Two Enzyme-linked Immunoassay Methods For The β-lactoglobulin Detection

Many studies have found that P-lactoglobulin is one of the major allergenic proteins in cow’s milk allergy. Therefore, establishment of detection system for dairy and related food containing cow’s milk β-lactoglobulin have great realistic meaning in the practice of preventing Cow’s milk allergy. A sandwich enzyme-linked immunosorbent assay (ELISA) and a indirect competitive enzyme-linked immunosorbent assay (ELISA) for β-lacto globulin were developed in this study.Rabbit antiserum of β-lactoglobulin was prepared successfully by using the β-lactoglobulin as immunogen and the titer was128000. The antibody which was purified with Protein A-sepharose4B was chosen for ELISA and acted as the first antiboy. The secondary antibody was labeled with horseradish peroxidase (HRP). A sandwich ELISA was developed to detect P-lactoglobulin by the optimization of experimental conditions:the optimal concentration of the coating antigen was0.5μg/well, the working concentration of antibody labeled with HRP was1:8000, select60℃,1h as the antigen-antibody reaction conditions, then got the regression equation:y=0.4564x+0.314, R2=0.9923and the most suitable detecting range:0.047-1.50μg/mL, the limit of detection was0.031μg/mL. Coefficients of variation of intra-assay and inter-assay were5.87%and8.88%respectively.Indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting β-lactoglobulin of cow’s milk was developed:the optimal concentration of the coating antigen was0.5μg/mL, the working concentration of polyclonal antibody against P-lactoglobulin and sheep anti-rabbit IgG was1:12000and1:10000respectively, select20℃,1h as the antigen-antibody reaction conditions, then got the regression equation:y=-1.5952x-0.0681, R2=0.9915and the most suitable detecting range:0.06-1.92μg/mL. Coefficients of variation of intra-assay and inter-assay were5.03%and5.78%respectively.Cross reaction between antibody and a-lactalbumin, casein, collagen, chicken ovalbumin as well as soy protein, protein powder were examined and the results indicated that the cross-reaction rate were low. Specificities of the both methods against P-lactoglobulin were determined.The P-lactoglobulin content of fresh milk, UHT milk, milk powder, yogurt and seasoning yogurt were detected by these two established detection method. Both methods had good specificity, sensitivity, accuracy and repeatability for the detection of P-lactoglobulin. The indirect competitive ELISA which does not require preparation of HRP was more convenient than the sandwich ELISA in operation.