Nonspecificity of 35 kDa protein: A proposed marker for the differential diagnosis of <i>M. avium</i> infection in the Indian population

Objective: The subunit vaccine strategies and development of various diagnostic reagents for Mycobacterium avium infection relies on the presence of secreted, species-specific mycobacterial antigens. The M. avium 35 kDa protein has been suggested as a candidate for vaccine/diagnostic reagent, specifically for M. avium infection. The present study was conducted to evaluate the diagnostic specificity of the M. avium 35 kDa protein in the Indian population. Materials and Methods: Culture filtrate proteins were isolated by growing the bacilli in modified Youman&#x2032;s medium. The 35 kDa protein was purified by high-resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blast search was carried out. Western blotting was performed with either monoclonal antibody CS-38 or serum samples of tuberculosis (TB) patients. The 35 kDa-specific immunoglobulin G antibody titer was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA). Results: Despite the absence of gene for the 35 kDa protein, the sera of TB patients and TB patient&#x2032;s contacts nonspecifically recognize it. Of 109 TB patients tested, the sera of 84 patients in ELISA (percentage recognition = 87.5&#x0025;) and 27 of 29 TB patients tested in western immunoblotting (percentage recognition = 93.10&#x0025;) recognized the M. avium 35 kDa protein, while with sera of TB patient&#x2032;s contacts, the recognition was 50&#x0025;. Conclusion: Contrary to Western studies, the M. avium 35 kDa protein does not seem to be a good candidate for the specific diagnosis of M. avium infection in the Indian population

Evaluation of Aro-Tal-AST Complex Protein as a Marker for Differential Diagnosis of Mycobacterium Avium Infection

Purpose: Conventional diagnostic techniques for detecting Mycobacterium avium infection are far from satisfactory. As serodiagnostic tests for M. avium infection have been shown to be simple and rapid, the present study was carried out to identify and evaluate M. avium secretory protein(s) of diagnostic potential. Materials and Methods: Initially, by differential immunoblotting, a specific protein band of 45-50 kDa was recognized. Anion exchange column chromatography was used for purification of proteins. After fractionation, blast search was carried out. Further immunoreactivity studies were done with M. avium and Mycobacterium tuberculosis infected mice sera. Clinical utilization was confirmed by conducting indirect enzyme-linked immunosorbent assay (ELISA) with serum samples from mycobacterial infected patients. Results: A complex of three proteins (Aro-Tal-AST) of molecular weight ~48 kDa, shown to be Aro A homologue (Aro), transaldolase (Tal) and aspartate transaminase (AST) by blast search was separated. Immunoreactivity studies of purified complex protein with mice sera confirmed it to be specific for M. avium infection. Indirect ELISA with patient samples further confirmed it to be M. avium infection specific. Conclusion: Aro-Tal-AST protein is specifically recognized by patients infected with M. avium and can be used as a marker for simple and rapid ELISA based tests for differential diagnosis of M. avium infection in patients with M. avium complex (MAC)

Evaluation of <i style="">Mycobacterium tuberculosis</i> specific RD antigens for delayed type hypersensitivity responses in guinea pig

117-123Tuberculin skin test (TST), an age old method is based on measuring delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). However, inspite of simplicity, ease and cost effectiveness, the usefulness of PPD test is limited due to its inability to distinguish among a protective immune response, latent infection and active tuberculosis disease. On the other hand, a skin test based on RD antigens would add advantages of a high specificity of antigens with the logistics of a skin test. However, except few reports, in vivo data of intradermal use of RD antigens for skin testing is limited. Therefore, in the present study, four M. tuberculosis (Mtb) specific antigens (ESAT6, CFP10, CFP21 and MPT64) were evaluated for their diagnostic utility based on DTH response. These antigens alone and their multiple combinations induced strong DTH response in Mtb infected guinea pigs and the response was negligible in BCG vaccinated and sham immunized animals