The objective of the proposed research is to developed a rapid, reliable, sensitive and clinically useful diagnostic test for mycobacterial infections, including tuberculosis, based upon a wholly novel methodology. The format of the assay to be used, termed an immuno-PCR, combines elements of an immunoassay with nucleic acid amplification by the polymerase chain reaction (PCR). By linking the enormous signal generating power of the PCR to an immunoassay detecting protein antigens, an exquisitely sensitive assay is created. An immuno-PCR has been reported to detect as few as 580 molecules of antigen (9.6 x 10.22 moles), making it probably the most sensitive method yet developed for detection of protein antigens. The sensitivity of this assay may allow it to be developed into a diagnostic and screening test for mycobacterial antigens in a variety of biological specimens, including ones containing relatively few organisms. It may even be possible to detect mycobacterial antigens in expired breath. Because of the global prevalence of tuberculosis, as well as the resurgence of the disease in the United States, there is a pressing need for a more rapid method of diagnosis. Based upon an innovative adaptation of the immuno-PCR format, and preliminary work applying it to the detection of mycobacterial antigens, the immuno-PCR shows great promise as the basis of a rapid and ultrasensitive diagnostic assay. The work proposed will involve collaborative by university researchers, a medical device research and development company, and clinicians. Thirteen specific tasks are proposed which will advance the assay through development, optimization, validation, testing on """"""""spiked"""""""" biological fluids, and testing with actual clinical samples. The immuno-PCR will be adapted for detection of tuberculosis and other mycobacterial antigens in a variety of biological fluids and in expired breath. Successful performance of these tasks will develop this novel assay into a rapid, reliable, sensitive and specific diagnostic test.
