Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection

Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected with Toxoplasma gondii Beverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection with T. gondii Beverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed

Humoral immune response in human tuberculosis: immunoglobulins G, A, and M directed against the purified P32 protein antigen of Mycobacterium bovis bacillus Calmette-Guérin

The P32 protein antigen of Mycobacterium bovis BCG, identified as antigen 85A in the BCG reference system, was used to investigate the humoral immune response in human tuberculosis (TB). Immunoglobulin G (IgG), IgA, and IgM directed against P32 were measured by an enzyme-linked immunosorbent assay. Mean IgG and IgA antibody levels differed significantly (P less than 0.001) between active-TB patients (50 untreated and 52 treated) and healthy control subjects (111 unvaccinated tuberculin negative, 38 unvaccinated tuberculin positive, and 72 recently BCG vaccinated). Mean IgG antibody levels, but not mean IgA antibody levels, were higher (P less than 0.05) in patients with positive microscopic examination for acid-fast bacilli than in patients with negative microscopic examination. A positive relation was found between mean levels and the extent of disease. There was no difference in mean IgM antibody levels between patients and controls. By setting the upper normal limit at the 95th percentile of the 221 healthy subjects, the sensitivities were 46% in untreated and 63% in treated patients for IgG and 30 and 50%, respectively, for IgA. Of the untreated patients, 56% were positive for either IgG or IgA antibodies. Among the untreated patients with negative direct smear, 35% were positive for IgG and 24% were positive for IgA. When both immunoglobulin classes were combined, the serological test was positive in 47% of those patients. Neither naturally acquired tuberculin hypersensitivity nor BCG vaccination affected positivity frequencies in healthy subjects. Only active TB seemed to induce significant anti-P32 antibody levels and to be associated with positivity. A serological test with P32 as the antigen might therefore be helpful for the rapid diagnosis of TB.</jats:p

Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt.) Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two- and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.</jats:p