Establishment Of ELISA Detection Technology For The Content Of Milk And Cow Whey Powder In Goat Milk And Goat Milk Powder

Goat’s milk and cow’s milk are rich in fat,protein,minerals and other nutrients,and their types and content are different.Goat’s milk has a high nutritional content,low sensitization and low yield,resulting in a much higher market value than cow’s milk..But the physical properties of the two are so close that it is difficult to distinguish them by senses.Protein in goat’s milk and cow’s milk is made up of casein and whey proteins.Casein mainly includesα-casein,β-casein andκ-casein,whey proteins includeα-lactoalbumin,β-lactoglobulin,lactoferrin and immunoglobulin.Although both milk and cow’s milk contain these proteins,there is a difference in the amount ofα-casein in goat’s milk,which is much lower than that in cow’s milk.More importantly,there are also differences in the molecular structure of various proteins in cow’s milk and goat’s milk,which can be specifically recognized by monoclonal antibodies.This is the basis for the establishment of an ELISA method for the detection of bovineα-casein and bovineβ-lactoglobulin based on the principle of antigen-antibody specific reaction.Therefore,in this study,bovineα-casein and bovineβ-lactoglobulin were used as antigens.Monoclonal antibody preparation technology and antibody purification technology were used to prepare antibodies,so as to establish an indirect ELISA technology for the detection of milk and bovine whey powder components and their contents in goat milk and goat milk powder.The results of this study are as follows:1.The concentration of bovineα-casein was 10μg/m L overnight at 4℃,the dilution of primary antibody and secondary antibody were 1:1000 and 1:5000,respectively,and the incubation time was 45 min.The optimal reaction conditions for the detection of bovineα-casein by indirect ELISA were obtained.2.The purified bovineα-casein monoclonal antibody was obtained with the concentration of 0.986 mg/m L and the antibody titer was 5?10~5.3.Using bovineα-casein monoclonal antibody as detection antibody,the standard curve for the detection of bovineα-casein was established according to the steps of indirect ELISA method,and the standard curve for the detection of the content of cow’s milk in goat’s milk and that of cow’s milk powder in sheep’s milk powder was established.4.When the concentration of bovineβ-lactoglobulin was 10μg/m L,the concentration of bovineβ-lactoglobulin was 10μg/m L,the dilution of primary antibody and secondary antibody were 1:1000 and 1:5000,and the incubation time was 45min,the optimal reaction conditions for the detection of bovineβ-lactoglobulin by indirect ELISA were obtained.5.A hybridoma cell line 5-D11 was obtained,which could secrete bovineβ-lactoglobulin monoclonal antibody with Ig G2b subtype andκlight chain type.The concentration of purified bovineβ-lactoglobulin antibody was 0.909 mg/m L,and the antibody titer was 5?10~5.6.Using bovineβ-lactoglobulin monoclonal antibody as detection antibody,the standard curve of bovineβ-lactoglobulin and the standard curve of bovine whey powder in sheep milk powder were established according to the indirect ELISA detection procedure.In this research,monoclonal antibodies against bovineα-casein and bovineβ-lactoglobulin were successfully prepared,and quantitative detection methods for the adulteration of goat milk,goat milk powder and bovine whey powder were established based on indirect ELISA technique,which has important value for the adulteration and differential detection of goat milk,milk and milk products.