Preparation And Purification Of Brucella Lps And Monoclonal Antibodies

Brucella， a gram-negative aerobic bacteria， causes brucellosis which results inserious economic losses to the livestock industry and great harm to human healtharound the world. As the brucellosis was not easy to cure currently， it is moreimportant to establish a useful detection method of brucellosis.In this study， the LPS was extracted from M5Brucella in hot phenol water.Then the BALB/c mice were immunized with purified LPS to measure itsimmunogenicity by Dot enzyme-linked immunoassay (Dot-ELISA). The resultsshowed that the LPS was highly purified and its immunogenicity was similar to thebrucella. The titer of immune serum can reach the ratio of1:106.On the basis， the spleen cells of immunized BALB/C mouse and SP2/Omyeloma cell were fused by PEG-4000to produce hybridoma. The hybridomas withpositive supernatants were screened by indirect enzyme-linked immunoassay (ELISA)for3times and cloned culture by limited dilution methods for3-4times. Finally， weobtained4cell lines which produced antibody steadily. The antibody titers of cellsupernatants determined by ELISA can reach the ratio of1:104，1:105，1:105and1:105， respectively. At the same time， the cell supernatants were identified byTricine-SDS-PAGE. The result showed that the target protein can be found in the cellsupernatants， while the target protein can not be found in the cell culture. At last，we identified the relative affinity and stability of the antibody. The results showed thatthe relative affinity of the antibody were about24.13μg/mL，24.65μg/mL，24.95μg/mL and23.16μg/mL， respectively.And the titers of antibody were stable.After obtaining the cell lines， the antibody was preliminary purified byammonium sulfate precipitation and further purified by affinity chromatography. Thenwe determined the titer of purified antibody by ELISA. The result showed that thetiter was a little lower than that in the cell supernatants. The results of theTricine-SDS-PAGE showed that molecular weight of light chain was approximately26ku and the heavy chain was approximately37ku. The results showed that4celllines with steady brucella antibody were obtained and it can be used to establish arapid diagnostic method for brucellosis.