Improved enzyme immunoassays using biotin-avidin-enzyme complex.

Using the characteristic of a high-affinity complex between enzyme-conjugated avidin and biotin, we improved the techniques of enzyme-linked immunosorbent assay and ultrasensitive enzymatic radioimmunoassay. The method included biotinylation of a specific antibody, complexing of a biotinylated antibody with avidin-alkaline phosphatase conjugate, and addition of the antibody-conjugate complex (with or without a standard amount of antigen for competitive or noncompetitive assays, respectively) to the microtiter wells containing antigen in the solid phase. Once biotinylated, antibodies could be stored for several months. Complexing of biotinylated antibody with avidin-alkaline phosphatase conjugate could also be performed once, and the stock solution could be used for several days. Thus, the steps of enzyme immunoassays were reduced to the addition of only antibody-conjugate complex and enzyme substrate. The improved assays, biotin-avidin-linked enzyme immunoassay and biotin-avidin-linked ultrasensitive radioimmunoassay, were simpler, quicker, less expensive, and more sensitive than their standard counterparts.