Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: Application to an enzyme immunometric assay

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly t o monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradia tion energy and wavelength. The best results were obtained at 254 (10 J/cm( 2), 45-min irradiation) and 312 nm (40 J/cm(2), 160-min irradiation:). The irradiation fully denatured both individual molecules (i.e., 17 beta-estrad iol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has c onsiderable potential. We used this original approach to develop a new enzy me immunometric assay of 17 beta-estradiol based on our previously describe d immunometric procedure, solid-phase immobilized epitope immunoassay, whic h uses chemical agents to cross-link haptens via amino groups to specific a ntibodies. The assay was specific (no cross-reactivity with other natural s teroids), precise, and sensitive (detection limit of 38 pg/mL in human seru m). It correlated well with two competitive commercial immunoassays when te sted on 40 human sera.