Preparation And Identification Of Monoclonal Antibody Against Caprine ?s1-casein

Goat's casein is the most important nutrient for breastfeeding lambs or humans who are fed with goat's milk.In addition,the level of synthesis and secretion of caprine's milk is also one of the important indicators reflecting the physiological function of goat's mammary epithelial cells.Therefore,the determination of goat's casein is not only for accurately assessing the dry matter content of goat's milk,determining the quality of goat milk processing products,but also has important theoretical and practical value for evaluating the physiological state of goat's mammary gland,and for identification and functional identification of mammary epithelial cells.Protein qualitative and quantitative assays using monoclonal antibodies as a tool are the most commonly used,most common,and most scientific techniques for protein assays.But the prerequisite for this technique is the development of monoclonal antibodies that detecting target proteins.In this study,goat's milk was used as the material to isolate the goat's casein by isoelectric point precipitation.The purified casein was used as the antigen to immunize 6-week-old BALB/c mice to establish an ELISA assay for casein antibody.The casein antibody titer of the immunized mouse is determined,and the positive hybridoma cells secreting anti-casein monoclonal antibody are screened by using the conventional monoclonal antibody preparation technology,and the mouse spleen cells and SP2/0 cells are prepared for screening.Monoclonal antibodies were identified according to conventional requirements.Finally,the feasibility of the developed monoclonal antibody of casein in some detection techniques was discussed.The study obtained the following results:1.Isolated caprine's casein and the corresponding immunized serum were obtained.2.Optimized coating concentration of goat casein protein is 10?g/mL,coating at 4°C for12h,immune serum incubation at 37°C for 60min,ELISA determination of anti-goat casein antibody can get stable results.3.After 4 immunizations in mice,the titer of immune serum can reach 1:10~5.4.A cell strain secreted by the antibody that specifically recognized goat's casein was named 5B,and the secreted antibody was negative for the identification of bovine casein.5.The titer of hybridoma 5B cell culture supernatant was 1:2~8,and the titer of unpurified ascites was 1:10~5.6.Western-blot results demonstrated the specificity of mAb 5B and recognize goat?s1-casein.This experiment successfully screened the monoclonal antibody 5B that can specifically recognize goat?s1-casein.