Research On The Brucella Strain Specificity Monoclonal Antibodies And Establishing Antigen-captured ELISA

The combination of monoclonal antibody and ELISA enhances greatly thespecificity of ELISA detecting pathogenic microorganism.This paper focus onB.abortus and B.melitensis which spreads in the word,and make monoclonal antibodiesof B.abortus 544A B.melitensis 16M,and estable a high specific and sensitive antigencapture ELISA.The first,this study select two smooth-Brucella strains of B.abortus 544A andB.melitensis 16M,and use two different methods to purify their LPS and O-chain.Thecomparison of two methods,the phenol-methanol get more pure productions than theheat phenol,however,the phenol-methanol get lower antigenic productions than the heatphenol.When purifying O-chain,we get two absorption peaks of ultraviolet,the agaroseimmunodiffusion (AGID) identificate that samples of the second absorption peak areO-chain.After electrophoresis of SDS-PAGE,we learn that the B.abortus 544A LPSmolecular weight is about 46KDa, B.melitensis 16M LPS molecular weight is about59KDa,and both of their O-chain molecular weight is about 6000～7000KDa.O-chain of smooth Brucella have 7 antigen sites,A and M is domination,othersinclude two C sites and three C/Y sites,M site is located mainly on the surface ofB.melitensis, A site is located mainly on the surface of B.abortus,C sites are specifiedto smooth Brucella,it is common antigen, C/Y sites are shared by Brucella and YersiniaO:9.If we would build up high specific ELISA,we should choose those monoclonalantibodies to M,A and C sites.We immunize respectively 5 female Balb/C mouse withB.abortus 544A and B.melitensis 16M.After 4 times immunifaction,we take mousespleen to make cell fusion.We use LPS andO-chain to detect positive cell pore,andclone positive pore three times,and make monoclonal antibodies ascites aftercultivating extending.Meanwhile,we analyze the charactor of monoclonalantibodies,and find that monoclonal antibodies of 4B8,1C9,1G9,1E2 aim directlyat A site of B.abortus 544A, 3C6 aim directly at M site of B.melitensis 16M, 2D10 and1E10 aim possiblly at C site of B.melitensis 16M and B.abortus 544A. Moreover,wefind that the content of antibodies in 4B8,2D10 ascites is the 2.1mg/ml and 2.4mg/ml,and their affinity quantity is the 8.99×108 M-1 and 5.30×107 M-1,so they aresuitable to be used to build up ELISA because of their high affinity quantity.We use 4B8 and 2D10 to build up sandwich ELISA,and optimize sandwichELISA by choosing monoclonal antibodies and groping various conditions, includingmulti-antibodies coating, monoclonal antibodies reacting,enzyme labeled antibodyreacting,substrate coloration and so on..Finally,we determine the procedure ofAC-ELISA.And then,we detect the repeatability of AC-ELISA,the data is analyzed bySPSS10.0,results of P1=0.107>0.05 and P2=0.173>0.05 show that the AC-ELISAhave qualified repeatability.The results of sensitivity detection show that theAC-ELISA of B.abortus 544A and B.melitensis 16M can not take place crossreactionwith E.coli O:157,Yersinia O:9,Salmonella and Actinobacillus.The minimum detectionof B.melitensis 16M AC-ELISA is 3.0×103CFU/ml to5.0×103CFU/ml, B.abortus544A AC-ELISA is 5.0×103CFU/ml to1.0×104CFU/ml.In laboratory,we make thesimulative samples,including water samples, oil samples and milk samples,and detectthese samples with AC-ELISA,the rate of coincidence is 100%,the 7 shares cow and8 shares ewe blood samples are gathered from TongLiao city Keerqin district ofinner-Mongolia,Plate agglutination detection show positive,and then AC-ELISAdetect these samples, the rate of coincidence is 100%.