A team of professors from Wayne State University has discovered new technology that will quickly and easily detect active antibodies to Mycobacterium tuberculosis (TB) infection. Their work, “Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics,” was published in a recent issue of Microbiological Spectrum, a journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, MD, a professor in the Center for Molecular Medicine and Genetics at the School of Medicine. Samavati is joined by Jaya Talreja, Ph.D, and Changya Peng, investigators in Wayne State’s Department of Internal Medicine.
Tuberculosis remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest World Health Organization report, tuberculosis is the 13th leading cause of death and the second biggest infectious killer after COVID-19. Latent tuberculosis infection (LTBI) is considered a reservoir for TB bacteria and individuals can progress to active TB. One-third of the world’s population is infected with TB and, on average, 5 to 10% of those infected with LTBI will develop active TB during their lifetime, usually within the first five years after initial infection.
The gold standard tests to determine whether an infection is active TB are sputum smear and culture tests. However, these methods require sputum collection, which is time-consuming, expensive, requires trained personnel, and lacks sensitivity. Current conventional tests that differentiate LTBI from uninfected controls — such as tuberculin skin tests (TSTs) and/or the interferon gamma release assay (IGRA) — do not differentiate between active and latent TB. Despite advances in rapid molecular techniques for TB diagnosis, there is an unmet need for a simple, inexpensive, rapid, non-sputum-based point-of-care (POC) test.
Samavati’s research team has worked for more than 15 years to develop technology to detect antibodies in various respiratory diseases. Her laboratory has developed a new technology that does not rely on sputum and has discovered several new immuno-epitopes that differentially bind to specific immunoglobulin (IgG) in people with TB. Serum levels of epitope-specific IgG can differentiate active tuberculosis from individuals with LTBI, healthy individuals, and other respiratory diseases. This technology can be used as a simple sputum-free serum POC-TB serology test, which is highly specific and sensitive to differentiate active TB from LTBI.
“Previously, we developed a T7 phage antigen display platform and after immunoscreening large sets of serum samples, we identified 10 clones that differentially bind to the serum antibody (IgG) of active TB patients that differentiate TB from other respiratory diseases,” said Samavati . “One of these high yielding clones had homology to the Transketolase (TKT) enzyme of tuberculosis bacteria which is a key enzyme required for the intracellular growth of the bacteria in a host. We hypothesized that the abundance of IgG in the sera against the identified new neoantigen which we termed as TKTμ can differentiate between active TB, LTBI and other non-TB granulomatous lung diseases such as sarcoidosis. We developed a novel direct peptide ELISA test to quantify IgG levels in serum samples against TKTμ. We designed two additional overlapping M .tb TKT peptide homologues with potential antigenicity corresponding to M.tb-specific transketolase (M.tb-TKT1 and M.tb-TKT3) and therefore standardized three peptide ELISAs (TKTμ, M.tb TKT1 and M.tb TKT3) for the serodiagnosis of tuberculosis.”
After developing and standardizing a direct peptide ELISA for three peptides, the research team tested 292 subjects and TKT peptide ELISA results show that TB patients have significantly higher levels of TKT-specific antibodies compared to healthy patients controls and with LTBI. Elevated levels of TKT-specific antibodies are probably associated with the growth of M.tb bacteria in active TB patients. TKT plays a key role in the transition from the dormant phase to the proliferative phase, and TKT-specific IgG can reveal the differences between active TB and LTBI. Thus, IgG-based TB serodiagnosis with TKT peptide ELISA is promising.
Currently, commercially available TB serological tests show low sensitivity and specificity. ELISA results obtained with the Wayne State group’s discovered TKT peptides yielded high specificity and sensitivity. Their results show that IgG antibodies against transketolase can distinguish active tuberculosis.
“Our TKT peptide ELISA test requires chemically synthesized TKT peptides to coat the wells on the ELISA plate, less than 100 µl of blood serum sample from a patient, detection reagents and an ELISA plate reader,” said Samavati. “We are extremely excited about our technology and the fact that with a simple test we can differentiate active TB from LTBI and other respiratory diseases. We believe that our method and the TKT peptide ELISA can meet the requirements of the World Health Organization and of the Centers for Disease Control and Prevention as a POC screening method.”
The research team has applied for a patent for its technology and is actively seeking companies interested in investing.
This research was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health, grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.