Limits of diagnostic accuracy of anti-hepatitis C virus antibodies detection by ELISA and immunoblot assay.

When human sera samples are tested for anti-hepatitis C virus (HCV) antibodies using different ELISA kits as well as immunoblot assay kits discrepant results often occur. As a result the diagnostics of HCV infection in such sera remains unclear. The purpose of this investigation is to define the limits of HCV serodiagnostics. Overall 7 different test kits of domestic and foreign manufacturers were used for the sampled sera testing. Preliminary comparative study, using seroconversion panels PHV905, PHV907, PHV908 was performed and reference kit was chosen (Murex anti-HCV version 4) as the most sensitive kit on the base of this study results. Overall 1640 sera samples have been screened using different anti-HCV ELISA kits and 667 of them gave discrepant results in at least two kits. These sera were then tested using three anti-HCV ELISA kits (first set of 377 samples) or four anti-HCV ELISA kits (second set of 290 samples) at the conditions of reference laboratory. In the first set 17.2% samples remained discrepant and in the second set - 13.4%. "Discrepant" sera were further tested in RIBA 3.0 and INNO-LIA immunoblot confirmatory assays, but approximately 5-7% of them remained undetermined after all the tests. For the samples with signal-to-cutoff ratio higher than 3.0 high rate of result consistency by reference, ELISA routing and INNO-LIA immunoblot assay was observed. On the other hand the results of tests 27 "problematic" sera in RIBA 3.0 and INNO-LIA were consistent only in 55.5% cases. Analysis of the antigen spectrum reactive with antibodies in "problematic" sera, demonstrated predominance of Core, NS3 and NS4 antigens for sera, positive in RIBA 3.0 and Core and NS3 antigens for sera, positive in INNO-LIA. To overcome the problem of undetermined sera, methods based on other principles, as well as alternative criteria of HCV infection diagnostics are discussed.

Performance of ELISA HBsAg Test Kits with Use of the Different Types of Panels Containing HBsAg.

Serological diagnostics of hepatitis B in clinical laboratories is mainly based on detection of HBs antigen (HBsAg) in human serum or plasma using commercial ELISA kits. In manufacturing and laboratory practice, sensitivity of ELISA kit is measured against either international or national reference standard. This approach is necessary, but limited as it does not take into account factors that influence on HBsAg detection kit potency at low HBsAg concentration and when HBsAg subtypes/variants are present in species. Several panels containing human HBsAg positive and HBsAg negative sera were prepared and then used for testing of commercial kits HBsAg detection efficacy either under the conditions of reference or clinical laboratories: (1) broadened panels containing 138 and 157 samples were used for lot-to-lot comparison of kits detecting HBsAg; (2) low titer panel containing 21 samples was used for the evaluation the detection limit of kits; (3) mini low titer HBsAg panels containing 17 samples were used for interlaboratory control in Moscow; (4) panel containing 38 diluted human sera (35 sera with "wild" type HBsAg and 3 abnormal sera with presumably mutant HBsAg) was used for the performance evaluation of kits. Variants of HBsAg were found in 11 sera among 132 HBsAg positive sera titrated in ELISA in the presence of monoclonal antibodies against a-determinant of HBsAg. For 4 of them the measured level of HBsAg varied 10-100 times with the change of the kit or type of monoclonal antibodies. The data obtained suggest that each control panel could be a useful tool for estimation of both the kit detection potency and laboratory assay efficacy.