Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum.
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.
Ohkuma
H
H
School of Pharmaceutical Science, Showa University, Shinagawa, Tokyo, Japan
Abe
K
K
Ito
M
M
Kokado
A
A
Kambegawa
A
A
Maeda
M
M
D000818:Animals; D001559:Benzhydryl Compounds; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007106:Immune Sera / Q000302:isolation & purification; D010636:Phenols / Q000097:blood*; D011817:Rabbits; D012680:Sensitivity and Specificity; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D007106:Immune Sera; D010636:Phenols; C006780:bisphenol A; D000098826:Bisphenol A Compounds