Study On Purification Of Membrane Protein By Immuno-affinity Chromatography And Indirect ELISA For Detection Of Mycoplasma Hyopneumoniae

Mycoplasma Pneumoniae is the etiological agent of Mycoplasma Pneumoniae of swine (MPS), which is a chronic respiratory disease of swine. MPS is characterized by cough, sneezing, high morbidity rates, low mortality rates, retarded growth, poor food conversion. The clinical suspected pigs were often difficult to make confirmed diagnosis. The membrane protein of Mycoplasma Pneumoniae is the main immunogens and it has important significance to diagnose the infection of MPS.The high activity and purity IgG had obtained from the anti-Mhp serum antibody which was purified by using caprylic acid and ammonium sulphate (CA-AS), and anion-exchange chromatography. The immunoadsorbent affinity chromatography of anti-Mhp were prepared by coupling the IgG to CNBr-activated Sepharose 4B. Membrane proteins which were extracted from Mycoplasma Pneumoniae by TritonX-100 and KI, adsorbed by such immunoadsorbent could be purified. The result of SDS-PAGE shows that the purified membrane proteins have high-purity that the molecular weights were 66KD, 48KD, 46KD, 40KD, 32KD and 24KD. The unpurified membrane proteins have low- purity and the range of molecular weights was 20KD to 100KD.An indirect enzyme-linked immunosorbent assay for detecting antibody Mycoplasma Pneumoniae was established by using purified membrane proteins as antigen for coating. Through repeatedly test and confirmed the optimization test conditions: the coating concentration of antigen was 5ug/ml, the optimum dilution of the tested serum should be 1:80, the sealed buffer was 1.5% BSA and blocking for 120 min, the action between antigen and antibody lasted for 90min, the optimum action period for the binding of antigen with enzyme-signed SPA which were diluted by 1:1000 was 60min, samples with an optical density (OD) value≥0.289 were scored positive base on the ELISA test.Coating the unpurified and purified proteins respectively in ELISA test, the results showed that the positive detection rate in sera from clinically pigs was raised to 7.5% by using purified membrane protein. 92% of agreement was obtained between our new ELISA and commercial IHA kit by simultaneously detecting 50 serum samples. The assay was further applied to detect 150 clinically collected at random serum samples. The detection rates in sera from postweaning piglets, growing pigs, finishing pigs were 44.2%, 41.2% and 17.9%. The result showed the prevalence of Mycoplasma Pneumoniae in pig herds and the significance of the new indirect ELISA in clinical diagnosis of MPS.