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 > or = 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.

FIG. 1.

Immunoaffinity chromatography-purified MAb WB8A11-reacting Ag. (A) Silver staining of a 12.5% polyacrylamide gel; (B) Western blotting with MAb WB8A11. A total of 2 μg of protein of purified Ag was loaded for both SDS-PAGE and Western blotting. The positions of the protein molecular mass markers are indicated on the left of each panel.

FIG. 2.

Expression of rICD2 in E. coli was assessed by Western blotting with anti-His tag MAb of lysates of TOP10/pBicd2 and TOP10/pBAD TOPO (negative control) induced with l-arabinose or uninduced. Lanes 1 and 2, whole-cell lysate of TOP10/pBicd2; lanes 3 and 4, TOP10/pBAD TOPO; lanes 1 and 3, uninduced; lanes 2 and 4, induced with l-arabinose. The positions of the protein molecular weight markers are indicated on the left.

FIG. 3.

Silver-stained two-dimensional gel (A) and two-dimensional Western blot of purified rICD2 with MAb WB8A11 (B), anti-His tag MAb (C), or anti-ICD-II polyclonal serum from a C57BL/6 mouse (D). The positions of the protein molecular mass markers are indicated on the left of each panel; the pI ranges are indicated at the bottom of each panel.

FIG. 4.

Recognition of rICD2 by IgG from human serum by Western blotting. A serum sample from a patient with TB was tested for recognition of purified rICD2 at dilutions ranging from 1:80 to 1:5,260 (lanes 1 to 6); lane 7, positive control (MAb WB8A11); lane 8, negative control (incubation in TBS-3% BSA followed by incubation with peroxidase-conjugated goat anti-human IgG). The positions of the protein molecular mass markers are indicated on the left.

FIG. 5.

Number of TB patients (TB) and healthy subjects (H) giving positive reactions for anti-rICD2 by Western blotting with different serum dilutions (indicated below). The purified rICD2 for which the results are shown in Fig. 3 was used in these experiments. ▪, positive result; □, negative result; ∗∗∗, P ≤ 0.001; ∗, P ≤ 0.05.