Development And Application Of Elisa Against Pmt Antigen And Antibody Derived From Toxic Pasteurella Multocide Infection

In today’s intensive farming industry,respiratory diseases of pigs are listed as one of the three major diseases in the breeding industry,and bacterial diseases account for a large proportion of respiratory diseases.Therefore,it is of great significance to make a rapid and accurate diagnosis of bacterial pathogen infection in the sick pigs,and it is possible to make quick countermeasures to reduce the loss.Porcine atrophic rhinitis(Swine atrophic rhinitis,AR)caused by toxotoxigenic Pasteurella multocida(toxigenic Pasteurella multocida,T+Pm)infection is one of the major causes of atrophic rhinitis in pigs.Pasteurella multocida toxin(Pasteurella multocida toxin,PMT)secreted by T+Pm is the main pathogenic factor of atrophic rhinitis.Detection of PMT antigen and antibody is the main method to diagnose AR.Therefore,it is of great significance to establish an immunologic method for rapid and accurate detection of PMT antigen and antibody.The specific contents of this study are as following:1.Development and application of blocking elisa against PMT antibody derived from toxic pasteurella multocide infecion.In this study,a blocking ELISA method was established based on a monoclonal antibody(MAb)against toxic Pasteurella multocida PMT protein.The conditions for each step were optimized,The optimal concentration of the coating antigen was 1 μg·mL-1;sera samples diluted 1 fold and incubated 2 h at 37℃;MAb-HRP dilution is 1500 fold and incubated 1 h at 37℃.The TMB substrate was added and incubated at 37℃ for 10min before terminated with stop solution.By statistical analysis of 50 PMT-negative sera blocking ELISA results,the criteria of this method were determined to be positive when the blocking rate PI≥36.8%,negative when PI≤31.47%,31.47%<PI<36.8%were considered doubtful.One hundred serum samples were tested by indirect ELISA and blocking ELISA,the sensitivity and specificity of blocking ELISA were 90%and 100%,respectively.Recombinant antigen had no cross reaction with theantibodies to HPS,E.coli,PRV,SVV,PCV2,PRRSV,gE and CSFV.In the repeatability test,both the intra-batch and inter-batches variation coefficients were lower than 10%.These results suggest that the blocking ELISA is specific,sensitive and reproducible.This method was used to detect 287 clinical serum samples from 7 provinces of China,resulting a positive detection rate was 41.09%,which proved that T+Pm infection was common in many farms across the country.The Blocking ELISA proved to be specific,sensitive and it showed high reproducibility and low variability.This method will be useful in clinical detection and epidemiological study on PMT.2.Development and application of sandwich elisa against PMT antibody derived from toxic pasteurella multocide infecion.In this study,a sandwich ELISA method was developedd by using an polyclonal rabbit antibody against PMT as capture antibody and anti-PMT MAb as capture antibody.The optimal conditions for the ELISA assays was as follows:The coating polyclonal rabbit antibody was diluted to 1:10,and incubated for 2 h at 37℃;The optimal blocking solution was 5%skim milk and the blocking time was 37℃ for 2h;the optimal dilution of the serum to be tested was 1:1 and incubated at 37℃ for 2h;the optimal dilution of the monoclonal antibody labeled with HRP was 1:500 and incubated 1h at 37℃;The incubation time of chromogenic liquid was 10 min.By statistical analysis of 30 PMT-negative sera sandwich ELISA results,the criteria of this method were determined to be positive when the OD450nm value was≥0.215,negative when the OD450nm value was less than 0.190,and suspicious when the OD450nm value was between 0.190 and 0.215.The results of the cross-reaction test were all negative.The lowest detection limit of antigen was 0.1225 μg/ml;The variant coefficient of the ELISA in a batch and between batches was less than 10%,which indicated that the ELISA method had high specificity,sensitivity and repeatability.By dilution of PMT standard,a standard curve was made in the working range from 0.1225 to 15.625 μg/ml wich had an correlation coefficient of R>0.99,which could be used for quantization of PMT.The method was used to detect the serum samples of pigs sent for clinical examination,and the qualitative and quantitative analysis of PMT antigen in the clinical serum was carried out.The establishment of this method and the development of subsequent detection kit will be used for swine atrophic rhinitis.Rapid diagnosis of swine atrophic rhinitis and epidemiological investigation lay the foundation.