Double-antigen sandwich time-resolved immunofluorometric assay for the detection of anti-hepatitis C virus total antibodies with improved specificity and sensitivity

Abstract

Current anti-hepatitis C virus (HCV) antibody screening immunoassays are routinely based on an indirect format. Although their use for anti-HCV antibody detection has achieved a very high specificity and sensitivity, false-positive results are still a problem especially among populations with a low prevalence of HCV infection. One strategy to obviate this problem is to adapt the assay from an indirect format to a double-antigen sandwich one to further improve its specificity. In this study, a double-antigen sandwich time-resolved immunofluorometric assay (DAS-TRIFMA) has been developed to detect total anti-HCV antibodies based on biotin-streptavidin interaction. For comparison, 1025 samples were analysed by the DAS-TRIFMA and three indirect anti-HCV antibody detection methods. For samples with discordant results, PCR-ELISA and Inno-LIA were employed as supplementary assays to analyse the presence of HCV antibodies. With regard to the 1025 clinical samples, the overall concordance between the DAS-TRIFMA and the three indirect methods was 99.41, 98.93 and 98.93 % for Ortho ELISA 3.0, WAT ELISA and I-TRIFMA, respectively. The specificity/sensitivity of the DAS-TRIFMA, Ortho HCV ELISA 3.0, WAT HCV ELISA and I-TRIFMA were 100/99.09, 99.34/98.18, 99.23/97.27 and 99.01 %/98.18 %, respectively. The DAS-TRIFMA was able to detect HCV antibodies at a concentration about 1/10 of that detectable by indirect methods. From the obtained results and their comparison, it is concluded that the DAS-TRIFMA is a more specific and reliable method for screening anti-HCV antibodies, and weakly positive S/Co values by the DAS-TRIFMA were more predictive of HCV infection than those by indirect methods.

Evaluation and Comparison of Three Different Anti-Hepatitis C Virus Antibody Tests based on Chemiluminescence and Enzyme-Linked Immunosorbent Assay Methods used in the Diagnosis of Hepatitis C Infections in Turkey

“…23 Owing to the high immunoglobulin G (IgG) concentration of human blood (generally > 5 mg/ml), there is a strong tendency for some IgG molecules to bind to the microwell surface by direct adsorption or by indirect capture via the surface molecules, which leads to signal generation and a falsepositive result. 23 This becomes more of a problem when the samples are from patients with systemic lupus erythematosus, portal cirrhosis, rheumatoid arthritis and some infectious diseases due to the very complex mixture and high concentration of immunoglobulin components in the blood of these patients. 23 Because HCV has yet to be cultured, natural HCV proteins are not available and, as a result, antibodies of a tertiary and quaternary nature, which can only be determined with natural antigens, are unlikely with the recombinant/synthetic antigens in the currently available kits.…”

Restricted Enzooticity of Hepatitis E Virus Genotypes 1 to 4 in the United States

“…Most of these EIAs employ for antibody capture antigens that are derived from HEV genotypes 1 and 2 and so may not be sufficiently sensitive to detect heterotypic antibodies generated in human and animal hosts infected by genotype 3 (19,24,45,48) or genotype 4 (2,23,62). Moreover, some indirect EIAs tend to generate false-positive reactivities (52,65) and therefore require, in order to validate the specificity of their reactivities, the extra steps of neutralization, immunoblotting, or prior production of genus-specific positive controls (1,12,35,45). To circumvent such problems inherent to indirect EIAs, DASAs have been developed.…”

Diagnosis of hepatitis C virus infection by enzyme‐linked immunosorbent assay and reverse transcriptase‐nested polymerase chain reaction: A comparative evaluation

“…Among them, recombinant immunoblot assay (RIBA), which identifies antibodies to individual HCV antigens (22), and also three generations of serodiagnostic anti-HCV antigen tests have been developed, with each new generation providing incremental improvements in the sensitivity to anti-HCV antibodies. The third-generation ELISAs are designed to detect antibodies to four recombinant HCV proteins, and it was reported to be more specific than their predecessors (23). Recently, a variety of home-brew or in-house PCR assays to test for the seropresence of HCV RNA are available.…”