Production and Purification of Monoclonal Antibody against ManLAM antigen from Mycobacterium Bovis

Lipoarabinomannan mannesylated (Man­LAm) is one of the lipoglycans which forms a considerable percent of Mycobacterium Bovis BCG’ cellular wall. Considering that generation of monoclonal antibody against Man­LAM antigen has many applications, specifically, in order to design the diagnostic kit for rapid diagnosing of tuberculosis, so the main objective of this study was based on production of monoclonal antibody against this antigen. The female BALB/c mice were immunized by regular injections of sonicated BCG and LAM antigen, then after each injection, circulation of generated antibodies were investigated. Spleen lymphocytes of immunized mice and SP2/0 myeloma cells were fused into each other. The cells were selected by HAT medium. Identification and selection of anti-LAM antigen producing clones were performed using ELISA test. After purification of anti-ManLAM, detection of ManLAM antigen and sonicated BCG carried out by western blot technique. The two H1-3D3, H2-3B9 clones, producing specified antibody were selected which had higher absorbance in ELISA test. The results of Western blotting showed that 30KDa band was related to sonicated BCG and ManLam antigen, using IgG3 IgM antibodies. The results indicated that using the fusion of hybridoma myeloma cells, it is possible to produce and purify monoclonal antibodies, and before performing diagnostic studies in order to have rapid diagnosing of tuberculosis in patient`s urine, and investigations for immunizing the BCG vaccine, conformation of specified reaction of mentioned antibody will be considered.