Reactivity patterns and infection status of serum samples with indeterminate Western immunoblot tests for antibody to human immunodeficiency virus type 1.

Serum samples with indeterminate Western blot (WB) tests from 61 individuals whose sera were positive by enzyme-linked immunosorbent assay (ELISA) were studied in order to characterize their putative reactions with the human immunodeficiency virus (HIV) proteins and to resolve the HIV infection status of these individuals. The reaction observed by WB could not be confirmed either by radioimmunoprecipitation assay and subsequent electrophoresis (RIPA) or by use of LiaTek (Organon Teknika, Turnbout, The Netherlands) in 28% of the samples. Of the 86 samples that were indeterminate by WB, 66 reacted with p24 by WB; this reaction was confirmed by RIPA in only 21 (32%) and by LiaTek in 49 (74%) of the 66 samples. On the other hand, none of the indeterminate samples that reacted with HIV envelope proteins by WB did so by LiaTek, while 50% precipitated at least some of these proteins in the RIPA. The sensitivities of the three methods for detecting the antibody reaction with the different HIV proteins, which were studied with serial dilutions of positive serum samples, were similar. Thus, a lower sensitivity of RIPA or LiaTek does not seem to be the cause for the lack of reaction of the WB-indeterminate samples by these two methods. Sequential samples from individuals whose serum samples reacted by the three methods gave reproducible results, but all showed low antibody titers. Peripheral blood mononuclear cells obtained from three of the four individuals with sequential samples that reacted with HIV env proteins by WB and RIPA were negative for HIV provirus DNA after amplification by the polymerase chain reaction.     

Secreted portion of glycoprotein g of herpes simplex virus type 2 is a novel antigen for type-discriminating serology

The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of >or=98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology.     

Detection of antibodies to human immunodeficiency virus in vaginal secretions by immunoglobulin G antibody capture enzyme-linked immunosorbent assay: application to detection of seminal antibodies after sexual intercourse.

In order to evaluate a commercial immunoglobulin G (IgG) antibody capture enzyme-linked immunoassay (ELISA) (Wellcozyme HIV1 + 2 Gacelisa; Murex Diagnostics Limited, Dartford, United Kingdom) for the detection of antibodies to human immunodeficiency virus (HIV) in vaginal secretion samples (VS) from HIV-seropositive and -seronegative women, serum samples (S) and VS were obtained from 129 African women living in the Central African Republic, a country of high HIV prevalence. Sera were tested for HIV by routine second-generation ELISA with confirmatory Western blot (immunoblot) (WB). By the Gacelisa IgG immuno-capture assay, 45 VS were positive and 84 were negative, whereas by WB, 44 VS were confirmed positive and 85 were confirmed negative. Considering WB as a reference, the IgG immunocapture assay in VS was 97.7% sensitive (43 of 44 positive samples) and 97.6% specific (83 of 85 negative samples). Of 42 HIV-seropositive women, 41 (97.6%) had S and VS that both were HIV positive (S+ VS+), and of 87 HIV-seronegative women, 83 (95.4%) had S and VS that both were HIV negative (S- VS-). Five women had discordant results for S and VS. One (S+ VS-) possibly had a false-negative VS result. Two (S- VS+) had similar indeterminate patterns for S and VS in WB. Two (S- VS+) had a typical HIV-positive pattern on WB of VS, whereas S results in WB were indeterminate in one case and negative in the other case; for both women, detection of prostatic acid-phosphatase was positive in VS, strongly suggesting recent sexual intercourse with an HIV-positive man. Because all HIV-infected men have detectable IgG antibodies to HIV in the seminal fluid, an HIV-seronegative rape victim with HIV-positive VS (S- VS+) should receive short-term antiviral therapy to prevent possible HIV transmission.     

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.     

Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.     

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.     

Flow cytometric indirect immunofluorescence assay with high sensitivity and specificity for detection of antibodies to human immunodeficiency virus (HIV)

A flow cytometric immunofluorescence assay (FIFA) was developed to detect antibodies to human immunodeficiency virus (HIV) using live cell indirect immunofluorescence and analysis by flow cytometry. A panel of 107 sera, previously tested for anti-HIV antibody with the Abbott Enzyme-Linked Immunosorbent Assay test and Western blot (WB), was rescreened by FIFA. Antibodies were tested on HIV-infected and uninfected H9 cells in the FIFA. Although ELISA results indicated seven false positive results by comparison with the WB, 46 of 46 FIFA positive results tested WB positive and 61 of 61 FIFA negative results were WB negative. The results of FIFA showed 100% sensitivity and 100% specificity compared with WB. This rapid, quantitative, relatively easy assay makes FIFA an alternate confirmatory test for the presence of HIV antibodies.     

Comparison of enzyme-linked immunosorbent and indirect hemagglutination assays for determining anthrax antibodies.

An enzyme-linked immunosorbent assay has been established to measure anthrax antibody titers. The protective antigen component of anthrax toxin was used as the capture antigen. Two types of conjugates (protein A-horseradish peroxidase and anti-human immunoglobulin G plus immunoglobulin A plus immunoglobulin M-horseradish peroxidase) were tested. Results from enzyme-linked immunosorbent assay testing were compared with those from indirect hemagglutination titers on serum from vaccinees. The overall trend of enzyme-linked immunosorbent assay and indirect hemagglutination titers was significant. The enzyme-linked immunosorbent assay offered speed, precision, and reduced cost per test.     

Detection of immunoglobulin M antibodies toTreponema pallidum in a modified enzyme-linked immunosorbent assay

The indirect enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies toTreponema pallidum in sera of syphilitic patients is complicated by false positive reactions due to the interference of IgM rheumatoid factor (IgM-RF) activity and the presence of treponemal IgG antibodies. Another source of error producing false negative results is the competition between treponemal IgG and IgM antibodies for the binding sites on the antigen. To avoid these complications in the indirectTreponema pallidum-specific IgM-ELISA, total IgG was immunoprecipitated from sera of syphilitic patients prior to the assay. The IgM-RF from non-precipitated sera reacted in an IgM-RF-ELISA and in theTreponema pallidum-IgM-specific ELISA with identical titers. After precipitation of total IgG no reactíon of the IgM-RF in the assay could be demonstrated. Competition between IgG and IgM antibodies can be prevented almost completely by the precipitation procedure. The sensitivity and specificity of theTreponema pallidum-specific IgM-ELISA after immunoprecipitation of total serum IgG were shown to be higher than 97 percent.     

Antiactin and antitubulin antibodies in canine visceral leishmaniasis

Visceral leishmaniasis, a chronic and often fatal disease, is caused by the protozoan parasite Leishmania donovani. Both specific and nonspecific antibodies are produced in the course of the disease, and autoantibodies may be involved in pathogenesis. Tubulin and actin have been found to be associated with L. donovani. To learn whether antiactin and antitubulin antibodies are present in visceral leishmaniasis, we tested sera from 263 infected dogs by enzyme-linked immunosorbent assay for antibodies to the antigens L. donovani, actin, and tubulin. All samples reacted positively with L. donovani, and a high percentage reacted positively with all three antigens. Sera from 202 uninfected dogs were also tested, none reacted with L. donovani antigen, although positive reactions were observed for 8 of the samples with actin or tubulin. It was found that the antibody-antigen reaction occurred at the Fab portion of the immunoglobulin molecule. Competitive enzyme immunoassays showed that the reaction was inhibited if the positive serum was first incubated with L. donovani antigen, actin, or tubulin and then tested by enzyme-linked immunosorbent assay. These results suggest that antiactin and antitubulin antibodies are present in the sera of dogs infected with visceral leishmaniasis.     

Neospora spp. and Toxoplasma gondii antibodies in horses in the Czech Republic

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had ≥50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.     

Comparison of Enzyme-Linked Immunosorbent Assay, Western Blotting, Microagglutination, Indirect Immunofluorescence Assay, and Flow Cytometry for Serological Diagnosis of Tularemia

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.     

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.     

GACPAT HIV 1+2: A simple,inexpensive assay to screen for,and discriminate between,anti-HIV 1 and anti-HIV 2

A simple and cheap assay suitable for screening for anti-HIV 1 and anti-HIV 2 and discriminating between them was evaluated. In it specimens are incubated in U-bottomed microplate wells coated with anti-human IgG for 30 min at room temperature. After washing, 100 μl of a 1 in 50 dilution of HIV 1-coated gelatin particles (Serodia-HIV 1/2, Fujirebio) are added. Settling patterns are read on the second day: A positive reaction is indicated by adherence of the particles and a negative by a button. The HIV 1 particles are then washed away and HIV 2 particles added. Anti-HIV 2 reaction patterns are read on the third day. To assess the performance of the modified “GACPAT HIV 1+2” assay a panel of 1,621 serum/plasma specimens was used. It comprised validated anti-HIV 1 positive (n = 220), anti-HIV 2 positive (n = 214), dual anti-HIV 1/anti-HIV 2 positive (n = 11), and anti-HIV negative (n = 1,176) serum/plasma specimens. All 434 specimens that contained anti-HIV 1 or anti-HIV 2 reacted positively with the homologous particles. The 11 dually positive specimens reacted positively with both HIV 1 and HIV 2 particles. Five (2.3%) anti-HIV 1 and five (2.3%) anti-HIV 2 positive specimens gave positive reactions with both particle types, but none of the five cross-reactive anti-HIV 2 specimens were dually reactive when the order of particle addition was reversed. One repeat false positive reaction was recorded with the HIV 1 particles and none with the HIV 2 particles, giving specificities of 99.9% and 100%, respectively. Type specificity was 98.8% after the results of the reverse assay had been taken into account. The assay was sensitive for anti-HIV 1 in seroconversion series and for anti-HIV 2 in highly diluted specimens. GACPAT HIV 1 +2 is an accurate anti-HIV assay applicable to serum/plasma. With few exceptions anti-HIV 1 positive specimens are reactive only on the second day and anti-HIV 2 positive specimens only on the third day. It thus discriminates anti-HIV 2 from anti-HIV 1, probably through depletion of cross-reacting antibodies by the reaction with the particles initially added. © 1995 Wiley-Liss, Inc.     

A comparison of three methods for detection of antibodies against the major core protein p24 of human immunodeficiency virus

The native major core protein p24 of the human immunodeficiency virus (HIV) was immunoaffinity purified by a monoclonal antibody and used to develop an indirect enzyme-linked immunosorbent assay (inELISA) for detecting p24 antibodies in human sera. Its ability to detect p24 antibodies was compared to that of the immunoblotting test (IBT) and a commercial available competition ELISA (compELISA) employing recombinant HIV core protein. In tests on 60 serum samples the overall agreement of the inELISA and the IBT was 93.3%. Fifty-two samples were p24 antibody positive in both the inELISA and the IBT and of these 24 (46.2%) were positive in the compELISA. All compELISA positive samples were derived from healthy individuals, whereas of the 28 (53.8%) compELISA negative samples 1 was from a patient with acute HIV infection, 18 from healthy individuals and 9 from ARC/AIDS patients. The compELISA was able to distinguish among healthy persons with normal or low T-helper cell count (P = 0.048), as was the inELISA when p24 antibodies were titrated (P = 0.027). The inELISA equals IBT in specificity and sensitivity, is convenient and is very suitable for titration of p24 antibodies.     

Sensitive enzyme immunoassay for early diagnosis of tuberculous meningitis

Cerebrospinal fluid from patients with tuberculous, pyogenic, and viral meningitis, as well as from appropriate control individuals, were assayed for immunoglobulin G and immunoglobulin M antibody activity to Mycobacterium bovis BCG by an enzyme-linked immunosorbent assay. BCG linked covalently to plastic disks served as the antigen in a classical indirect enzyme-linked immunosorbent assay. A significant difference was found between the tuberculous meningitis group and the nontuberculous meningitis and control groups. All samples from the tuberculous meningitis group gave a positive reaction, and none of the known negative samples gave false-positive reactions. Because of its sensitivity, specificity, and predictive value, this test may be useful in the early diagnosis of tuberculous meningitis.     

Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies.     

A simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is rapidly gaining worldwide importance as one of the most economically significant diseases of swine. The antibody of Porcine reproductive and respiratory syndrome virus (PRRSV) is detected currently by the combined use of an enzyme-linked immunosorbent assay, serum neutralization test, immunoperoxidase monolayer assay, indirect immunofluorescent antibody test. These methods are time-consuming and require specialized equipment operated by trained technicians. The purpose of this study was to evaluate a simple strip assay (based on a chromatographic and immunogold system) for specific detection of PRRSV antibody in swine sera. This "immunochromatographic strip" test uses Escherichia coli-expressed viral recombinant membrane protein antigen in combination with recombinant nucleocapsid protein as capture protein for detecting antibodies against PRRSV. In this study, the performance of this assay was evaluated with sera from both clinical samples and experimentally infected piglets. Detection by immunochromatographic strip test was compared with detection by a standard, available commercially, indirect enzyme-linked immunosorbent assay and an immunoperoxidase monolayer assay. The immunochromatographic test strip detected antibodies in sera known to contain antibodies to PRRSV in 95.7% sensitivity of samples from pigs infected experimentally and 98.6% sensitivity of clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was 97.8% and 98.2% for clinical and experimental serum samples, respectively.     

Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract.     

Competition ELISA using a human monoclonal antibody for detection of antibodies against human immunodeficiency virus type 1.

A novel competition ELISA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. coli-produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.