Peptide-based antibody detection for tuberculosis diagnosis

Abstract

Tuberculosis (TB) is a major cause of morbidity and mortality, especially in developing countries. Despite significant limitations, microscopy remains the cornerstone of the global TB control strategy. As the TB epidemic escalates, new diagnostic methods that are accurate and also economical and simple to manufacture and deploy are urgently needed. Although several promising antigens have been identified and evaluated in recent years, the reproducible production of high-quality recombinant mycobacterial proteins with minimal batch-to-batch variation is difficult, laborious, and expensive. To determine the feasibility of devising a synthetic peptide-based diagnostic test for TB, we have delineated the immunodominant epitopes of three candidate antigens, Ag85B, BfrB, and TrxC, that were previously identified to be immunogenic in TB patients. The results demonstrate that combinations of carefully selected synthetic peptides derived from highly immunogenic proteins can be the basis for devising an immunodiagnostic test for TB.

FIG. 1.

Epitope mapping of Ag85B. (A) Overlapping peptides of Ag85B were screened for their reactivities with sera from TB patients and healthy PPD⁺ and PPD⁻ subjects. Peptides that were recognized by none of the healthy control sera are depicted as minimal bars (1% reactivity). (B) Validation of reactivities of peptides identified to be recognized by >50% of the serum samples from TB patients during initial screening. AR, additive reactivity obtained with the six validated peptides.

FIG. 2.

Reactivities of individual serum samples from PPD⁻ healthy subjects (•), PPD⁺ healthy subjects (▪), and TB patients (▴) with peptides A25 (A), B13 (B), and T11 (C). OD values obtained from one representative experiment are shown. OD values above 1.0 are depicted as 1.0. There were no statistically significant differences between the OD values obtained with sera from PPD⁺ or PPD⁻ individuals with any of the peptides. The OD values for the sera from TB patients were statistically different from those for the PPD⁻ and the PPD⁺ healthy subjects. OD405, OD at 405 nm.

FIG. 3.

Validation of reactivities of BfrB peptides (A) and TrxC peptides (B) which were identified to be recognized by the sera from TB patients during the initial screening. AR, additive reactivity obtained with the validated peptides.