Establishment Of An Antibody Blocking Elisa Against Recombinant Mnsod Protein Of Glaesserella Parasuis

As an opportunistic pathogen,Glaesserella parasuis(G.parasuis)is usually colonized in the upper respiratory tract of pigs.When the hosts are in stress conditions or immunity dysregulation,some serovars of G.parasuis infection can be occurred and lead to Gl(?)ser’s disease.Gl(?)ser’s disease is a worldwide pig disease with high mortality and high incidence rate.It has brought huge economic losses to the increasingly intensive farming enterprises.To date,15 standard serovars of G.parasuis have been identified.However,due to a large genetic heterogeneity among the serovars of G.parasuis,it is difficult to screen the common antigens with immune protection,which leads to the lack of common antibody detection methods for each serovar infection and hinders the infection monitoring and antibody evaluation of G.parasuis infection.So far,indirect ELISA is commonly used in the diagnosis of G.parasuis in China.However,the sensitivity and specificity of ELISA are affected by the purity and immunogenicity of antigens,and homology with other organisms.In addition,it has been reported that blocking ELISA can greatly improve the specificity and sensitivity of detection in pathogen diagnosis.Therefore,in this study,stably expressed manganese superoxide dismutase(Mn SOD)with good immunogenicity in serovars 4,5 and 12 of G.parasuis was designated as target antigen in blocking ELISA on the basis of the metabolic pathway of G.parasuis and previous proteomics and related researches.Based on this,the specific research of this paper is as follows:1.Screening target antigens of Blocking ELISAIn this study,recombinant Mn SOD and serovars 4,5 and 12 of G.parasuis were used as coating antigens to detect 200 clinical pig sera by indirect ELISA.The ELISA results showed that the coincidence rate of i ELISA coating with Mn SOD with that of coating with whole bacteria was 98.5%,especially,Mn SOD results showed strong reactivity.The results of western blot showed that Mn SOD was expressed in 15 serovars of G.parasuis,and which did not react with the mouse polyclonal antibodies of A.pleuropneumoniae,E.coli,P.multocida,B.bronchiseptica and S.suis.In addition,the highest expression level of Mn SOD is in serovar 13,the medium levels are in serovars 9,11 and 15,and the lowest in serovar 7.There was no correlation between the expression level of Mn SOD and the virulence of the serovars.This study confirmed the potential of recombinant Mn SOD as a target antigen for the establishment of blocking ELISA and laid the foundation for the subsequent establishment of blocking ELISA detection method.2.Preparation,identification and HRP labeling of monoclonal antibody against recombinant Mn SOD proteinRecombinant Mn SOD was used an immunogen to immunize Balb/C mice and cell fusion was used to prepared specific monoclonal antibody against recombinant Mn SOD.After three subclones and i ELISA detection,five hybridomas were obtained.The results of ELISA addition test showed that the five Mc Abs targeted the same epitope.Western blot showed that Mn SOD reacted specifically with supernatant of hybridomas,but not with that of SP2/0.The continuous passage showed that the antibody titer of 2C2-Mc Ab was 25600 and could secrete antibody stably when it was passaged to the 15 th generation.The results of Mc Ab subtype identification showed that 2C2 belonged to Ig G2 a and light chain was kappa type.The purified ascites was detected by i ELISA and analyzed by SDS-PAGE.The titer of ascites antibody was more than 204800,and the purity was more than 85%.The Mc Ab prepared in this experiment can be used as the detection antibody in the subsequent blocking ELISA method.3.Establishment and evaluation of blocking ELISAusing recombinant Mn SOD proteinIn this study,by optimizing the coating concentration of antigen,sera dilution,blocking solution and time,sera incubation time,HRP-Mc Ab incubation concentration and time,TMB substrate color development time,a blocking ELISA method were established with recombinant Mn SOD protein as coating antigen and 2C2 HRP-Mc Ab as diagnostic antibody.After the cut-off value was determined,the reproducibility,sensitivity and specificity of the blocking ELISA were tested.The optimal conditions for blocking ELISA reaction were as follows: the coating concentration of antigen was 0.25 μg/ml,0.5% BSA was incubated for 2 h,the tested serum was diluted 1:1 and incubaed for 2 h,HRP-2C2 Mc Ab was diluted as 1:2000 and incubated for 0.5 h,and TMB was developed for 15 min.Fifty negative sera results determined that PI≤38.22% was negative and PI≥46.59% was positive.Compared with i ELISA,blocking ELISA is high sensitivity and specificity,which can better differentiate negative and positive sera.The results of repeatability showed that the coefficients of variation within and between batches were less than 10%.In the conclusion,the Mn SOD-blocking ELISA method established in this study can be used for sera epidemiology investigation of G.parasuis infection in the future,and has the potential for serological monitoring of G.parasuis infection and evaluation of the efficacy of whole cell inactivated vaccine.