Establishment of Eu3+ /Sm3+ dual-label time-resolved fluoroimmunoassay for measurement of antibodies to hepatitis C virus and its clinical application

Abstract

Objective To establish a time-resolved fluoroimmunoassay (TRFIA) system for simultaneous measurement of immunoglobulin (Ig)M and IgG antibodies to HCV. Methods Recombinant HCV antigen was fixed on microtiter plates to detect serum HCV antibodies. Eu3+ -labeled anti-human IgM and Sm3+ -labeled anti-human IgG were prepared. HCV-IgM and HCV-IgG TRFIA were established using indirect assay and further optimized and evaluated. The one-sided 95th-percentile was used to calculate the cut-off (CO) values. Results Defining 1 sample/CO (S/CO) as measuring unit, the detection limit was 0.06 S/CO for HCV-IgM and 0.15 S/CO for HCV-IgG. When diluted a strong-positive specimen from 1∶12.5 to 1∶51 200, there was a good liner range within 1∶12.5 to 1∶12 800 for HCV-IgM and 1∶25 to 1∶6 400 for HCV-IgG. The average intra-assay CV of HCV-IgM and HCV-IgG were 3.37% and 3.66%, respectively, and the average inter-assay CV were 6.52% and 6.75%, respectively. Compared with enzyme-linked immunosorbent assay (ELISA) kits, the positive conformity rate, the negative conformity rate and total conformity rate were 100.0%(20/20), 90.0%(18/20), 95.0%(38/40) for HCV-IgM TRFIA, and were 100.0%(20/20), 95.0%(19/20), 97.5%(39/40) for HCV-IgG TRFIA, respectively. Additionally, the established HCV-IgM and HCV-IgG assay kits presented good stability with a decline in the value of fluorescence of 11.1% and 9.5% respectively after being stored at 37 ℃ for 7 d. Conclusions The established HCV-IgM and HCV-IgG TRFIA could simultaneously measure HCV-IgM and HCV-IgG antibodies at one step. The method has wider detectable range and may be a more sensitive, stable, and reliable method for diagnosing HCV infection. Key words: Hepatitis C antibodies; Fluoroimmunoassay; Europium; Samarium