The Detection And Dynamic Change Of IgA In Milk During Cow's Lactation Period

Secretory immunoglobulin A(SIgA) performs a very important role in mucosal immunity and ecto-exudate. As an important antibody of mucosal immunity, SigA expel pathogenic microbe in order to pretect the body from the infection through by immune clearance, immune defence and neutralization. It becomes an important part of mucosal immune systerm, because SIgA is the main antibody that generated by mammary gland. SigA in cow's milk may prevent young stock from gastrointestinal infection.In this study, separation and purification IgA directly from cow; s colostrum were performed by gel filtration and DEAE laminar analysis. Then it was identified by SDS-PAGE. Rabbit were inoculated with the purified IgA. The antibody of rabbit are labeled with HRP and rabbit anti-cow IgA enzyme labelled antibody were prepared. In order to investigate mucosal immunity and the disease correlate to mucosal immune system deeply. The representative samples were collect from JiLinChunGuang and JiLin Agriculture Univercity dairy farm, and detected by DAS-ELISA.The purpose of this study was to establish a double antibody sandwich-enzyme linked immunosorbent assay(DAS-ELISA) to detect the IgA antibody in cow's milk. The mouse anti-cow IgA antibody derived from mouse serum was used as coated antigen. Test protocol as follow: wells of a microtitration enzyme immunoassay plate were coated with 100ul of 30ug/ml of the antigen in carbonate buffer and incubated 3h at 37℃. Wells were rinsed by flooding them 3 times (3 minutes each) with a wash solution containing 0.15M NaCl and 0.05% Tween-20. Test cow's milk was diluted 1:2 in ELISA buffer (0.15M NaCl, 0.01M PB, 0.05% Tween-20) and 100μl was applied to each well. Plate were incubated for 60 minutes at 37℃and rinsed 3 times. Horseradish peroxidase-conjugated rabbit anti-cow IgA antibody was diluted 1:400 in ELISA buffer (0.15M NaCl, 0.01M PB, 0.1% bovine serum albumin, and 0.05%Tween-20), and 100ul was added to each well. The plate were incubated for 60 minutes and rinsed as before, 100μl substrate containing OPD-H₂O₂ was added to each well. Plate were laid in 37℃protect from light for 20 minutes. The reaction were termination with a drip of stop buffer (2M H₂SO4) each well. Plate were read by enzyme linked immunosorbent assay instrument.688 uninfected mastitis cow's milk samples were collected from four masto-section of 178 cows, and examined by DAS-ELISA. Result as follows: The disparation of IgA content in four masto-section is not significant at the same time. Then, 941 uninfect mastitis cow's milk samples are examined by DAS-ELISA. Result shows: the IgA antibody is permanently existence during cow's lactation period. From the OD figure of DAS-ELISA and recurrence equation y=1.1985·x^2.1819 (R²=0.9927), the IgA content of bovine colostrum and 70d-190d's milk is significant higher than other period.