Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum

Abstract

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.