Establishment And Application Of Blocking Elisa Detection Method Using Pasteurella Multocida Toxin Monoclonal Antibody

Toxigenic Pasteurella multocida(T+Pm)is a bipolar staining Gram-negative Brevibacterium,which is an important pathogen of livestock and poultry.The bacterial infection can not only cause disease in many animals,but also infect humans.Progressive atrophic rhinitis(PAR)in pigs is mainly caused by T+Pm,and its damage to pig turbinates is irreversible.Since Pm was first discovered in the 1830s,it has become widespread in many countries and regions where the swine industry is well developed.Because it can work synergistically with many pathogenic bacteria,the morbidity and mortality caused by respiratory diseases in swine will also increase.The secretion of Pasteurella multocida toxin(PMT),encoded by ToxA gene of T+Pm,is one of the main cause for the induction of PAR.By inoculating PMT alone,the symptoms of PAR can be fully replicated in experimental pigs.It mediates PAR by inhibiting osteoblast differentiation and promoting bone regeneration.The current method of diagnosing PAR is mainly the detection of PMT antibodies.However,due to the unstable and easily degradable nature of PMT whole protein,it has a bad effect on the development of subsequent kits.As early as 1991,Petersen and Nielsen et al.analyzed the immunogenicity of different truncated PMTs.They found that the C-terminus of PMT is an essential component of its immunogenicity.Therefore,rPMT-C-terminal protein truncated expression was used to immunize mice to prepare monoclonal antibodies in this study.rPMT-C and HRP labeled monoclonal antibody were used to establish an blocking ELISA for PMT antibodies.The specific research is as follows:1.Preparation of monoclonal antibody against rPMT-C terminal truncated proteinIn this study,glycerol bacteria containing the plasmid pET32a-toxC stored in the laboratory were used to express a large amount of rPMT-C terminal proteins after resuscitation,purified recombinant protein was emulsified with freund's adjuvant and used to immunize BALB/c mice.After three immunizations,the mice with highest antibody titer were screened by indirect ELISA for stimulating immunization.After fusion of the mouse spleen cells with SP2/0 cells and subcloning several times,9 hybridoma cell lines capable of stably secreting monoclonal antibodies were screened and named 3E3,4H2,7A3,4H6,11B2,9G1,12F4,8G5 and 6A3 respectively.The supernatant titers are 1:12800,1:12800,1:6400,1:12800,1:12800,1:3200,1:1600,1:800 and 1:6400,respectively.Superimposed ELISA showed that the 9 monoclonal antibodies targeted the same epitope.Western blot results showed that rPMT-C-terminal protein could specifically react with the monoclonal antibody 3E3 supernatant but not with SP2/0,which indicaties 3E3 has good specificity.3E3 monoclonal antibody has good stability within 10 generation passages.The subtype identification results showed that the monoclonal antibody 3E3 belongs to the IgG1 subclass and the light chain is a kappa chain.Mice treated with autoclaved sterilization liquid paraffin injection one week in advance were inoculated with monoclonal antibody 3E3 to prepare ascites.The antibody titer for ascites was 1:102400.The collected ascites was sent to the company for purification and HRP labeling to obtain an enzyme-labeled primary antibody that could establish an ELISA method for the detection of Pasteurella toxin.2.Establishment and preliminary application of blocking ELISA for detection of antibodies against PMTIn this study,the recombinant rPMT-C terminal protein was used as the coating antigen,and HRP-labeled 3E3 was used as the detection antibody.By optimizing the conditions,a method of blocking ELISA for the detection of antibodies to Pasteurella toxin was established.ELISA microtitre plates were coated with 100?L of purified PMT/well diluted in 0.05 mol/L carbonate buffer(pH=9.6)and incubated 2h at 37?.Plates were washed three times with PBST and then blocked with 200?L/well of PBST containing 2%BSA for 3 h at 37?.After three washes with PBST,diluted sera were added and incubated for 0.5h at 37?.After the plates were washed three times,100?L of diluted HRP-labelled McAb 3E3 was added and the plates were incubated at 37? for 0.5 h.The plates were then washed thoroughly,and color development was achieved by adding 100?L/well of tetraethyl benzidine substrate solution and stopped after 10 min incubation at 37? by adding 50?L/well of 2M H2SO4.Using this method,50 clinical PMT-negative sera were tested and statistically analyzed.It was determined using this method that the serum samples were positive for PI?51.51%and negative for PI?44.89%,samples with PI values lie between 44.89%and 51.51%were considered for suspension.This method was used to test 74 clinical samples that had been confirmed positive by indirect ELISA.The results showed that the sensitivity of blocking ELISA was 90.5%.This method was used to detect clinical HPS,Bb,APP,ASFV,PRV,PCV2,PRRSV,and CSFV positive samples,the PI values were all negative,which indicating that the method is specific.Intra and inter-assay repeated CVs were less than 10%.It can be seen that the detection method has good specificity,high sensitivity and repeatability.This method was used to test the sera of some farms in six provinces and one city in East China.The positive rate was 16.16%.Among of them,the positive rate of detection in a pig farm in Fujian Province was as high as 76.47%,indicating that there is still a serious T+ Pm infection in China.All in all,the preparation of PMT-C terminal protein monoclonal antibody and the establishment of a subsequent blocking ELISA detection method are of great significance for the rapid and large-scale detection of T+ Pm-infected swine in clinical practice,and also provide important scientific support for the following-up control and eradication of PAR.