Development of the ELISAs for detection of hormone-disrupting chemicals

Abstract

Six kinds of enzyme-linked immunosorbent assay (ELISA) systems were developed for the quantitative analysis of hormone-disrupting chemicals (HDCs), such as estrogen (ES: the total amount of estrone (E1), 17 β-estra (E2) and estriol (E3)), E2, bisphenol A (BPA), alkylphenol (AP), phthalate esters (PE) and chlorophenols (CP). To generate specific monoclonal antibodies against BPA, AP, PE, CP, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with carboxylated derivatives, while anti E2 monoclonal antibody was selected from those available on the market, and anti ES monoclonal antibody was purchased from Teikoku Hormone Mfg Co. Ltd. The detection limits of ES, E2, BPA, AP, PE and CP ELISAs were 0.1, 0.1, 5, 10, 200, 10 μg/L, when E2, E2, BPA, Nonylphenol (NP), Dibutylphthalate (DBP), 2,4-CP were used as standard, respectively, and the specificity of each ELISA was confirmed with the cross-reaction test using several compounds which have structural resemblance to the compounds of interest.

Immunochemical determination of xenobiotics with endocrine disrupting effects

“…Goda et al [76,77] reported the production of monoclonal antibodies recognizing both APEs and APs (NP and OP) to the same extent. The chosen immunizing hapten retained the alkyl chain (the nonyl chain) intact and was derivatized keeping five ethoxylates groups (EO) in the molecule (see hapten 1A in Scheme 1).…”

“…The magnetic properties of these particles permit the easy separation of the immobilized reagent from the media in different sequential steps. Competitive assays have been carried out using APEs as the target analyte to obtain limits of detection which were quite good (LOD=6.6 µg L -1 ) (see Table 1) compared with the direct ELISA [76]. The system also studied the pattern of cross-reactivity with AP itself and APEs individually and the same profile was observed as in commercial indications of the direct ELISA (i.e., APEs are better recognized than APs).…”

“…On the other hand, Goda et al [76] have developed a direct ELISA using specific monoclonal antibodies. By using DBP as standard, an assay with a limit of detection of 200 µg L -1 has been obtained.…”

“…A Hapten used by Ius et al [94] to obtain polyclonal antibodies. B Hapten used by Goda et al [76] to obtain monoclonal antibodies hapten. In this case the spacer arm has been introduced in both esters, blocking the two important epitopes in the molecule (structure 2B in Scheme 2).…”

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

“…Comparison between the result of indirect and competitive ELISA suggested that the IgG titre difference might be deceptively greater in the competitive ELISA. A possible explanation is that the latter assay is more sensitive as has been reported in other studies (Goda et al 2000, Reddington et al 1991, Wreghitt et al 1986). Mice sera used in both assays may contain non-immune IgGs and other serum factors, which can bind nonspecifically to the immobilised antigen and consequently cause background interference.…”