Development Of Lyophilized Hemagglutination Inhibition Test Antigen And Establishment Of Indirect ELISA Method For Detecting Mycoplasma Synoviae Antibodies

Mycoplasma synoviae(MS)is an important pathogen in poultry breeding industry.The poultry will be in a state of long-term bacteria schlepping after infection,which causes substantial economic losses from decreased performance.At present,the main methods for detecting MS antibody are hemagglutination inhibition test(HI)and enzyme-linked immunosorbent assay(ELISA).Among the advantages of HI test are its ease of operation and high specificity,but there is a lack of commercial antigens that can be preserved stably for a long time.ELISA can be used for a large number of samples with high sensitivity;therefore,the cost of detecting is high due to the commercial ELISA kits are expensive.To provide technical supports for the prevention,control,and purification of MS infection in China,this study aimed to develop a lyophilized MS HI test antigen which can be preserved stably.Meanwhile,the recombinant MSPB antigen(rMSPB)expressed in genetic engineering was used as the coating antigen for establishing an indirect ELISA detecting MS antibody which can replace the foreign similar products1.Development and application of lyophilized MS hemagglutination inhibition test antigen.A strain JS2018-4,with strong replication ability and stable hemagglutination property,was selected from the MS epidemic strain library established by our laboratory.A stable and specific MS lyophilized HI test antigen was developed through optimization of inactivation technology,concentration technology,and freeze-drying technology,and then the HI test method for detecting MS antibody was optimized.The HI test was used to detect 148 reference serum samples(including 47 negative serum samples of SPF chickens and 101 positive serum samples from SPF chickens infected with MS).Based on the results of the receiver operating characteristic curve analysis,the judgment standard was determined as follows:titers of≥1/80 are considered to be positive,titers of 1/40 to 1/80 are strongly suspicious and titers of<1/40 are negative.The inspection results showed that lyophilized MS hemagglutination inhibition antigen had good specificity and stability since it had no cross-reactions with the positive serum of Mycoplasma gallisepticum(MG),Avian influenza virus(AIV),Newcastle disease virus(NDV)and Infectious bronchitis virus(IBV)and its HA titer could keep invariant after storage in 4℃for 12 months.The HI test and commercial ELISA Kit were used to detect 453 clinical serum samples and 40 clinical serum samples with different antibody titers,and the results showed that the negative and positive coincidence rates were 100%and 77.07%respectively and the trends of antibody increase and decrease were consistent.The above results showed that the HI test method established in this study had high clinical application value.2.Establishment of a recombinant protein rMSPB-based indirect ELISA for detecting MS antibodies.In this study,the vlhA nucleotide sequence and amino acid sequence of JS2018-4 strain were analyzed by Protean software and two fragments:MSPB(6aa-312aa)and MSPA(485aa-720aa)which have strong antigenicity were selected for PCR reaction.Two fusion proteins with His tags were expressed by pET prokaryotic expression system.The results of SDS-PAGE identification showed that the two fusion proteins were expressed in BL21(BE3)as inclusion bodies and the weights of two recombinant proteins were consistent with that expected,which were 54 kDa and 46 kDa,respectively.The results of Western-Blot identification showed that both of them couldbe recognized specifically by MS single factor positive serum and were named as rMSPB and rMSPA,respectively.Recombinant proteins with high purity were obtained as the coating antigen after denaturation,purification,and renaturation.rMSPB was used as coating antigen because it had a much better detection performance for MS antibody than rMSPA and an indirect ELISA method for detecting MS antibody was established after optimizing condition.Then we prepared MS standard serum and control samples for ELISA test.676 reference serum samples were detected by this indirect ELISA and the ROC curve analysis showed that a S/P value of 0.45 was the optimal cut-off value.Then,65 reference serum samples were detected by ELISA endpoint assay,the regression analysis showed that the linear equation between the S/P value at the dilution of 1:800 and antibody titer was:Log10(Antibody titer)=1.148×Log10(S/P value at the dilution of 1:800)+3.247,(R2=0.9030).Thus,quantification of MS antibodies quickly was realized in the indirect ELISA method by this equation.The identification results showed that the indirect ELISA had good specificity and reproducibility since it had no cross-reaction with Mycoplasma gallisepticum(MG),Avian influenza(AIV),Newcastle disease(NDV)and Infectious bronchitis(IBV)positive serum,and the intra-batch and inter-batch repetition coefficients of variation were all less than 10%.178 clinical serum samples and 207 serum samples of different ages from the same chicken flock were detected with the indirect ELISA established in this study and commercial antibody detection kits,the results showed that the negative and positive coincidence rates of this method and commercial kit were 99.04%and 98.65%respectively,and the trends of antibody growth and decline from this indirect ELISA were consistent with that obtained by commercial test kits.According to the results,this method had good performance for detecting MS antibodies.