Graduation Year

2006

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology

Major Professor

Daniel V. Lim, Ph.D.

Keywords

Autopsy, Biosensor, Immunoassay, Lung Tissue, Tuberculosis

Abstract

There is no rapid diagnostic technique at medical examiners' offices to determine if a decedent is infected with Mycobacterium tuberculosis. Present diagnostic testing requires at least one month for results. A biosensor-based sandwich immunoassay for the detection of M. tuberculosis was developed in this study. M. tuberculosis polyclonal antibody was used for target antigen capture and detection in the immunoassay. Live attenuated M. tuberculosis (ATCC 25177) suspended in phosphate-buffered saline with 0.1% Tween 20 was used as the antigen in the detection assay. The Analyte 2000 was the initial biosensor platform. Initial testing was of Freund's adjuvant complete. M. tuberculosis was detected 50% of the time at 1,000,000 CFU/ml and 100% of the time at 10,000,000 CFU/ml and 100,000,000 CFU/ml. Live attenuated M. tuberculosis was also tested using the Analyte 2000 biosensor. Detection was obtained 87.5% of the time at 1,000,000 CFU/ml and 100% of the time at 10,00

0,000 CFU/ml and 100,000,000 CFU/ml. The RAPTOR, an automated, portable instrument, was then tested as the fiber optic biosensor platform. Positive biosensor detection was obtained 75% of the time at cell concentrations of 1,000,000 CFU/ml, 95% of the time at 10,000,000 CFU/ml, and 99% of the time at 100,000,000 CFU/ml. Live attenuated M. tuberculosis suspended in PBST and seeded into decedent lung tissue was tested using the RAPTOR. Positive detection was obtained 21% of the time at cell concentrations of 1,000,000 CFU/ml, 86% of the time at 10,000,000 CFU/ml and 100% of the time at 100,000,000 CFU/ml. Antibody specificity studies using ELISA were performed to determine the anti-M. tuberculosis antibody's cross reactivity with microorganisms other than M. tuberculosis. M. tuberculosis actively growing in the lung of an individual is found at levels of 10,000,000 to 1,000,000,000 CFU in the lesions of the lung. This study determined that the RAPTOR biosensor assay was capable of

detecting the presence of M. tuberculosis in lung tissue homogenate within three hours when the concentration of M. tuberculosis was 10,000,000 to 1,000,000,000 CFU/ml.

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