BACKGROUND: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouse IgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity. METHODS: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli, and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step. RESULTS: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot. CONCLUSION: The purification method used is rapid, simple, and specific and can be easily scaled up.
BACKGROUND: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouseIgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity. METHODS: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli, and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step. RESULTS: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot. CONCLUSION: The purification method used is rapid, simple, and specific and can be easily scaled up.
Authors: Surinder M Singh; Aparna Sharma; Arun K Upadhyay; Anupam Singh; Lalit C Garg; Amulya K Panda Journal: Protein Expr Purif Date: 2011-09-20 Impact factor: 1.650
Authors: R Reljic; S O Clark; A Williams; G Falero-Diaz; M Singh; S Challacombe; P D Marsh; J Ivanyi Journal: Clin Exp Immunol Date: 2006-03 Impact factor: 4.330
Authors: Yamilé López; Daniel Yero; Gustavo Falero-Diaz; Nesty Olivares; María E Sarmiento; Sergio Sifontes; Rosa L Solis; Jorge A Barrios; Diana Aguilar; Rogelio Hernández-Pando; Armando Acosta Journal: Int J Med Microbiol Date: 2009-01-20 Impact factor: 3.473