Preparation Of The Monoclonal Antibody Against S-LPS And Development Of Competitive ELISA For Detection Of Antibodies Against Brucella

The serological responses following infection with smooth Brucella species are directed predominantly against the smooth lipopolysaccharide (S-LPS). Thus, in humans as well as in animals, the diagnosis of brucellosis is usually based on the detection of specific antibodies against S-LPS.The O polysaccharides of the smooth lipopolysaccharide is a homopolymer of 4,6-dideoxy-4-formamidoct-D-mannopyranose residues. Up to now,There were four types of antigen has been recognized : The A antigen, the M antigen, the common antigen (C) specificity for smooth Brucella and the common antigen crossreacting with Y.enterocolitica O:9 (C/Y).A problematic aspect of traditional serological detection was false-positive serological reactions (FPSRs). These FPSRs find their origins in the cross-reactivity observed between the S-LPS of Brucella species and the LPSs of some other Gram-negative bacterias, In particular Y.enterocolitica O:9. Whether the traditional detections such as tube agglutination test, rose bengal plate test and complement fixation test, or indirect ELISA with S-LPS as coating antigen, the FPSRs can not be ruled out .Based on the problem of cross-reaction may be solved by the monoclonal antibody aginst C epitopes of Brucella can solution, The B.melitensis M5-90 were inactivated to immune BALB/c mice, hybridomas were obtained by standard cell fusion techniques in the study. Extraction, Purification and Identification of LPS from Brucella vaccine strains M5-90, S-19 and Y.enterocolitica O:9 and Two cell strains secreting monoclonal antibody (McAb) against S-LPS, named 4G6 and 16C5, were screened by I-ELISA using three kinds of LPS .Specificity analysis showed that McAb 4G6 has no cross reaction with Gram-negative bacterias except Y.enterocolitica O:9 while McAb 16C5 with high sensitivity has no cross reaction with all of other bacterias Involved . Ascites of McAb 16C5 was purified by protein G and used in competitive ELISA (C-ELISA) as detect antibodies to develop the method for detection of antibodies against Brucella. A large number of clinical samples was detected to optimize reaction conditions and evaluat the detection methods by Compared with the rose bengal plate agglutination test, IDEXX indirect ELISA Kit, Sweden Svanova competition ELISA Kit and indirect ELISA kit produce by OIE recommended technology .Serum samples from cattle (n=507) were tested by the rose Bengal plate test (RBT) and IDEXX indirect enzyme-linked immunosorbent assay (IELISA) kits and the data demonstrated that the coincidence between C-ELISA and RBT ,C-ELISA and IDEXX IELISA kit was 84.7% and 91.2% respectively. One hundred and fourty-two of the 507 serum were tested and the results showed that the coincidence between C-ELISA and PINGHE IELISA , C-ELISA and Svanova C-ELISA was 94.4% and 97.2% respectively. The results of the C-ELISA are in good agreement (κ>0.5) with those of RBT, IDEXX IELISA kit, PINGHE IELISA kit, Svanova C-ELISA kit, which concluded that the C-ELISA has good reliability. Thus, the CELISA was a valuable asset to the diagnosis of brucellosis and could eliminate false-positive serological reactions that caused by Y.enterocolitica O:9, E. coli O:157 and so on.