Preparation Of Monoclonal Anitbody To Brucella Melitensis And The Detection Of Clinical Isolates

Brucellosis is a zoonotic disease caused by Gram-negative bacteria from thegenus Brucella. It features more routes of infection, general susceptibility topeople,difficulty in curing, and worldwide distribution.Also,brucellosis is a kind ofB class infectious diseases of zoonoses according to 《Infectious Diseases PreventionLaw of the People’s Republic of China》.The prevalence and treatment of brucellosis isespecially important,because the prevalence of it has seriously bad impact on theeconomic development and the health of residents.And because of lacking specificclinical manifestations, laboratory diagnostic methods become the main basis fordifferential diagnosis of brucellosis. Beside,compared with traditional laboratory tests,the monoclonal antibody has higher specificity and becomes one of the primarymeans of differential diagnosis of brucellosis.Objectives: To prepare the monoclonal antibody with high specificity applyingfor differential diagnosis of Brucella and other Gram-negative bacteria (salmonellatyphimurium, enterocolitica O:9, Escherichia coli O157, lislefia monocylogenes),which can lay the foundation for the later experiments.Beside,it also can further applyto the detection of the epidemic strains of Songyuan in Jilin Province and providereliable laboratory evidence for the early diagnosis of local brucellosis.Methods: In order to create a high sensitivity of the iELISA, the most suitableconcentration of Enzyme-labeled goat anti-mouse antibody was determined by usingSquare matrix titration optimization to select the best package concentration..Firstly, a mixture of Brucella melitensis M5suspension and Freund’s completeadjuvant or Freund’s incomplete adjuvant we used to immunizate healthy femaleBALB/c mice. Next, mice with high titer were screened by iELISA, and thesplenocyte from these mice were fused with SP2/0cells in logarithmic growth phaseratio of5:1. At the same time, positive fusion cells were subcloned by limiting antibody ascites were prepared by the body induced germinal. Saturated ammoniumsulfate and AKTA purification device were used to purify monoclonal antibody. AndSDS-PAGE Electrophoresis analysis was used to detect purification and antibody titer.Finally,detecting monoclonal antibody on the aspect of specificity, subtype and abilityto secrete antibodies stably.The iELISA established was used to detect the cross-reaction betweenmonoclonal antibody and isolates of Songyuan.Results: The most suitable concentration of coating antigen and Enzyme-labeledgoat anti-mouse antibody were determined, respectively1:10and1:4000. And theiELISA with high sensitivity also was determined. The cell lines6D3that had nocross-reaction with B.abortus, B.suis and some Gram-negative bacteria, highspecificity, and ability to secrete antibodies stably were obtained by cell fusion. Andthe identification of subtybe was IgG3for monoclonal cell lines6D3. Beside, therewere no significant changes of monoclonal antibodies in the ascites which wereextracted crudely by saturated ammoninm sulfate with33%and50%. Also, theresultes of crude extraction showed that there were still o lot of impurity proteinbands, and that the antibody titer was1:640. However, monoclonal antibodies hadtwo clear bands and no non-specific bands after purified by puriication device. Andthe results of puriication showed that the heavy chain was located between46KD and58KD,while the light chain was located around25KD, and that the purified antibidytiter was1:1280.After MLVA analysised on the10clinical isolates, there were8clustering toB.melitensis type2, while S95and S178clustering to the M5branch. The result ofcross-reaction were positive between monoclnoal antibodies secreted by6D3andS95(one of the10isolates), and between monoclnoal antibodies was positive.the P/Nwas15.67,other resuits were negative. The result that the monoclonal antibodiessecreted by6D3and S476(one of the12isolates), but others were negative. Besides,the values of P/N were respectively15.6and7.43.Conclusions: By hybridoma technology, the cell lines6D3obtained with highspecificity and ability to secrete antibodies stably can distinguish not only between Brucella and other Gram-negative bacteria, but also between B.melitensis, B.abortus,B.suis, and can provide a reliable basis for laboratory testing for brucellosis.Monoclonal antibody is initially applied to detect the22isolates of Songyuan in Jilinprovince, the positive cross-reaction only with S95and S476strains,Withtype-specific, can be used for pathogen detection.