Studies On Immunochemistry For Analysis Of Bisphenol

Two haptens mono-carboxylic methyl ether (MCME) and mono-carboxylic propyl ether (MCPE) were synthesized. The haptens were covalently conjugated to carrier proteins BSA and OVA by the method of active ester or mixed anhydride to prepare artificial antigens MCME-BSA and MCPE-BSA, and coating antigens MCME-OVA and MCPE-OVA. The effect of different molar ratios of reactants on conjugate ratio of haptens to protein was investigated. The conjugates were confirmed and their molar ratios of haptens to carrier protein were determined by UV spectrophotometry. The results showed that 60:1 of molar ratio for reactants(haptens to protein)would be appropriate, and the molar ratios of haptens to protein were 20³5:1 for the resultant conjugates.The antisera were prepared by using MCME-BSA and MCPE-BSA as immunogens to immunize New Zealand white rabbits. After the titers of anti- MCME-BSA serum (for antibodyâ… ) and anti-MCPE-BSA serum (for antibodyâ…¡) reached to 32:1 by agar double immunodiffusion, the polyclonal antibodies were separated from antisera by the method of salting out with 35% saturated ammonium sulfate and freeze-dried in a high vacuum.The purified antibodyâ… a nd antibodyâ…¡were conjugated to HRP. The MCME-OVA (coating antigenâ… ) and MCPE-OVA (coating antigenâ…¡) were used to coat micro-plate respectively and the direct competitive ELISA for BPA was developed. The results showed that antibodyâ… had specific affinity to BPA, while the affinity of antibodyâ…¡to BPA was higher. The concentration of coating antigenâ…¡and HRP-antibodyâ…¡were selected by phalanx test and the optimized coating concentration is 4.0μg/mL and the HRP-antibodyâ…¡was diluted two hundred times. The effects of pH, ionic strength and organic solvent on the affinity of coating antigenâ…¡to HRP-antibodyâ…¡and the inhibition rate of BPA to antibody-antigen reaction were evaluated respectively. The most appropriate pH value was 7.2 for antigen-antibody reaction. Ionic strength of the buffer had slightly effect on the affinity of coating antigenâ…¡and HRP-antibodyâ…¡. Low concentration of acetone can promote the combination of coating antigenâ…¡and HRP-antibodyâ…¡, but the affinity of coating antigenâ…¡to HRP-antibodyâ…¡decreased while the concentration of acetone was higher than 3%, the inhibition rate of BPA to antibody-antigen reaction decreased apparently as the concentration of methanol increased. Under optimized conditions, the standard ELISA inhibition curve of BPA to antibody-antigenâ… reaction was developed and the regression equation was I=16.059LogC+44.574, r=0.9949, the half-maximal inhibition (IC₅₀) was 16.07ng/mL with relative standard deviation (RSD) was 6.27 % (n=5) and the limit of detection (IC₂₀) was 0.28ng/mL. The standard ELISA inhibition curve of BPA to antibodyâ…¡-antigenâ…¡reaction was also developed and the regression equation was I=20.967LogC+40.169, r=0.9903, the IC₅₀ was 2.68ng/mL with RSD of 8.8% (n=5) and the limit of detection (IC₂₀) was 0.13ng/mL.The standard ELISA inhibition curve of Diethylstilbestrol(DES), an analog of BPA, to antibodyâ…¡-antigenâ…¡reaction was found. The regression equation of the standard ELISA inhibition curve was I=25.59LogC-9.214, r=0.9967, IC₅₀=206.1 ng/mL. The cross reaction (CR%) of DES to antibodyâ…¡was less than 1.3%.After the BPA was added into natural water at the level of 3ng/mL, the spiked water was detemined by the direct competitive ELISA, the recovery ranged from103.4% to128.8%, the average recovery was 110.6% with RSD of 9.4% (n=5).