Preparation Of Anti Pig SIgA Polyclonal Antibody And Detection And Verification In Intestinal Epithelial Tissues

Secretory immunoglobulin A(SIgA)is very important immunoglobulins in milk and is the main effector of gastrointestinal mucosal immune,but also has immune clearance,immunomodulators,start immunological activity and other functions,can resist the surface adhesion of pathogenic bacteria,improve the ability of local anti-infection and neutralize virus.Changes of SIgA content and antibody activity will cause the generation of many diseases,infant susceptibly infect pathogenic E.coli if reduction of SIgA in milk;SIgA reduction will cause the flora disequilibrium;mucosal SIgA reduction may cause the recurrent respiratory infections and other diseases in gastrointestinal tract and respiratory tract.Therefore,accurate and effective detection of SIgA content in serum and exocrine fluid has an important significance for early diagnosis and treatment of most diseases.The study aimed at the large number of secretory immunoglobulin A in porcine colostrums,using two step salting-out sedimentation to extract the SIgA of porcine colostrum,then gel filtration and Cellulose chromatography were used to purified SIgA;secretory immunoglobulin A quantitative detection kit and BCA protein concentration determination kit then were used to detect and identify the purified SIgA;New Zealand white rabbits were immunized by the SIgA to prepare Rabbit anti pig SIgA polyclonal antibody.After the separation,purification and identification,Rabbit anti pig SIgA antibody was obtained.Antibody obtained in this study can be used for detection of existence of intestinal mucosa mainly secretory factor---immunoglobulin SIgA which laid the material foundation for the study of porcine intestinal mucosal immunity.The results are as follows:1.30%,50%two saturated ammonium sulfate salting-out method,Sephadex G-200 gel chromatography and ion exchange chromatography were used to separate and purify SIgA in porcine colostrum,after ammonium sulfate crude protein Sephadex G-200 gel chromatography column,eluted,column chromatography,identified by the method of SDS-PAGE and the porcine SIgA quantitative detection reagent box,SIgA protein concentration was 0.792±0.015mg/mL,BCA protein assay kit measured the total protein concentration was 0.850±0.021 mg/mL,the purity of protein>90%,proved to be purified with high purity SIgA isolated from porcine colostrum.2.SIgA contains heavy chain,light chain and secretory piece by identification of porcine colostrum,the molecular weights were 56 KD,27 KD,70 KD.3.New Zealand white rabbits were immunized by the SIgA to prepare Rabbit anti pig SIgA polyclonal antibody.The serum antibody titer of 1:32000,the agar double immunodiffusion measured serum antibody titer of 1:32,standards-compliant.4.The presence of SIgA in porcine small intestinal mucosa tissue was detected by the prepared Rabbit anti pig SIgA polyclonal antibody combined with immunohistochemical staining method.