| Mycoplasma hyopneumoniae can attach to the pig airway epithelium, stimulates the mucosal immune system to produce specific antibodies, SIgA is the main effective factor among them, and plays an important role in mucosal immunity. SIgA antibodies on the surface of respiratory tract play an important role in mucosal immunity. A SIgA-indirect enzyme-linked immunosorbent assays (ELISA) method with P97R1protein as the envelope antigen and goat anti-swine IgA-HRP was developed in this study to evaluate the mucosal immune response after infection or vaccination.In the experiment, seven pigs were infected with Mhp field strain in lung homogenate. The nasal swab samples were collected on1,2,4,6,8,12,16and21day post-infection(dpi) to detect specific IgA titers against P36, P46and P97R1proteins by the indirect ELISA methods. The IgA titers were normalized by the total protein concentration of nasal swab samples. The secretion features of three specific IgA antibodies against P36, P46and P97R1proteins in the respiratory tract were compared within21days. The results showed that the levels of three sorts of specific IgA antibody start to rise at the6dpi and peak at the12dpi without significant differences (P>0.05), The titers could keep high until21DPI. The results showed that P36, P46and P97R1proteins could induce the specific IgA antibody in the respiratory tract with the same trends and titers without significant differences during the early stage of post-infection. According to the pathogenesis and the main antigen protein research background of Mhp, Mhp P97R1protein was selected as SIgA-ELISA kit envelope antigen.After determining the envelope antigen, P97R1recombinant protein was expressed in E.coli expression system by using the positive P97R1plasmid, and the protein was purified with chromatography. The results of Western-blotting showed that P97R1is a protein with species specificity. The preparation method of P97R1are optimized and standardized, in order to maintain the stability and specificity of the antigen in kit.By screening the protective agent in the SIgA-ELISA kit reagents, the SIgA-ELISA kit preparation technology was determined according to ELISA detection conditions, and three batches of kit were assembled. Cut-off value (CV value) of kit was also determined at0.261according to the results from detecting amounts of nasal swab samples with infected, uninfected and vaccine immunization. Then, the sensitivity and specificity of kit were determined as95.1%and92.3%after detection of150positive nasal swabs and87negative ones, respectively. The lowest detectable ratio of kit was tested by the strong positive sample with dilution of1:160, and the weak positive sample with that of1:80. No positive response was detected when anti-Mhr and anti-Mf antibody were added as samples. Repeatability test of three batches of kits was confirmed with CV value is3.1%-8.76%between different holes,4.26%-8.06%intra-batches and4.64%-14.5%inter-batches. Also, the diagnostic sensitivity and specificity of kit were not changed when SIgA-ELISA kits were stored at4℃for more than six months.In addition, secretory component (SC) is a specific integral part of SIgA. Detection of SC can exclude the interference of other immune globulin, and the SIgA levels could be measured more accurately. In this study, SC protein was also extracted and purified from pig colostrum with gel filtration chromatography and ion exchange chromatography. Monoclonal antibody of SC was also produced by subcutaneously immunizing4week old BALB/c mice with purified SC protein. Then, one hybridoma obtain cell line secreating the stable SC antibody, was named2A6. The results of western blot showed that McAb2A6can reacted with SC protein, has no reaction activity with porcine IgG protein. ELISA titers which reached with SC of ascites of2A6was1:204800. |
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