A hypersensitive biotin-avidin-TRFIA for quantitative detection of ANA-Ig(GAM) and its clinical application

We demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) with high sensitivity and wide range for quantitative detection of ANA-Ig(GAM) antibodies using a biotin-avidin amplification system. The immunoassay was conducted by following procedures for a typical sandwich immunoreactions with cell nucleus form Hela and the Eu(3+)-labeled biotin combined with biotinylated mouse anti-human Ig(GAM) served as the solid nuclear antigen for ANA and the tracer, respectively. The sensitivity, specificity, and stability of the kit were evaluated and comparison with the classical enzyme-linked immunosorbent assay (ELISA) kit was also made. The average intra-assay and interassay CVs detected by the established ANA-Ig(GAM) biotin-avidin-TRFIA were 4.21% and 6.34%, respectively. The lower detection limit was 2.24 U/mL, and the mean recovery rate was 100.74%. The good measurable range of the established biotin-avidin-TRFIA was within 1.95-64,000 U/mL, while it was only within 32.5-4000 U/mL using an ELISA kit. The values determined by the biotin-avidin-TRFIA and ELISA correlated well (R2 = 0.989). The positive rate of healthy volunteers and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC), Sjögren's syndrome (SS), scleroderma, and mixed connective tissue disease (MCTD) was 0, 100%, 18.5%, 100%, 37.9%, 90.9%, and 92%, respectively. We conclude that the biotin-avidin-TRFIA we developed gives promise for greater sensitivity and accurate detection for ANA-Ig(GAM) in diagnosing and monitoring autoimmune disorders.