Preparation Of Monoclonal Antibody Against P97 Protein Of Mycoplasma Hyopneumoniae And Preliminary Establishment Of Blocking ELISA

Mycoplasma hyopneumoniae(Mhp)is the causative pathogen for Mycoplsma pneumoniae of swine(MPS).After infection by Mhp,the pigs manifests a clearly clinical symptoms such as dry cough,lower daily weight gain and feed conversion,which causes enormous financial losses for the pig industry.At the same time,the mucosal damage caused by the Mhp upon infection will lead to the secondary infection with other pathogens,which results in a higher mortality rate in pigs.In order to effectively control the prevalence of the disease,it is best to find out and eradicate the infected pigs as earlier as possible.Enzyme-1inked immunosorbent assay(ELISA)is the most commonly method used to detect antibodies in serum,of which the blocking ELISA has a higher specificity because of no disturbence by other proteins.Previously,our laboratory found a main immunogenic region,P97CR1,on P97 protein used yeast display technology.In this study,the specific primer pairs were designed according to the P97CR1 gene.The P97CR1 fragment was obtained by PCR and cloned into the prokaryotic expression vector pET-28a to obtain the recombinant prokaryotic expression vector pET28a-P97CR1.P97CR1 protein waspurified after expression in the prokaryotic expression system and subjected toimmunize six weeks old of BALB/c mice after emulsification with Freund's adjuvant.When the titer of serum reached 1.28�10~5,splenic cells from the immunized mice were fused with SP2/0 myeloma cells.A positive hybridoma cell line A3 stably secreting the MAbs against P97CR1 protein was successfully screened by indirect ELISA.The identification results indicated the heave and light chain of A3 MAb were IgG1 and?subclass,respectively.The A3 MAb was able to specifically recognize the P97CR1 protein expressed in E.coli BL21(DE3)using western blotting assay,and recognize natural P97 protein expressed on the Mhp with flow cytometry analysis.The epitope sequence recognized by the A3 MAb was identified as LDDNLQ by pepscan technique,and is highly conservative among the P97 proteins of all Mhp strains.A3 hybridoma cell line was used to prepare ascitic fluid and monoclonal antibodies in the ascitic fluid were purified and labeled with horseradish peroxidase(HRP).The titer of both ascitic fluid and HRP-labeled antibody was 5.1�10~5 by indirect ELISA.Using P97CR1 protein as coating antigen and HRP-labeled monoclonal antibody as secondary antibody,a blocking ELISA detecting antibodies specific for Mhp was preliminarily established.All optimized conditions in this ELISA were as following:The optimal coating antigen and blocking protein are a final concentration of 20 ng/ml and 5%skimmed milk,respectively;The optimal dilution ratio of pig serum was 2 times;optimal dilution of HRP-labeled monoclonal antibody is 16,000 times;The optimal reaction time for blocking,reaction with serum,HRP-labeled monoclonal antibody and TMB substrate were 120 min,60 min,30min and 10min,respectively.The determination criteria of this method were determined according to the test results of 51 negative serum samples:when the sample PI value was?44.94%,it was judged to be positive;when the sample PI value?38.86%,it was judged to be negative;when the PI value is between 38.86%and 44.94%,it was judged to be dubious;When the experimental result is dubious,it needs to be tested again.If it is still dubious,it will be judged as positive.Specificity test showed that only the Mhp positive serum has a high blocking rate.Meanwhile,the sera from pigs infected by the Mycoplasma hyorhinis(Mhr),Haemophilusparasuis(HPS),Streptococcus suis(SS),Porcine circovirus type 2(PCV2),Porcine epidemic diarrhea virus(PEDV),Actinobacillus pleuropneumoniae(APP)or Pasteurella multocida(PM)had no blocking effect,indicating this method has good specificity.The coefficient of variation was less than 10%in both intra-batch and inter-batch repeatability experiments,which showed a good repeatability.The coincidence rate of this method with commercial IDEXX kit is 91.2%.These results provide a foundation to explore a blocking ELISA kit to detect Mhp-specifc antibodies in sera with a good prospect for application in the future.