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Subcellular Distribution of Monoclonal Antibody Defined Epitopes on Immunodominant Mycobacterium tuberculosis Proteins in the 30‐kDa Region: Identification and Localization of 29/33‐kDa Doublet Proteins on Mycobacterial Cell Wall
Author(s) -
RAMBUKKANA A.,
DAS P. K.,
CHAND A.,
BAAS J. G.,
GROOTHUIS D. G.,
KOLK A. H. J.
Publication year - 1991
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1991.tb02551.x
Subject(s) - epitope , monoclonal antibody , mycobacterium tuberculosis , antigen , biology , cytosol , mycobacterium bovis , antibody , microbiology and biotechnology , tuberculosis , virology , biochemistry , immunology , enzyme , medicine , pathology
Two different groups of monoclonal antibodies (MAbs) directed to different epitopes on 30‐kDa region proteins of Mycobacterium tuberculosis were isolated; MAbs 5F9, 5D5 and 5D2 reacted with a single 33‐kDa protein band, whereas MAb 3A8 readied with A distinct 29,33‐kDa doublet when analysed by immunoblotting. The present paper describes the distribution of MAbs defined epitopes in the 29 33‐kDa region proteins in well‐characterized subcellular fractions: cytosol, plasma membrane, cell wall as well as culture filtrate of M. tuberculosis. MAbs 5F9, 5D5 and 5D2 reactive epitopes were found in cytosol, whereas 3AS epitope is distributed in all cellular compartments of the mycobacterium as well as in the culture filtrate. Localization of these epitopes by indirect immunoflourescence and immunogold‐labelling demonstrated that only 3A8 epitope is present on the cell surface of the mycobacterium. Both immunoblotting and EL[SA showed that only MAh 3A8. and not 5F9. 5D5 and 5D2, reacted with secreted BCG 85 antigen complex of Mycobacterium bin‐it BCG. Furthermore, using an MAb 3AS‐coupled affinity column, we purified antigen 3A8 from the cytosol fraction of M tuberculosis. All these MAhs reacted with antigen 3AK with varying degrees of intensity, thus suggesting that they are directed to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and BCG‐85 complex suggests that these epitopes might have been lost during the processing of the same 33‐kDa protein on its way out from cytosol to the cell wall or when the protein is secreted out into the culture filtrate. Our results demonstrate, for the first time, direct evidence of the presence of a 30‐kDa region protein not only in secreted antigens but also in the cell wall and on the cell surface of the mycobacterium.