Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Abstract

Tuberculosis is emerging as a major public health problem in developing and developed wodd. Early and precise diagnosis is of prime importance in successful control of Infection. Indirect ELISA with penicillinase as marker was developed using purified M. tuberculosis excretory-secretory (EST-DEt) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE t antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb and Mtb , as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE 1 antigen.

Excretory Secretory Proteins Released during Growth of Mycobacterium tuberculosis (H37Ra), With Diagnostic Potential in Pulmonary and Extra Pulmonary Tuberculosis

“…The 2nd, 6th, 7th and 10th SDS-PAGE fractions from ESAS proteins were isolated and screened for antigenic activity. ESAS-6 antigen on fractionation on resource Q (1 ml) anion exchange column by FPLC, gave ESAS-6A containing 43 kDa protein [20]. ESAS-7 antigen on fractionation on resource S (1 ml) cation exchange column by FPLC, gave ESAS-7F (31 kDa) protein fraction having ES-31 antigen [21,22].…”

“…The bound antibodies were eluted using 1 M acetic acid and neutralised with tris buffer (0.1 M, pH 8.6). The eluents were concentrated by ultra-filtration [20] and stored at -200˚C till its use.…”

“…In short, indirect ELISA for detection of antibodies in serum samples of pulmonary as well as extra pulmonary TB cases was performed using cellulose acetate membrane (CAM) sticks [20]. In brief 5 μl of optimally diluted specific antigens, antihuman IgG penicillinase conjugate and freshly prepared starch-iodinepenicillin-V substrate was used.…”

“…Complete decolourizations or decolourization with slight tinge of substrate colour denoted a positive reaction. Similarly Sandwich ELISA was performed for antigens [20]. For convenience and bulk sample analysis, PVC microtitre plates were used.…”

Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of mycobacterium tuberculosis

“…The bacteria was pelleted by centrifugation and washed thdce with buffer A. The protein was precipitated with 10% Trichloroacetic acid (TCA) (Sigma) (12). The precipitated protein was dissolved in a buffer of 20mmol/l Tris-HCI (pH 7.4), containing l mg/l benzamidine-HCI (Sigma), 0.2mmol/I phenyl methyl sulphonyl fluodde and lgm/I Triton X -100 (Sigma).…”