Establishment Of ELISA Method For Detection Of Convalescent Serum Antibody Of Mycoplasma Hyopneumoniae

Mycoplasmal pneumonia of swine?MPS?,also known as swine enzootic pneumonia,is a chronic,highly contagious infectious disease caused by Mycoplasma hyopneumoniae?M.hyopneumoniae?,with cough and wheezing as the main clinical symptoms.The disease is widely distributed worldwide,and the prevalence is from 38%to 100%.At present,the main prevention and control measure of the disease is immunization with inactivated vaccine.The ELISA method is the most widely used method for the antibody detection of M.hyopneumoniae clinically.However,the commercial IgG ELISA kits cannot distinguish between antibodies produced by inactivated vaccines or natural infections.Mhp366 protein is a membrane surface lipoprotein with specific antigenicity.The expression of this protein in M.hyopneumoniae cultured in vitro is very low,and cannot stimulate to produce humoral immune response.Nevertheless,it can be expressed at a high level in vivo and stimulated to produce a strong humoral immune response.In this study,we want to establish two ELISA methods.One is to develop a method to differential diagnosis of hyperimmune serum antibody and convalescent serum antibody of M.hyopneumoniae,andanother is to develop a method to differential diagnosis of negative sera and convalescent sera of M.hyopneumoniae.1.Prokaryotic expression and purification of Mhp366-N protein.Objective:Prokaryotic expression and purification of Mhp366-N protein.Method:Specific primers were designed by using Primer Premier 5.0.The fragment of mhp366gene from 1 to 837 nucleotide composed of amino acid sequence 68-88?68QKENSQKNDVVNSQNKTEKTE88?which can distinguish between hyperimmune serum antibody and convalescent serum antibody was amplified.This gene fragment was linked to the prokaryotic expression vector pET28a?+?and transformed into BL21?DE3?.The recombinant protein was induced in recombinant bacteria culture by IPTG at a final concentration of 1 mmol/L,and purified by Ni²⁺affinity chromatography to obtain high-purity Mhp366-N protein.Result:The recombinant Mhp366-N protein could be expressed in soluble and inclusion body forms.The expression of soluble recombinant Mhp366-N protein is about 5%of the whole bacterial protein.The purity of the purified Mhp366-N protein was over 90%by Ni²⁺affinity chromatography.Conclusion:Highly purified Mhp366-N protein provided material for the subsequent ELISA assay.2.Development of an ELISA method for differential diagnosis of hyperimmune serum antibody and convalescent serum antibody of M.hyopneumoniae.Objective:Establishment of an ELISA method for differential diagnosis of hyperimmune serum antibody and convalescent serum antibody of M.hyopneumoniae.Method:The optimal reaction conditions of the ELISA method for the differential diagnosis of hyperimmune serum antibody and convalescent serum antibody of M.hyopneumoniae were determined,including optimal antigen coating concentration,optimal blocking buffer,optimal blocking time,optimal dilution ratio of sera,optimal incubation time of primary antibody,optimal dilution ratio of HRP-labeled rabbit anti-porcine IgG,optimal incubation time of HRP-labeled rabbit anti-porcine IgG,optimal colorimetric reaction time.A cut-off value was determined,and intra-assay repeated assay,inter-assay repeated assay,specificity and sensitivity assays were performed.Finally,clinical samples were tested and the results were compared with other kits'results.Result:The optimal antigen coating concentration was determined to be 0.25?g/mL,the optimal blocking buffer was 2.5%skim milk,the optimal blocking time was 0.5 h,the optimal dilution of sera was 1:1 000,and the optimal incubation time of primary antibody was 0.5 h,the optimal dilution ratio of HRP-labeled rabbit anti-porcine IgG was 1:10 000,the optimal incubation time of HRP-labeled rabbit anti-porcine IgG was 2 h,and the optimal colorimetric reaction time was 10 min.The cut-off value is 0.319.The coefficient of variation for both intra-assay and inter-assay trials was less than 7%.This method has good specificity and sensitivity.Conclusion:The method differential diagnosis of hyperimmune serum antibody and convalescent serum antibody of M.hyopneumoniae was established.3.Development of an ELISA method for differential diagnosis of negative and convalescent sera of M.hyopneumoniae.Objective:Establishment of an ELISA method for differential diagnosis of negative and convalescent sera of M.hyopneumoniae.Method:The optimal reaction conditions of the ELISA method for the differential diagnosis of negative and convalescent sera of M.hyopneumoniae were determined,including optimal antigen coating concentration,optimal blocking buffer,optimal blocking time,optimal dilution ratio of sera,optimal incubation time of primary antibody,optimal dilution ratio of HRP-labeled rabbit anti-porcine IgG,optimal incubation time of HRP-labeled rabbit anti-porcine IgG,optimal colorimetric reaction time.A cut-off value was determined,and intra-assay repeated assay,inter-assay repeated assay,specificity and sensitivity assays were performed.Finally,clinical samples were tested and the results were compared with other kits'results.Result:The optimal antigen coating concentration was determined to be 0.25?g/mL,the optimal blocking buffer was 2.5%skim milk,the optimal blocking time was 1 h,the optimal dilution of sera was 1:500,and the optimal incubation time of primary antibody was 0.5 h,the optimal dilution ratio of HRP-labeled rabbit anti-porcine IgG was 1:10 000,the optimal incubation time of HRP-labeled rabbit anti-porcine IgG was 2 h,and the optimal colorimetric reaction time was 15 min.The cut-off value is 0.297.The coefficient of variation for intra-assay is less than 8%,and the coefficient of variation for inter-assay is less than 6%.This method has good specificity and sensitivity.Conclusion:The method differential diagnosis of negative and convalescent sera of M.hyopneumoniae was established.