Abstract: To optimize the indirect enzyme-linked immunosorbent assay(ELISA) conditions based on prepared antiserum was studied.Antiserum titre was determined by ELISA methods.Optimal working concentrations of DES fold antigen and antibody were determined by phalanx method.Then the immunoreagent conditions,such as dilution,pH and incubation time and others,were optimized.Four similarities were choosed to calculate the cross-reactivity.Results showed that the dilute rate of antiserum can reach 1:102400.The optimal working concentrations of DES fold antigen and antibody by phalanx method are 1:200 and 1:4000 respectively.Standard curve is established through ELISA methods.This inhibitory curve well expresses the specificity of the antibody with good linear relationship.The value of IC50 is 1.89 ng/ml.The value of the LOD is 0.08 ng/ml.During the four similarities,the cross-reactivity is 3.1% for dienestrol while the other similarities are less than 0.1%.The immunoreagent conditions are thus optimized and the research provids appropriate method of ELISA for diethylstilbestrol assay.This can provide a base of the pack of DES ELISA kit.

Key words: diethylstilbestrol, indirect enzyme-linked immunosorbent assay, optimize, antiserum