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Journal of Clinical Microbiology, July 2001, p. 2485-2493, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2485-2493.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Serological Expression Cloning and Immunological Evaluation of MTB48, a Novel Mycobacterium tuberculosis Antigen

Michael J. Lodes,1,* Davin C. Dillon,1 Raodoh Mohamath,1 Craig H. Day,1 Darin R. Benson,1 Lisa D. Reynolds,1 Patricia McNeill,1 Diana Pedral Sampaio,2 Yasir A. W. Skeiky,1 Roberto Badaro,2 David H. Persing,1,3 Steven G. Reed,1,3,4 and Raymond L. Houghton1

Corixa Corporation1 and Infectious Disease Research Institute,3 Seattle, Washington 98104; Department of Pathobiology, University of Washington, Seattle, Washington 981954; and University of Bahia, Salvador, Brazil2

Received 22 November 2000/Returned for modification 27 March 2001/Accepted 23 April 2001

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

* Corresponding author. Mailing address: Corixa Corporation, 1124 Columbia St., Suite 200, Seattle, WA 98104. Phone: (206) 754-5797. Fax: (206) 754-5715. E-mail: lodes{at}corixa.com.

Journal of Clinical Microbiology, July 2001, p. 2485-2493, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2485-2493.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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