to vaccine-induced antibodies and a high baseline rate of false positives (12 of 83) among nonvaccinated high-risk volunteers. Assays containing only gp41 and p24 solid-phase components are compatible with gp120-based vaccines but are unlikely to be useful in a similar role for vaccines containing gp160, gp41, or gp120 plus p24 antigens. Efficacy trials must be designed in concert with available diagnostic screening assays to avoid problems caused by vaccine-induced seroconversion in high-risk populations.     

Diagnostic detection of human immunodeficiency virus type 1 antibodies in urine: a brazilian study

We evaluated, for the first time in Latin America, the performance of a commercial enzyme immunoassay (EIA) (Calypte Biomedical Corporation, Berkeley, Calif.) that detects human immunodeficiency virus type 1 (HIV-1)-specific antibodies in urine in comparison to standard serological assays (two commercial EIAs and a commercial Western blot [WB] assay). Paired serum and urine specimens were collected from two different groups of Brazilian patients: 225 drug users with unknown HIV status who attended drug treatment centers in Rio de Janeiro, Brazil, and 135 subjects with known HIV status. Patients showing positive results in the serum EIAs and/or in the urine EIA were serologically confirmed by WB assay. For 135 individuals with known HIV status, the urine EIA showed 100% sensitivity (74 positive samples) and 95.1% specificity (58 of 61 negative specimens). For 225 drug users, the test showed 100% sensitivity (2 positive samples) and 98.7% specificity (220 of 223 negative samples) compared to WB-confirmed serological EIA results. Thus, in a total of 360 samples, the urine EIA correctly identified all 76 HIV-positive samples and 278 of 284 negative samples (100% sensitivity and 97.9% specificity). Detailed analysis of the urine EIA results indicates that an increase of the recommended cutoff value might raise the specificity of the assay without affecting its sensitivity. Our results suggest that the HIV-1 urine EIA is a good screening test suitable for developing countries like Brazil. However, as for all other HIV screening tests on the market, it is not specific enough to be used as a one-step test and therefore requires confirmation.     

Evaluation of the Recombigen HIV-1 Latex Agglutination Test.

The Recombigen HIV-1 Latex Agglutination (LA) Test was recently licensed by the U.S. Food and Drug Administration for use as a rapid screening assay for human immunodeficiency virus type 1 (HIV-1) antibodies. However, its performance in various settings and in different populations has not been firmly established. Consequently, we evaluated the test in the Cleveland Clinic Retrovirus Laboratory, a regional reference laboratory for HIV diagnostic testing and a testing laboratory for the Ohio Department of Health Anonymous HIV Testing and Counseling Program. Serum samples from 93 individuals presumed to be at high risk for HIV infection were evaluated. The sera were initially tested for HIV antibodies by enzyme-linked immunosorbent assay (ELISA). All repeatedly reactive sera were subjected to confirmatory Western blot (WB; immunoblot) testing. Of 97 serum specimens tested (5 were from one seroconverter), 44 were repeatedly reactive by ELISA and 53 were nonreactive. Of the reactive serum specimens, 31 were confirmed positive and 12 were indeterminate by WB. All of the sera were coded and then retested by the LA test. Of 53 serum specimens nonreactive by ELISA, 51 were also nonreactive in the LA test. Of the 44 serum specimens reactive by ELISA, 16 were nonreactive by LA; however, 3 of the latter were WB positive. No serum specimen with an ELISA ratio (specimen optical density/cutoff optical density) of less than 2.1 scored reactive in the LA test. The LA test was positive for only two of five consecutive serum specimens from a seroconverter despite the fact that all but the earliest of these were ELISA reactive and WB positive. Although the LA test appears to be an adequate first-line screening test when appropriately used according to the directions of the manufacturer, our data suggest that occasional sera with low levels of reactivity by ELISA may not be readily detected as reactive by the LA test.     

A simple semi-rapid HIV-1&2 confirmatory immunoassay using magnetic particles

The Bionor HIV-1&2 Confirmatory Test is a semi-rapid simple immunoassay based on magnetic particles for the confirmation of serological status to human immunodeficiency virus (HIV). The specificity and sensitivity of this assay was evaluated by comparison with the Diagnostic Biotechnology HIV-1 Western blot (WB) 2.2 and the HIV-2/SBL-6669 WB. Bionor's confirmatory test demonstrated 98% specificity when testing sero-negative blood donors and false positive sera in screening tests compared to 81.5 and 71.6%, respectively, using the HIV-1 WB. The sensitivity of this assay for HIV-1 antibody positive sera was 97.9% compared to the WB which was 99.5%. When testing confirmed HIV-2 antibody positive samples, 2/100 scored negative using this confirmatory test similar to other HIV-2 peptide-based line immunoassays available commercially, whilst 8/100 were indeterminate reacting to HIV-2 membrane antigens only. Bionor's confirmatory test detected HIV-1 seropositivity earlier than the WB in longitudinal seroconversion panels and could discriminate between HIV-1 and -2 infection. The number of indeterminate responses was generally reduced significantly using Bionor's confirmatory test compared to the HIV-1 WB. The greater specificity, speed and ease of interpretation of Bionor's confirmatory test renders it an attractive and cost effective alternative to the WB for confirming HIV serological status worldwide.     

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.     

Amino acid sequence of the region of beta 2-glycoprotein 1 (gp1) which mediates binding of autoantibodies to the cardiolipin-gp1 complex in humans.

Anticardiolipin antibodies (ACA) in sera from patients with autoimmune and infectious diseases were tested for binding to beta 2-glycoprotein 1 (gp1) in order to determine whether human gp1 acts as a cofactor for the binding of ACA to cardiolipin (CL) or as an antigen recognized by ACA. While none of the ACA-positive sera tested recognized gp1 by itself, gp1 was necessary for the binding of ACA to CL in sera from four patients with autoimmune diseases. In three of the four sera the presence of lupus anticoagulant (LA) was detected by prolonged partial thromboplastin time (PTT). Examinations using the bovine equivalent of human gp1 contained in fetal calf serum (FCS) and adult bovine serum (ABS) showed that the human protein can be replaced by the bovine equivalent in the enzyme-linked immunosorbent assay (ELISA). Using affinity-purified antibodies directed against the CL-gp1 complex it was shown that the binding of these antibodies is dependent on the concentration of the bovine gp1 equivalent contained in the formed complex. Similar results found with the human gp1 confirmed this assertion. In order to find out which region of gp1 might mediate the binding between ACA and cardiolipin, we examined to what extent selected oligopeptide sequences of gp1 can substitute for the protein. Peptide P2 (representing the amino acids at positions 268-278 of the gp1 molecule) and gp1 showed about the same binding capacity. Histidine in this peptide seems to be essential for the binding to CL as we found decreased binding with peptides modified in this position. Conclusions from this work show that gp1 does not act as a relevant antigen for ACA, but occupies an essential function in the complex formed with cardiolipin for a certain group of ACA.     

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.     

Multicenter evaluation of a fully automated screening test, VIDAS HIV 1 + 2, for antibodies to human immunodeficiency virus types 1 and 2.

A multicenter study was done to evaluate the sensitivity, specificity, and efficiency of a new screening test for the simultaneous detection of human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) antibodies. The VIDAS HIV 1 + 2 (bioMérieux, Marcy l'Etoile, France) is a fully automated enzyme-linked fluorescent immunoassay that uses synthetic peptides from immunodominant regions of gp41 of HIV-1 and gp36 of HIV-2 as antigens. A total of 2,984 samples were evaluated with this system in six different laboratories, and the results were compared to those obtained with other enzyme-linked immunosorbent assays. The VIDAS HIV 1 + 2 assay showed a very good performance in terms of sensitivity (100%) and specificity (99.6%), requiring minimal manipulation and short incubation time (32 min) to give results similar to or better than those of the other enzyme-linked immunosorbent assays used for screening.     

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.