Antigen- versus antibody-immobilized ELISA procedures based on a biotinyl-estradiol conjugate

A biotinyl-6 alpha-estradiol derivative (Bio-E2) was synthesized and used as the key component in antigen- and antibody-immobilized ELISA techniques, and the relative merits of the two methods were compared. A precise and reproducible antigen-immobilization was achieved in avidin-coated microtiter plates with Bio-E2. This assay, when completed by the incubation with primary antibody and second antibody-peroxidase conjugate, has a very low detection limit (6 pg/ml estradiol) but required a long incubation time with primary antibody to reach equilibrium. At non-equilibrium conditions, using a high antibody concentration, the assay could be very fast and sensitive. In the antibody-immobilized assay, the Bio-E2 was added to compete with the estradiol present in the calibrator or sample and visualized with a streptavidin-peroxidase conjugate. The detection limit is higher (34 pg/ml), but the specificity was superior and the incubation time to reach equilibrium shorter as compared to the antigen-immobilized assay. Therefore, the antibody-immobilized assay appeared to be ideal for the classical ELISA technique, whereas the antigen-immobilized method seemed to be best suited for automated assay systems using antibody in excess.