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Research

Disease State Differentiation and Identification of Tuberculosis Biomarkers via Native Antigen Array Profiling^*,S

Mark J. Sartain, Richard A. Slayden, Krishna K. Singh, Suman Laal^,¶ and John T. Belisle^,||

From the Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80521 and Department of Pathology, New York University School of Medicine and ^¶ Veterans Affairs New York Harbor Health Care System, New York, New York 10010

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients’ sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients’ sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients’ sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.

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