Development Of Monoclonal Antibody Against Bovine Brucellaellosis And Its CELISA Kit

Brucellaellosis is one of the worldly commonst zoonosis caused by Brucella strains, especially B.abortus and B.melitensis. Brucellaellosis has been listed as necessary-report pathogen by CDC, its popularity among many countries,especially in China, except a few of free-Brucellaellosis countries. In resent years, Brucellaellosis has the trend of recover and cause to the huge economic loss of public health and development of animal husbandry, it’s serious of Brucellaellosis prevention. Because there is no ideal medicine for Brucellaellosis, the key point of prevention is accurate test. Brucellaella detection methods comprising traditional serological diagnosis, like agglutination test (SAT), rose bengal plate agglutination test (RBPT), complement fixation test (CFT), polymerase chain reaction (PCR),fluorescence polarization immunoassay(FPIA). However, Brucellaella is similar with some other Gram negative bacteria, especially Yersinia’s 09 and E.coli’s 0157 in LPS’s structure, the false-positive serological reaction exist in these serological diagnosis because of this cross-reactivity, especially in SAT and RBPT. Considering this problem, this study has developed the anti-B. abortus specific monoclonal antibody (mAb) and a competitive ELISA (cELISA) was developed with this mAb to test Brucellaella antibodies in animal serology, compared with the commercial cELISA test, the result showed that the cELISA in this study could eliminate the false-positive serological reaction that caused by Yersinia’s 09 and E. colis 0157. All these data demonstrate the developed cELISA here can exert important function in eradication program of Brucellosis.1. Development and identification of monoclonal antibody against BrucellaIn this study, inactivated bovine species Brucella strains S2308 was injected as immunity antigen for BALB/c mice, intensive injection was conducted after the three common injection, the spleen cells from the immunized mice were fused with SP2/0 myeloma cells. Bovine species Brucella A99, Yersinia O9 and E.coli O157 were coated to screen antibodies by iELISA respectively, two cell plants, named Brucella-5C10 and Brucella-4D2, could secret MAb against Brucella stably were obtained. Identification of the Brucella-5C10 showed that subgroup was IgG3/λ and ascitic fluid level was 1:64000; Identification of the Brucella-4D2 showed that subgroup was IgG3/λ. and ascitic fluid level was 1:32000. The SAT results showed that two MAbs performed intensive agglutinations with B.abortus 2308, B.abortus A99 and B.melitensis 16M; on the contrary, there is no obvious agglutinations with Y. enterocolitica 09 and E. coli 0157. The results of Western-blot showed that two MAbs performed obvious reactions with Brucella but no reactions with Y. enterocolitica 09 and E. coli 0157. These two MAbs in this study provide well biological materials for the development of cELISA kit for testing antibodies against Brucella in animals.2. Development of the cELISA kit for testing Antibodies against BrucellaTo test Brucellaellosis’s antibody in samples, Bovine species Brucella A99 was used as coated antigen in the ELISA plates, Brucella-5C10 was used as a competitive type antibody, HRP-conjugated goat anti-mouse IgG was used as the second antibody for this cELISA test kit. The optimized assay results showed that the diagnosis was the most reasonable when Bovine species Brucella A99 was coated as 4μg/hole; MAb was diluted as 1:4000; samples were diluted as 1:20; HRP-conjugated goat anti-mouse IgG was used as 1:10000. After ROC curve analysis, inhibition rate of 30% was selected in this cELISA method; the sensitivity was 93.3% and the specificity was 96.2% under 95% confidence interval. Clinical samples of 320 cattle and 147 sheep serum were tested by the developed cELISA and commercial cELISA, the results showed that 167 samples were positive and 139 samples were negative by both cELISA tests,14 samples were different between the two methods, with an overall agreement of 95.6%; 112 were positive and 24 were negative in the sheep sera,11 samples were different between the two methods, with a compatibility of 92.5%. Then the 25 controversial samples between the two cELISAs were comfirmed by the CFT, the agreement of the CFT test was calculated to be 85% (21/25) for cELISA in this study. Otherwise, the cELISA kit in this study could not appear the false-positive reactions when the sera against Y. enterocolitica 0:9 were below 200 IU/ml titer.Statistics show that the developed cELISA kit has nice sensitivity and specificity, especially the characteristic of elimination of false-positive caused by the classic cross-reaction bacterium Y. enterocolitica 09 and E. coli 0157. This test provides an effective tool for accurate serological diagnostic of Brucellaella.