Development of a time-resolved fluorescence immunoassay for Epstein-Barr virus nuclear antigen 1-immunoglobulin A in human serum

Enzyme‐linked immunosorbent assay (ELISAs) specific for Epstein–Barr virus nuclear antigen 1 (EBNA1)‐immunoglobulin A (IgA) are most commonly used in the clinical diagnosis of EBV infection. But they have a low sensitivity and the enzyme‐labeled antibodies are unstable. In this study, a novel immunoassay based on an indirect time‐resolved fluoroimmunoassay (TRFIA) was developed. Microtiter plates were coated with recombinant EBNA1. We used Eu3+‐labeled anti‐human IgA as probe. The precision, sensitivity, specificity, and stability were evaluated, and comparison with traditional and commercially available ELISAs was also made. The cut‐off value for our TRFIA was 2.7. Intra‐ and inter‐assay coefficients of variation for the TRFIA were 1.56–4.99% and 3.92–6.95%, respectively; whereas those for the ELISA were 4.54–8.16% and 7.07–10.52%, respectively. Sensitivity was obviously better than traditional ELISA when diluted positive samples serially. Additionally, stability, specificity test and comparison of sensitivity and specificity between the TRFIA and commercial ELISAs all proved satisfactory. In conclusion, the results demonstrated that EBNA1 IgA TRFIA was a sensitive immunoassay and had potential value in large‐scale screening of human serum samples in developing countries. J. Med. Virol. 87:1940–1945, 2015. © 2015 Wiley Periodicals, Inc.