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 second-antibody (S-Ab) as the separation agent, we observed a significant measurement 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-T4 McAb. Our results indicate that the matrix interference was removed and the assay performance was improved with the use of streptavidin-biotin separation technique. In our method, microwells were first coated with biotinylated BSA and then a streptavidin solution in the presence of 1% BSA was added to allow streptavidin to be immobilized via the pre-coated BSA-biotin. Surface streptavidin prepared 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 biotinylated 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 isolated from the free trace by washing. Serum TT4 TRFIA based on surface streptavidin was accurate, precise and economic, maintained all the merits of the directly immobilized surface antibodies.
Wu
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Laboratory of Immunoassay, Department of Isotope, China Institute of Atomic Energy, 102413, P.O. Box 275-39, Beijing, People's Republic of China. WuFb@etang.com
He
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YF
Han
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D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004867:Equipment Design; D015200:Fluoroimmunoassay / Q000379:methods; D006801:Humans; D007103:Immobilization; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D013974:Thyroxine / Q000097:blood*
D000911:Antibodies, Monoclonal; D020099:Coated Materials, Biocompatible; D012710:Serum Albumin, Bovine; D001710:Biotin; D019809:Streptavidin; D013974:Thyroxine