Abstract: Abstract Objective: to establish a double antigen sandwich enzyme linked immunosorbent assay (ELISA) to detect the specific antibody in milk allergic patient serum. Methods: main components of milk allergen were used to coat microwell plate and prepare enzyme conjugate antigen by labelling horseradish peroxidase (HRP). The specific antibody in test serum was combined with coating antigen and enzyme conjugate antigen simultaneously to form the double antigen sandwich compound. After enzyme catalyzed coloration and reaction termination, the absorbance at 450 nm (A450) was read and results were judged by setting 2.1 A450 of negative control as the cut off value. Results: the coefficient of variations (CV) in the group and between groups of the developed method were 3.7%-13.6%, and its sensitivity, specificity, accuracy were 91.7%, 94%, 93.3% respectively. Conclusion：the double antigen sandwich ELISA established in this study can detect the specific antibody in doubtful milk allergic patients serum，showing its feasibility in vitro diagnosis of milk allergy.

Key words: milk allergy, ELISA, double antigen sandwich method, specific antibody