Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of Mycobacterium bovis BCG in Serodiagnosis of Tuberculosis

Abstract

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of >1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.Tuberculosis (TB) remains a major cause of death and disabilities in developing countries, where over 90% of global cases occur, and is now also a cause for growing concern in industrialized countries, where the incidence of the disease has also increased (6). Diagnosis of TB in developing countries mainly relies on examination of chest X rays and/or examination of smears under a microscope for detection of acid-fast bacilli. However, only about 50% of the patients with pulmonary TB are smear positive, and chest X rays can detect advanced pulmonary TB only after extensive damage of lung tissues has already occurred (22). At present, the most reliable method for diagnosis of TB is still isolation of organisms by culture and biochemical identification of the tubercle bacilli, but because of the slow growth rate of Mycobacterium tuberculosis, results are not seen until several weeks after specimen collection. The time to detection might be shortened to 1 week by the use of radiometric systems and nucleic acid probes, but these techniques are still too expensive for use by laboratories in developing countries. A simple and inexpensive diagnostic assay might be of great benefit for global control of the disease (9). Considerable efforts have been made to evaluate assays based on specific recognition of selected mycobacterial antigens (Ags) in vitro by serum antibodies (Abs) (16,17,25,34) or the release of gamma interferon by peripheral blood mononuclear cells stimulated with M. tuberculosis-specific Ags (33, 34). In the latter case, the use of cocktails of immunodominant Ags of M. tuberculosis has been indicated to increase the sensitivity of the assay significantly without affecting the specificity of the assay (33), and the same strategy has been suggested for use in the diagnosis of TB based on detection of specific Ab responses (13,14). As the pattern of Ag recognition by patient Abs may be influenced by the stage of the disease (15, 28) and b...

Identification of immunogenic proteins of Candida parapsilosis by serological proteome analysis

“…Its homologous protein was identified as antigen of pathogenic fungus A. fumigatus (Singh et al 2010) and Gram-negative bacterium Helicobacter pylori (Mcatee et al 1998). Idh2p was also shown to elicit strong B cell response and was a potential immune marker for serodiagnosis of tuberculosis (Florio et al 2002). Importantly, this study showed that the recombinant Idh2p reacted specifically with pooled sera from mice infected with C. parapsilosis on immunoblot, therefore validating its antigenicity.…”

“…Idh2p was also shown to elicit strong B cell response and was a potential immune marker for serodiagnosis of tuberculosis (Florio et al . ). Importantly, this study showed that the recombinant Idh2p reacted specifically with pooled sera from mice infected with C. parapsilosis on immunoblot, therefore validating its antigenicity.…”

Variant tricarboxylic acid cycle in Mycobacterium tuberculosis : Identification of α-ketoglutarate decarboxylase

“…Many TCA cycle and related enzymes in Mtb have been studied individually in recent years, such as isocitrate lyase (8), malate dehydrogenase (26), and isocitrate dehydrogenase (26,27). However, to our knowledge, there have been no reports in which KDH activity was purified from Mtb lysates or reconstituted by using recombinant Mtb proteins.…”

PPE Antigen Rv2430c of Mycobacterium tuberculosis Induces a Strong B-Cell Response

“…The picture remained unaltered when we used PPD in place of Hsp10. It is pertinent to note that although several antigens have been tested for use in serodiagnosis, no test with a single antigen has proved able to achieve sensitivity and specificity in a study population that is suitably large and heterogeneous (3,5,9,11,13,14,16,17,18,19,21,22). The factors responsible for this include (i) the stage of the disease, (ii) the location of the infection, and (iii) the genetic background.…”