ABSTRACT
Objective/background: the development of new tools capable of targeting Mycobacterium tuberculosis [Mtb]-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system [Mtb lipid-CD1b complexes]. Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2' -sulfate-alpha-alpha'-o-trehalose [Ac[2]SGL] is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac[2]SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac[2]SGL complexes
Methods: a synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac[2]SGL using CD1btransfected THP-1 cells loaded with Ac[2]SGL
Results: one clone, D11- a single, light-variable domain [kappa] antibody [dAb[kappa]11]-showed high relative binding to the Ac[2]SGL-CD1b complex
Conclusion: a ligand recognizing the Ac[2]SGL- CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications
ABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis , Immunoglobulin A , ChromatographyABSTRACT
In order to determine the IgA levels in secretions, the authors engaged themselves to obtain monoclonal antibodies against this immunoglobulin and to show their possible utilization in the development of quantitation methods. 6 monoclonal antibodies were obtained: one against the secretory component (SC) and five against the heavy-chain (HC) of IgA. IgA purified from human colostrum was used as immunogen (HC). The immunoglobulin isotypes (IgG2a and IgG1) were determined in 2 monoclonal antibodies, one against SC and the other against HC. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulphate and b-mercapto-ethanol. Immunotransference was also carried out to determine their specificity. Secretory IgA was detected in saliva specimens. The anti-SC monoclonal antibody obtained in-the laboratory of the "Pedro KourÝ" Institute of Tropical Medicine (IPK) was compared with a commercial one (Sigma Chemical Co.), and a similar behaviour was observed. The monoclonal antibody against SC (IPK) was used to detect IgA specific to LPS ogawa in persons vaccinated with an attenuated strain of Vibrio cholerae. Marked differences were found between the levels before and after immunization. The anti-HC monoclonal antibody was utilized to make an ELISA-like simulated system. The detection limit was 9.89 ng/mL.