Matrix interference in serum total thyroxin (T4) time-resolved fluorescence immunoassay (TRFIA) and its elimination with the use of streptavidin-biotin separation technique

In our development of total serum thyroxin TRFIA using an immobilized secon d-antibody (S-Ab) as the separation agent, we observed a significant measur ement bias caused by a matrix interference when the immobilized S-Ab had a relatively low binding capacity for the primary anti-T4 monoclonal antibody (McAb). Therefore, we employed a new separation system based on the highly active surface streptavidin and biotinylated anti-T-l McAb. Our results in dicate that the matrix interference was removed and the assay performance w as improved with the use of streptavidin-biotin separation technique. In ou r method, microwells were first coated with biotinylated BSA and then a str eptavidin solution in the presence of 1% BSA was added to allow streptavidi n to be immobilized via the pre-coated BSA-biotin. Surface streptavidin pre pared in this protocol expressed a significantly increased binding capacity for the biotinylated anti-T4 McAb, compared to the passively adsorbed S-Ab for binding the original anti-T4 McAb. The immunoreactions between the bio tinylated anti-T4 McAb and the T4 in the standard or sample or the europium -labeled T4-BSA conjugate mainly occurred in liquid solution, and then the immune complex was specifically trapped by the surface streptavidin and iso lated from the free trace by washing. Serum TT4 TRFIA based on surface stre ptavidin was accurate, precise and economic, maintained all the merits of t he directly immobilized surface antibodies. (C) 2001 Elsevier Science B.V. All rights reserved.