Immunochemistry For Analysis Of Diethylstilbestrol

Two haptens diethylstilbestrol-mono-carboxylic methyl ether (DES-MCME) and diethylstilbestrol-mono-carboxylic propyl ether (DES-MCPE) with different length of spacers 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 DES-MCME-BSA and DES-MCPE-BSA, and coating antigens DES-MCME-OVA and DES-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 50:1 of haptens to protein would be appropriate molar ratio for reactants, and the molar ratios of haptens to protein for conjugates were 10~30:1.The antisera were prepared by using DES-MCME-BSA and DES-MCPE-BSA as immunogens to immunize New Zealand white rabbits. After the titers of anti- DES-MCME-BSA serum (for antibodyⅠ) and anti-DES-MCPE-BSA serum (for antibodyⅡ) were higher than 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.DES-MCME-OVA(coating antigenⅠ) and DES-MCPE-OVA(coating antigenⅡ) were used to coat micro-plate respectively and the indirect competitive ELISA for DES was developed. The results showed that antibodyⅠhad no specific affinity to DES while antibodyⅡhad. The concentration of coating antigenⅡand antibodyⅡwere selected by phalanx test and the optimized concentration were 1.5μg/ml both. The effects of pH, ionic strength and organic solvent on affinity of coating antigenⅡto antibodyⅡand on inhibition rate of DES to antibody-antigen reaction were evaluated respectively. It was shown that low pH value of the medium was better than high pH value, the most appropriate pH value was 6.6 for antigen-antibody reaction. Ionic strength of the medium had slightly effect on the affinity of coating antigenⅡand antibodyⅡ, the most appropriate ionic strength was 0.15mol/L, the inhibition rate of...