Establishment And Preliminary Application Of Indirect ELISA For Detection Of Serum Antibodies Against Mycoplasma Mycoides Subsp Mycoides

As the pathogen of Contagious Bovine Pleuropneumonia(CBPP),Mycoplasma mycoides subsp Mycoides(Mmm)has very high infectivity.The main symptoms of infected cattle are elevated body temperature,missed fever,dyspnea and other symptoms,which have caused huge economic losses to our country.Although CBPP has been eliminated in China,the disease is still prevalent in surrounding countries.According to the requirements of OIE,China carries out nationwide serological surveillance every year.Enzyme-linked immunosorbent assay(ciELISA)is the only commercial kit for CBPP detection in the world.Because of the large sample size,expensive commercial kits and complete dependence on imports,these factors hinder the prevention and control of CBPP in China.Therefore,it is of great significance to develop a domestic CBPP serum antibody kit.In this study,five kinds of membrane-related lipid proteins,namely P35,P42,P46,P47 and P50,were selected according to the genome sequence of Mmm and analyzed by bioinformatics software.The five proteins were expressed and purified by prokaryotic expression system,and then their specificity and reactivity were studied.Specificity study showed that recombinant protein P35(rP35)could distinguish CBPP positive serum from positive serum of Mycoplasma Bovirhinis,Mycoplasma agalactiae,and Mycoplasma Bovis,Reactivity study showed that CBPP positive serum was used as an anti-Western blot and Dot-blot test.Dot-blot test showed that rP35 was positive,suggesting that rP35 might be a conformation-dependent immune-related egg specific to Mmm.White and reactive.The rP35-iELISA method was established with rP35 as coating antigen.The optimum reaction conditions were as follows: coating concentration 2.5 ug/ml,dilution of serum sample 1:80,encapsulation time 60 min,encapsulation condition,reaction condition of second antibody,colouring condition,cut-off value and so on.The optimum conditions of the second antibody were 1:10000 dilution at 37 for 30 min,and the optimum time of colour development was 10 min.In order to evaluate the performance of rP35-iELISA,the sensitivity,specificity,repeatability of the method,the coincidence rate of rP35-iELISA and ciELISA for clinical samples were evaluated comprehensively.The results showed that the sensitivity and specificity of rP35-iELISA were 90% and 89%,respectively.The maximum dilution ratio was 1:640.There was no cross-reaction between rP35-iELISA and the positive sera of six common bovine diseases,such as Mycoplasma bovis.Repeated tests showed that the intra-and inter-batch variability coefficients of rP35-iELISA were less than 10%.The total coincidence rate between rP35-iELISA and commercial kits was 82.99%.In summary,this method has good sensitivity,specificity,repeatability and coincidence rate,and is expected to provide a new detection tool for CBPP diagnosis.