Study Of Indirect Enzyme-linked Immunosobent Assay For Detecting Antibody Of Haemophilus Parasuis

In this study,whole-cell antigen and supersonic antigen were both extracted from Haemophilus parasuis(Hps).Indirect ELISA was developed to detect the species specific antibody by these two kinds of antigens respectively.At the same time,heat-stable antigens was extracted from Hps 1-15 serotype.Indirect ELISA was developed to detect the serotype specific antibody.The purpose is to fred out the best species-specific antigen through the cross-reaction test between whole-cell antigen and supersonic antigen,we establish indirect ELISA with the heat stable antigens as the diagnosis antigen,which could detect a part of serotype,and can provide informations for identifying the Hps serotype.1.Preparation of antigensThree kinds of antigen were preparated in this study:(1).The whole-cell antigen:H.parasuis grown on a TSB plates overnight at 37℃was harvested into PBS which has 2%formaldehyde 37℃1d.Then bacterium was washed by PBS and harvest ed into 3ml PBS.Concentration was 3.33×10~9CPU/ml.(2).The supersonica antigen:H,parasuis grown on a TSB plates overnight at 37℃was harvested into PBS,after supersonicaing(160W 20min) the supematant was centrifuged.The pellet was resupended in PBS,and concentration was 2.84 mg/ml.(3).The heat stable antigen:H.parasuis grown on a TSB plates overnight at 37℃was harvested into PBS.The suspensions were pooled and autoclaved for 2h at 121℃.After autoclaving,the suspension was centrifuged and retained,Concentration was 1.33 mg/ml.2.Establishment of three kinds of indiret ELISAThe indirect ELISA were developped by the whole-cell antigen,the supersonic antigen and the heat-stable antigen respectively.Through the optimization of the reaction conditions,The optimal coating concentration of supersonic antigen,the whole-cell antigen and the heat-stable antigen were dilution of 1:100,1:200 and 1:100 for the standard suspension of three antigens and concentration were 28.4μg/mL,1.665×10~7CPU/ml and13.3μg/mL respectively;2%milk was determined to be the best blocking reagent of the supersonic antigen and the heat-stable antigen,37℃90min and 120 min were the best reaction condition respectively.10%calf serumin was determined the best blocking reagent of the heat-stable antigen,37℃90min was the best reaction condition;A screening dilution of 1:320 for serum was confirmed to be the best dilution;The optimal dillution of HRP were 1:6000 and 37℃30min for three kinds of ELISA;The blocking test indicated the the three antigens can both block the reaction between antisero and corresponding coating antigens.The three kinds of indirect ELISAs specificity were detected respectively.The results showed that Actinobacillus pleuropneumoniae positive sero(-),Streptococcus suis positive sero(-),Pasteurella multocida positive sero(-) and Bacillus coli positive sero(-).The indirect ELISA was confirmed to have good reproducibility by the repeating test.The whole-cell antigen,the supersonic antigen and the heat-stable antigen from Hps were used as coating antigens respectively in indirect ELISA in order to detect positive sero of all Hps serotype.The results of cross-reaction shows that the whole-cell antigen and the supersonic antigen had cross-reaction with all Hps serotype.The heat-stable antigen from Hps serotype4 had without cross-reaction with serotype 10.Serotype 8 had without cross-reaction with serotype 4,5 and 14.Serotype 10 had without cross-reaction with serotype 3,4,6,8 and 12.3.Detection of clinical serumThe two kinds of indirect ELISA with the whole-cell antigen and the supersonic antigen as the diagnosis antigen respectively detected Haemophilus parasuis antibody of 162 serum which is from four swine farms in SiChuan and ChongQing province,at the same time HIA was the contrast test.Coincidence of two indirect ELISAs and HIA were 98.15%and 97.53%respectively.Therefore,the indirect ELISA with the whole-cell antigen was better than others.The positive Clinic pig serums were detected indirect ELISA by Hps 1-15 serotype as diagnosis antigen respectively,the results show that The result is that 27 samples were showed positive of Hps 4,3 samples were the Hps 5or 14.Although there were crossreaction among some serotype,could detect some serotype.This study could diminish range of diagnosis.