Abstract: This experiment is aimed to develop a ELISA method to detect the precise content of protein in the milk specificly. Immune the rabbit with pure casein,the polyclonal antibodies against casein were obtained. The best working concentration of antigen and antibody was determined using the checkerboard test, and the reaction conditions of each steps were optimized. The 1% BSA was served as blocking agent, and its incubation time was 120 min. The optimal dilution and incubation time of serum sample and enzyme-labeled second antibody were 1∶12000 for 90 min and 1∶3000 for 30 min respectively. The detection range of casein was 0.063 to 0.500 μg/mL. The method had good specificity, accuracy, repeatability, the range of regression rate was 97.6% and 123%.The ELISA method for detecting the protein content of milk was successfully established.

Key words: milk protein; casein; ELISA; quantitative detection