Development Of Immunogolloidal Gold Strip Assay For Detecting The Mycoplasma Hyopneumoniae

Mycoplasma Pneumoniae(Mhp) 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. And it brought great economy loss in swine industry. There are lots of different methods for detecting MPS. However most of them are still difficult practice in clinic. In this paper an instant method is successfully established to detect MPS using gold immunochromatography assay.Membrane proteins which were extracted from Mycoplasma Pneumoniae by TritonX-100 and KI, purified by affinity chromatography assay could be purified. The result of SDS-PAGE shows that the purified membrane proteins have high-purity that the molecular weights were 83KD,66KD,40KD,36KD,34KD and 27KD. The purified membrane proteins were inoculated into the 60 d white rabbit with alhydrogel adjuvant. The titer of the antiserum reaches to 1:32 proved by double agar diffusion test. Rabbit anti-MHP IgG was purified using caprylic acid and ammonium sulphate (CA-AS).Colloidal gold of 20nm was prepared by trisodium citrate method. The particles were detected by spectral scanning (400nm-700nm), which had good uniformity in shape. We labeled the purified membrane proteins using colloidal gold, were compared and found that pH 8.2 and purified membrane proteins in 9μg/ml could get best results. Purified membrane proteins and rabbit anti-MHP IgG were coated in turns on the nitrocellulose membrane as test line and control line. Assembled them and incised into immmocolloidal gold strip. It was found that 2mg/ml for test line and 4mg/ml for control line on the nitrocellulose membrane could get best results. 54 specimens were detected by immunocolloidal gold strip and ELISA, the results showed that 90.7% Mhp positive rate were by the ELISA and 83.8% Mhp positive rate were by the strip. And the coincidence was 92.6%, in respectively. The result showed that the Mhp immunogolloidal gold strip is a kind of sensitive, rapid, and specific method. Therefore, it is playing an important role in clinical application.