ANTIGEN IDENTIFICATION FOR SERODIAGNOSIS OF TUBERCULOSIS

Project Details

Description

The dramatic re-emergence of tuberculosis has rendered present diagnostic
methods inadequate. The PPD skin test detects infection by Mycobacterium
tuberculosis, so infection and active disease are not distinguished.
Additionally, the PPD test can often produce unreliable results:
immunocompromised patients with active tuberculosis are frequently PPD-;
healthy, BCG vaccinated people are PPD+, and exposure to non-pathogenic
mycobacteria produce a positive PPD result. Tests that diagnose active
tuberculosis ultimately depend on recovery of M. tuberculosis in culture
and thus are extremely slow. Microscopic examination of stained sputum
specimens remains the most rapid method of detecting active tuberculosis.
However, this method often produces false negative, or equivocal results.
It is thus clear that new diagnostics are necessary. Serology
constitutes an attractive alternative technique for tuberculosis
diagnostics, because it does not require the presence of infecting
organisms in the clinical specimens, is rapid, simple and inexpensive.
Lack of success in establishing a sensitive and specific ELISA for
tuberculosis to this date is primarily due to limited availability of pure
antigens and insufficient knowledge of humoral immune response to
tuberculosis in humans. Our overall goal is to identify M. tuberculosis-specific antigens that are
recognized by human immune sera and to obtain them in a readily
purifiable form, in order to develop ELISA-based serodiagnosis of
tuberculosis. Antigens will be identified by screening an M. tuberculosis
recombinant phage expression library with human sera from various
individuals (HIV+, HIV-, latent infection, active disease, during anti-
tubercular therapy, different age groups). Positive clones will be
characterized by restriction enzyme analysis and nucleotide sequencing,
and on the basis of their reactivity with monoclonal antibodies directed
against known mycobacterial antigens. Screening selected clones with M.
tuberculosis DNA isolated by genomic substraction with BCG DNA will help
identify antigens that are unique to M. tuberculosis. Analysis of
selected antigen-expressing clones will establish a panel of antigens that
will be used for screening with large numbers of sera by ELISA to begin to
establish their diagnostic value. DNA fragments containing full-length
genes will be cloned in recombinant gene expression vectors that allow
high-level expression as well as simple one-step protein purification.
During the period of requested funding, we expect to identify antigens
with different specificities: antigens that specifically react with sera
from infected versus diseased individuals, that detect infection/or
disease in children, that are recognized by individuals coinfected with M.
tuberculosis and HIV, that are recognized by antibodies that wane during
successful antitubercular treatment, and that are recognized by antibodies
that are maintained at detectable serum levels well past completion of
therapy.
StatusFinished
Effective start/end date9/30/949/30/04

Funding

  • National Institutes of Health: $398,054.00
  • National Institutes of Health: $57,916.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $422,145.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $409,922.00

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)

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