Immunosorbent Assay For Detecting α-lactalbumin And β-lactoglobulin From Cow' Milk

Cow's milk allergy(CMA) is one of the most common food allergies and becoming a security risk cannot be ignored during the growth of infants and children. CMA not only affects the growth and development of infants,but also can cause death when severe allergic response occurred.Many studies have been foundα-lactalbumin andβ-lactoglobulin are the major allergenic protein inducing CMA, even at trace levels.Therefore,establishment of dairy and related food Cow's milkβ-lactoglobulin andα-lactalbumin detection system have great realistic meaning in the practice of prevention CMA.β-lactoglobulin andα-lactalbumin were isolated and purified by saturate ammonium sulfate salting out and DEAE-52 anion-exchange chromatogram.The result of SDS-PAGE revealed the purity of obtainedβ-lactoglobulin andα-lactalbumin both reach the desired purity of the standard antigen.The purifiedβ-lactoglobulin andα-lactalbumin were used as the antigen to immunize rabbit,then the anti-βLg and anti-αLA polyclonal antibody were prepared, titer of the anti-serum determined by indirect ELISA both were 1:12800.The result of western blotting showed that the antibody high specificity was successfully obtained by immunization of rabbits withβ-lactoglobulin andα-lactalbumin can be used to detect enzyme-linked immunosorbent assay.After optimizing the condition of ELISA, stablishment of indirect competitive enzyme-linked immunosorbent assay for detectingβ-lactoglobulin andα-lactalbumin from cow'milk.Establishment of indirect competitive enzyme-linked immunosorbent assay for detectingβ-lactoglobulin from cow' milk:the optimal concentration of the coating antigen was 1μg/L,the working concentration ofβ-LG-PcAb and sheep anti-rabbit IgG was 1:1600 and 1:800 respectively,then got the regression equation:y＝-28.64x+122.5,R~2=0.991 and the most suitable detect range:10-1000 ng/mL ng/L.Through evaluated this method,gained the coefficients of variation of intra-assay and inter-assay were 6.25%%and 8.15%;All the index accorded with the demand of indirect competitive ELISA,Can be used forβ-lactoglobulin Detection in dairy and related food.Establishment of indirect competitive enzyme-linked immunosorbent assay for detectingα-lactalbumin from cow'milk:the optimal concentration of the coating antigen was 5μg/L,the working concentration ofα-LA-PcAb and sheep anti-rabbit IgG was 1:1600 and 1:800 respectively,then got the regression equation:y=-43.24x +166.88,R~2=0.9844 and the most suitable detect range:50-1000 ng/mL.Through evaluating this method,gained the coefficients of variation of intra-assay and inter-assay were 5.05%and 8.92%.All the index accorded with the demand of indirect competitive ELISA,Can be used forα-lactalbumin Detection in dairy and related food.