| Liu Mao Ke(Microbiology) Directed by Prof.Xiong Yan(Sichuan Agricultural University)Immunity at the mucosal surfaces is the first line of defence against pathogenic microorganismessential. In addition, Secretory immunoglobulin A (SIgA) is the most abundant immunoglobulin present at all mucosal surfaces,where it plays crucial roles in mucosal protection. Therefore,the diagnose of the SIgA concentration in the mucosal surfaces are important for evaluating animal mucosal immunity.The paper was conducted on intestinal mucus of piglet. The improved method of ammonium sulfate was employed to isolate SIgA from the intestinal mucus.Then SIgA was purified by High Q Anion exchange chromatography,and 0.5 mg SIgA per ml intestinal mucus had been obtained.SDS-PAGE indicated that the molecular weight of heavy chains,light chains and joining chains of SIgA were about 57.1 KD,28.9KD and 14.1KD,respectively.The purified SIgA was inoculated into the 60 d white rabbit. Rabbit anti-PIG (SIgA) IgG was purified using caprylic acid and ammonium sulphate. The titer of the Rabbit anti-PIG (SIgA) IgG reaches to 1:24 proved by double agar diffusion test. Rabbit anti-PIG(SIgA)IgG was labeled using horseradish peroxidase(HRP) by the sodinm periodate reaction. Enzyme labeling rate and molar ratio of Rabbit anti-PIG (SIgA)IgG-HRP were 36.2%,and 2.69,respectively.An direct ELISA for detecting SIgA was established. Optimum conditions were developd as follows:the coating protein concentration of specimen was 10ug/ml, working concentration of Rabbit anti-PIG (SIgA)IgG-HRP were 1:200.the reaction between specimen and Rabbit anti-PIG (SIgA)IgG-HRP lasted for 60 min. Specimen with an optical density (OD) value=0.449 were scored positive. The inter-and intra-assay variation coefficient of the direct ELISA were 6.63% and 8.99%,respectively.The linear interval range of SIgA concentration standard curve were 90-1400ng/ml. When the concentration of purified SIgA added to mucus were 100-400ng/ml,recoveries from mucus were in the range of 85.85-133.88% and the coefficient of variation were in the range of 13.12-17.67%.A total 15 d 36 piglets were average assigned randomly to three treatments. Treatmentâ… :At 21 d,piglets were vaccinated with Mycoplasma pneumonia (Mpn) inactivated vaccine, and were secondary immunized at 42 d. At 61 d, these pigs were challenged with a wild Mhp strain;â…¡:pigs were challenged with a wild Mhp strain at 61 d;â…¢:pigs neither immunized against Mhp nor challenged with a wild Mhp strain. Serum, lung fluid and intestinal mucus of piglets in each treatment were collected at 15,35,56 and 75 d. The serumal pecific anti-Mpn IgG(SP value)was measured using an indirect ELISA,and the SIgA concentration in lung fluid and in intestinal mucus was measured using the direct ELISA which was established in this research. In general,no difference in antibody level was observed among treatments at the beginning of experiment(15d). However, after basic immunization (35 d),the serumal IgG SP value of the piglets in treatmentâ… increased from 0.166 to 0.308,which were significantly higher than that in treatmentâ…¡,â…¢(P<0.05).In addition,no difference in the SIgA concentration in lung fluid of the piglets among treatments were observed at this time point. After booster (56 d), the serumal IgG SP value of the piglets in treatmentâ… were 0.503.Moreover, the SIgA concentration in lung fluid of the piglets in treatmentâ… were 153.89ng/ml which approximately 31.81ng/ml higher than the piglets ofâ…¡and 22.78ng/ml higher than the piglets ofâ…¢. After challenged(75d),the serumal IgG SP value were 0.408 in piglets of treatmentâ… , and the SIgA concentration in lung fluid were 205.81 ng/ml in treatmentâ… , which were significantly higher than that inâ…¡(147.56 ng/ml),and that inâ…¢(124.37 ng/ml.)In the other hand, no discrepancy in the SIgA concentration of intestinal mucus between these treatments were obtained throughout the trial.During the entire experiment,the range of SIgA concentration in intestinal mucus were 49.69-70.19ng/ml. |
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