Homogeneity of antibody responses in tuberculosis patients

Abstract

The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936-3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.

FIG. 1

Reactivities of sera with fractionated lipoarabinomannan-free CFP of M. tuberculosis. Lanes: 1 to 6, PPD-negative healthy controls; 7 to 18, PPD-positive healthy controls; 19 to 25, smear-negative, noncavitary TB patients; 26 to 31, smear-positive, noncavitary TB patients; 32 to 50, cavitary TB patients; 51, murine monoclonal antibody IT-23 (anti-38-kDa protein); M, molecular mass (in kilodaltons). Each lane contains 10 μg of antigen, and all sera were tested at a 1:100 dilution.

FIG. 2

Reactivities of sera from four PPD-positive healthy controls (diluted 1:200) with 2-D polyacrylamide gel-fractionated, lipoarabinomannan-free CFP of M. tuberculosis. Thirty micrograms of antigen was fractionated on each blot. Antigens recognized by healthy individuals are circled in blue. Molecular masses (in kilodaltons) are indicated on the left of each panel.

FIG. 3

Reactivities of sera from four non-HIV-infected, smear-negative, noncavitary TB patients (diluted 1:50) with 2-D polyacrylamide gel-fractionated, lipoarabinomannan-free CFP of M. tuberculosis. Thirty micrograms of antigen was fractionated on each blot. Antigens recognized by sera from controls also are circled in blue. Antigens recognized by sera from patients but not from control individuals are circled in green. Only antigens recognized by at least two of the four patients are circled. Antigens recognized primarily, and strongly, by sera from cavitary TB patients (see Fig. 4) are circled in red. 2-D blots and lanes corresponding to Fig. 1: panel A, lane 22; panel B, lane 23; panel C, lane 19, and panel D, lane 21. Molecular masses (in kilodaltons) are indicated on the left of each panel.

FIG. 4

Reactivities of sera from five non-HIV-infected, smear-positive, cavitary TB patients (diluted 1:200) with 2-D polyacrylamide gel-fractionated, lipoarabinomannan-free CFP of M. tuberculosis. Thirty micrograms of antigen was fractionated on each blot. Antigens recognized by sera from controls also are circled in blue. Antigens recognized by sera from noncavitary and cavitary TB patients but not from control individuals are circled in green. Antigens recognized primarily, and strongly, by sera from cavitary TB patients are circled in red. 2-D blots and lanes corresponding to Fig. 1: panel A, lane 47; panel B, not known; panel C, lane 48, panel D, lane 41; panel E, not known. Molecular masses (in kilodaltons) are indicated on the left of each panel.

FIG. 5

Reactivities of sera from four HIV-infected, noncavitary TB patients (diluted 1:200) with 2-D polyacrylamide gel-fractionated, lipoarabinomannan-free CFP of M. tuberculosis. (A and B) Smear-positive patients; (C and D) smear-negative patients. Thirty micrograms of antigen was fractionated on each blot. Antigens recognized by sera from controls also are circled in blue. Antigens recognized by sera from non-HIV-infected, noncavitary and cavitary TB patients but not from control individuals are circled in green. Antigens recognized primarily, and strongly, in cavitary TB patients are circled in red. Molecular masses (in kilodaltons) are indicated on the left of each panel.