Cross-Linking of 17β-Estradiol to Monoclonal Antibodies by Direct UV Irradiation: Application to an Enzyme Immunometric Assay

Ultraviolet irradiation was used to cross-link 17β-estradiol directly to monoclonal anti-17β-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation 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., 17β-estradiol and monoclonal anti-17β-estradiol antibody), but 17β-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17β-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17β-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.