Development of a new indirect ELISA method for detection of anti-tuberculosis antibodies in human serum

Tuberculosis is a crucial health problem. Establishing a rapid, reliable and still inexpensive diagnostic method for tuberculosis seems to be substantial in developing countries where TB has very high incidence rate. An Indirect Enzyme-linked immunosorbent Assay [ELISA] was established to detect serum antibodies against Mycobacterium tuberculosis. Three kinds of antigens were used to prepare the solid phase for antibody assay including: purified protein derivative [PPD], M. tuberculosis Bacilli, and Mycobacterium bovis Bacillus Calmette Guerin [BCG]. Sera of two main following groups were investigated in this study: sera samples from smear-positive, culture-positive and Tuberculin Skin Test-positive TB patients and sera samples from smear-negative, culture negative and TST-negative healthy individuals. Among the antigens used, BCG produced higher sensitivity and specificity in the assay. With PPD as the solid phase, higher sensitivity, but lower specificity was observed in comparison with BCG. Both, low response and noise [non-specific binding] were observed with TB bacilli as the solid phase in the assay. Using BCG solid phase system in this method resulted in higher sensitivity in comparison to single antigen solid phase systems. In addition, we were able to circumvent the problem of non-specific bindings in more popular multi-antigenic solid systems such as PPD. By using this new indirect ELISA, a rapid, reliable and still inexpensive diagnosis of tuberculosis might be possible. Although, further investigations are required to our result

Cloning, expression, purification and immunoreactivity analysis of gag derived protein p17 from HIV-1 CRF35 in fusion with thioredoxin from human subjects

So far, recombinant antigens of HIV-1, the etiologic cause of Acquired Immunodeficiency Syndrome [AIDS], have been widely used for the diagnosis and vaccine development. P17 or the matrix protein formed by the proteolytic cleavage of gag is strongly antigenic and is as conserved and immunogenic as p24. In some cases, antibodies to p17 are more prevalent than antibodies to p24 and the decline in the level of p17 antibodies is an earlier prognostic marker for disease progression than decline in the level of antibodies to p24. The aim of this study was to clone and express the gag derived p17 protein in soluble form in E. coli and then assess the immunoreactivity of produced recombinant p17. DNA sequence encoding p17 matrix protein was cloned from PTZ-gag53-IR vector that has the complete gag polyprotein sequence. The T7-promoter-based expression system used in this study was TOPO directional cloning strategy that expressed p17 matrix protein in fusion with thioredoxin in E. Coli. Purification of produced recombinant protein has been done using Ni-NTA nickel chelating agarose beads. Sequencing result of the cloned sequence showed that it belongs to CRF35_AD subtype of HIV-1 which is highly prevalent in Iran and Afghanistan. The immunoreactivity of produced recombinant p17 to sera from infected individuals showed 93.8% sensitivity and 100% specificity