Aim
To develop a new immunoassay based on the time-resolved fluorescence immunoassay (TRFIA) system for the simultaneous measurement of IgM and IgG antibodies to HCV.
Conclusion
We established a dual-label HCV-IgM/IgG TRFIA assay with a wide detection range, high specificity, high sensitivity, good stability, and good clinical value for the simultaneous measurement of HCV-IgM and HCV-IgG titers in a single test.
Methods
Coated recombinant HCV antigens and Eu3+ -labeled IgM and Sm3+ -labeled IgG antibodies were prepared. HCV-IgM/IgG TRFIA was established and optimized, followed by a methodological assessment. Data were expressed as ¯xx¯<math> <mover><mrow><mi>x</mi></mrow> <mrow><mo>¯</mo></mrow> </mover> </math> +SD and analyzed using the SPSS 13.0 software. The percentile method was used to calculate cutoff values.
Results
The detection sensitivities of HCV-IgM and HCV-IgG were 0.06 S/CO and 0.15 S/CO, respectively. There was a good linear response from 1:40 to 1:40 960 for HCV-IgM and 1:20 to 1:40 960 for HCV-IgG, when samples strongly positive for HCV-IgM and HCV-IgG were serially diluted from 1:10 to 1:81 920. The average intra-assay coefficients of variation (CV) for HCV-IgM and -IgG were 3.45% and 3.71% and the inter-assay coefficients of variation (CV) were 6.49% and 6.79%, respectively. When HCV-negative/positive sera were tested by ELISA and using established kits, the negative, positive, and total conformity rates for HCV-IgM were 93.3% (28/30), 100% (25/25), and 96.4% (53/55), and those for HCV-IgG were 93.3% (28/30), 100% (35/35), and 96.9% (63/65), respectively. Additionally, the established kit exhibited good stability, with declines in fluorescence values to 11.1% and 9.5%, respectively, after storage at 37°C for 7 days.