Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics

Despite advances in rapid molecular techniques for tuberculosis (TB) diagnostics, there is an unmet need for a point-of-care, nonsputum-based test. Previously, through a T7 phage antigen display platform and immunoscreening, we identified that the serum IgGs of active TB patients differentially bind to several antigen-clones and that this immunoreactivity discriminates TB from other respiratory diseases. One of these high-performance clones has some homology to the transketolase of Mycobacterium tuberculosis ( TKT). In this study, we developed a direct enzyme-linked immunosorbent assay (ELISA) detecting IgG against the TKT antigen-clone (TKTμ). Through sequence alignment and analysis, we designed two more peptides with potential antigenicity that correspond to specific transketolase ( TKT1 and TKT3) epitopes. After the development and standardization of a direct peptide ELISA for three peptides, we tested 292 subjects, including TB ( = 101), latent tuberculosis infection (LTBI) ( = 49), healthy controls ( = 66), and sarcoidosis ( = 76). We randomly assigned 60% of the subjects to a training set to create optimal models to distinguish positive TB samples, and the remaining 40% were used to validate the diagnostic power of the IgG-based assays that were developed in the training set. Antibodies against TKT3 yielded the highest sensitivity (0.845), and these were followed by TKTμ (0.817) and TKT1 (0.732). The specificities obtained by TKTμ, TKT3, and TKT1 on the test sets were 1, 0.95, and 0.875, respectively. The model using TKTμ obtained a perfect positive predictive value (PPV) of 1, and this was followed by TKT3 (0.968) and TKT1 (0.912). These results show that IgG antibodies against transketolase can discriminate active TB against LTBI, sarcoidosis, and controls. There is an unmet need for a point-of-care, nonsputum-based TB test. Through the immunoscreening of a novel T7 phage library, we identified classifiers that specifically bind to IgGs in active TB sera. We discovered that one of these clones is aligned with Mycobacterium tuberculosis transketolase (TKT). TKT is an essential enzyme for Mycobacterium tuberculosis growth. We designed three TKT epitopes (TKTμ, TKT1, and TKT3) to detect TKT-specific IgGs. After the development and standardization of three different ELISA-utilizing TKT peptides, we tested 292 subjects, including active TB, LTBI, healthy controls, and sarcoidosis. Rigorous statistical analyses using training and validation sets show