Development Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Lps Antigen Of B. Melitensis

The object of this study to produce specific monoclonal antibody (mcAb) against lipopolysaccharide (LPS) of B.melitensis. The LPS antigen was purified from B. melitensis strain 16M by using hot phenol extraction method. Six to eight-week-old female BALB/c mice were immunized with LPS in Freund's complete adjuvant and inactivated B. melitensis (strain 16M) in Freund's incomplete adjuvant successively. The mice were boosted with LPS intraperitoneally when their sera ELISA antibody titer reach 1:10000 after the fourth immunization. Fusion of SP2/0 myeloma Cells with spleen cells from immunized mice was conducted three days later.Twelve hybridoma cell lines, named 4A11, 6B8, 3H7, 4D4, 3E3, 3F9, 6E3, 6F2, 2C3, 5D11, 4C3 and 5H3, were developed and identified. Corresponding mAbs from 3E3 and 6E3 were of IgG1 isotype, mAbs from 3F9, 2C3, 5D11, 3H7 and 4D4 were of IgG3 isotype, while mAbs from 6F2, 5H3, 6B8, 4A11 were of IgM isotype. Chromosome analysis of two hybridoma cells(4D4 and 3H7)indicated that average chromosome number were 92~108 , which is consistent with chromosome number sum of SP2/0 cell and spleen cell. The result demonstrated that acquired antibody-secreting cells were hybridoma cells. The specificity assay showed that mAbs from 6B8,5H3 and 3H7 had non-cross-reactivity with antigens from Colibacillus O157, Salmonella pullorum, Flexneri Shigella and LPS of Gallibacterium anatis, only reacted with the inactivated thalli of B. melitensis (strain 16M). Rose-Bengal plate and tube agglutination assays indicated that the acquired mAbs could react with standard antigen of B.melitensis, which further demonstrated that the mAbs from 6B8,5H3 and 3H7 had strong specificity. The ELISA titers of cell cultures supernatant and ascites fluids of all 12 hybridoma cell lines ranged from 1:1000 to 1:10000, and1:10000 to 1:160000 respectively. Stability detection of five hybridoma cell lines, 6B8, 3E3, 3F9, 6E3, and 6F2 indicated that the growth of hybridoma cell was still vigorous after being cultured 30 passages and frozen and thawed three times. Even the ability of secreting antibody of several hybridoma cell lines decreased a little bit, they still can secret mAbs with high titer. This showed that established hybridoma cell lines could secret mAbs consistently.A sandwich ELISA for detecting B.melitensis was developed by using the mcAbs produced from hybridoma cell lines. Simulative samples, including water samples(S1,S2, milk samples(S3,S4)and soil samples(S5,S6)were tested with the developed sandwich ELISA. Results showed that this ELISA had very high accuracy.