Establishment Of Colloidal Gold Immunochromatographic Assay For Detecting The Mycoplasma Hyopneumoniae Antibodyinserum And Mucosa

Mycoplasma hyopneumoniae of swine(MPS)is a chronic respiratory disease characterized by high morbidity and low mortality,which caused by Mycoplasma hyopneumoniae(Mhp).After infecting Mhp,the ability of swine respiratory system to defend diseases would be destroyed and other respiratory diseases would be easy to infect.As a result,the development of pig industry will be restricted seriously.In the initial stage,a pig who has been suffering from Mhp always presents subclinical and choric infection so that it is difficult to detect.Purification of Mhp is very difficult to farms.Therefore,it is important to diagnosis the disease as early as possible.At present,ELISA and PCR are particular to monitor the Mhp,which need long time,expensive equipments and technical staff Both technologies are not convenient for clinical test.Colloidal gold immunochromatographic assay(GICA)comes into being,which is fast,simple,accurate and inexpensive.GICA can be used to clinical rapid diagnosis.Therefore,this study established a colloidal gold immunochromatographic assay for the detection of Mhp antibodies by preparing two high purity of Mhp dominant antigens:P97R1 and P46.The results are as follows:1.According to the literature,recombinant E.coli was induced to express specific Mhp antigens:P97R1 and P46.Both antigens were purified by Ni-NTA affinity chromatography column.We prepared colloidal gold particles with a concentration of 0.02%and a diameter of 20?30 nm by trisodium citrate reduction method P97R1 and P46 proteins were labeled on colloidal gold respectively.Two kinds of immune colloidal gold solutions were prepared.The results showed that P97R1 and P46 proteins with high purity were obtained.Western blot showed the good antigenicity of the two proteins.The prepared immune colloidal gold solution can be used for colloidal gold immunochromatographic test strips.2.Followed by the selection of the best combination of colloidal gold test strips,we optimized the test strips.In the end,this study established a colloidal gold immunochromatographic assay to detection Mhp antibodies in serum.We can obtain the results within 5?10 min.The sensitivity,specificity and reproducibility of the test strips showed excellent as well.A total of 76 serum samples were tested by the GICA and commercial ELIS Atest kit at the same time.The results showed that coincidence rate ofthe GICA and ELISA was 96.1%.In conclusion,the newly developed test strips can be used for clinical rapid detection of Mhp antibodies in serum.3.Based on the assay of colloidal gold immunochromato graphic for detecting Mhp antibody in serum,the optimal chromatography mode of colloidal gold chromatographic test paper was selected and the condition was optimized,the study established a colloidal gold immunochromatographic assay to detect sIgA in nasal swabs and bronchoalveolar lavage fluid(BALF)samples.We can obtain the results within 5?10 min.A total of 40 nasal swab samples were tested by the GICA and ELISA test assay at the same time.The results showed that coincidence rate of GICA and ELISA was 87.5%.However,the sensitivity of the GICA for detecting sIgA in nasal swabs remains to be improved.