Improved Sensitive Sandwich Enzyme Immunoassay for Secretory Immunoglobulin a in Human Serum

To overcome the IgA interference in enzyme immunoassay for serum secretory IgA (SIgA), a new specific, simple and more sensitive sandwich enzyme immunoassay, fully free of the serum IgA interference, was developed. SIgA standards or samples were added into the wells of polystyrene plate coated with rabbit IgG antibody to human secretory component. After incubation, the wells were washed and then, horseradish peroxidase-labeled Fab′ fragment of goat IgG antibody to human α-chain was added and incubated. The wells were washed again to remove the unbound labeled antibody, and the enzyme activity specifically bound to the well was assayed using 3,3′, 5,5′ - tetramethylbenzidine and H 2 O 2 as substrate. The enzyme reaction was stopped by addition of 2M H 2 SO 4 . The SIgA concentration was determined from the absorbance at 450 nm. The minimun detectable sensitivity was 6ng/ml. The assay system had very good selectivity overcoming the interference of IgA. As the result of high sensitivity, only small amount of sample (2 μ1 for serum) was needed for analysis. In this assay, no cross reactivity was found with purified human IgG, mIgA, IgM or free secretory component (FSC). The recovery of SIgA mixed with human sera or biles was 99.6-108.1%. The coefficients of within-assay and between-assay variation were 5.8-9.3% and 6.2-9.2% respectively. It correlated well with a liquid phase competitive radioimmunoassay for human serum SIgA (r=0.96, n=30, P