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Infect Immun, February 1998, p. 717-723, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

Morten Harboe,1,* Adam S. Malin,2 Hazel S. Dockrell,2 Harald Gotten Wiker,1 Gunni Ulvund,1 Arne Holm,3 Mikala Clok Jørgensen,4 and Peter Andersen5

Institute of Immunology and Rheumatology, University of Oslo, N-0172 Oslo, Norway1; Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London, United Kingdom2; and Chemical Institute, Royal Veterinary and Agricultural University,3 and Clinical Biochemistry Department4 and TB Research Unit,5 Statens Seruminstitut, Copenhagen, Denmark

Received 15 May 1997/Returned for modification 26 June 1997/Accepted 10 July 1997

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

* Corresponding author. Mailing address: Institute of Immunology and Rheumatology, University of Oslo, Fr. Qvams gate 1, N-0172 Oslo, Norway. Phone: 47 22 03 31 70. Fax: 47 22 20 72 87. E-mail: morten.harboe{at}labmed.uio.no.




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