4,4'-Isopropylidenediphenol, bisphenol A (BPA), was derivatized to BPA-carboxymethylether (BPA-CME), BPA-carboxypropylether (BPA-CPE) and BPA-carboxybutylether (BPA-CBE), and then linked to bovine serum albumin (BSA). The BPA-BSA conjugates were injected into female New Zealand White rabbits, which then generated six kinds of polyclonal antibodies. In addition, BPA and bisphenol B (BPB)-enzyme conjugates were derivatized to BPA-CME, BPA-CPE, BPA-CBE, BPA-carboxyphenylether (CPhE) and BPB-CPE, and then linked to horseradish peroxidase (HRP). An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of BPA standards. Although anti-BPA antibodies cross-reacted with BPB by more than 13.6% at all dilutions used, cross-reaction with phthalates and phenols occurred only less than 0.1%. The combination with the highest sensitivity was obtained using anti-BPA-CME-BSA antibody and BPA-CPhE-HRP conjugate. ELISA successfully detected BPA in human serum at concentrations as low as 0.3 ng mL(-1), and over a measurable range of 0.3-100 ng mL(-1). Recovery tests were carried out by adding BPA to three kinds of human serum, and ranged from 89.7 to 97.3%, from 85.4 to 94.9% and from 81.9 to 97.4%, respectively. The correlation between the results from ELISA and gas chromatography-mass spectrometry (GC-MS) for BPA in spiked serum was r2 = 0.990, indicating that the proposed method is a potential tool for screening a large number of human serum samples.

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A sensitive and simple chemiluminescent assay (CL) for alkaline phosphatase (ALP) using dihydroxyacetone phosphate or its ketal (DHAP or DHAP-ketal) was developed. New substrates were transformed to dihydroxyacetone (DHA) after they were hydrolysed by ALP, which reacts with lucigenin and produces strong chemiluminescence. Under the optimum assay condition, the detection limits were 3.8 x 10(-19) and 1.5 x 10(-18) moles of ALP, respectively. The coefficients of variation (CV) at each points on the standard curve were 0.8-5.4% and 1.8-7.1% (n = 6), respectively. The mechanism of lucigenin CL with DHA was investigated by ESR spectrometry using the spin-trapping method. The mechanism was speculated as follows: the O2(-) generated by the reaction of DHA and O2 in alkaline solution reacts with lucigenin, and then emit light. The proposed CL assay was applied to the enzyme immunoassay of 17beta-oestradiol, using ALP as a label enzyme. The measurable range of 17beta-oestradiol was 15-4000-pg/mL, and the proposed method was four times more sensitive than the colorimetric assay for ALP by using 4-nitrophenyl phosphate as substrate.

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