Development of novel IC-ELISA as a primary high throughput screening for various estrogen molecules

Abstract

An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) has been developed for detecting estrogen molecules in environmental samples.For generating anti-estrogen monoclonal antibody (mAb), BALB/c mice were immunized with 17β-estradiol (E2)-BSA and 5760 hybridoma cell lines were established. Through the optimization processes, a mAb (4BSA-e 3C11) and estriol (E3)-BSA were finally selected as a primary antibody and a coating antigen, respectively. The IC 50 value for a standard estrogen (17β-E2) was 6.26 ng mL 1 and the detection range (20-80% B/B 0 ) was 0.01-377.92 ng mL 1 . The developed IC-ELISA showed some cross-reactivities (CRs) to various estrogen analogues, such as estrone (E1) (1.79%), E3 (77.34%), 16-epiestriol (27.54%) and 16 keto-17β-E2 (2.02%). On the other hand, the assay showed a negligible CRs to other steroid hormones (CRs⁄0.063%), suggesting the specificity of the assay to estrogen molecules. For assay validation, the developed IC-ELISA was compared side by side with high performance liquid chromatography (HPLC), which showed no significant difference in their performances between the two methods. The sensitivity of our IC-ELISA was approximately 100 fold higher than that of HPLC. The estrogen contents (Estrogen Equivalent Concentrations; EEC) in field samples were determined using the IC-ELISA, including swine sewage effluents (7.043 0.023 ng-EEC mL 1 ), bovine feces (0.013 0.001 ng-EEC mL 1 ), and avian feces (0.017 0.001 ng-EEC mL 1 ). Conclusively, we have developed an IC-ELISA that is highly sensitive to estrogens as well as can detect various estrogen analogues at the same time. This assay can be used as a primary screening for a large number of field samples before the instrumental analysis that is laborintensive and time-consuming.