A team of faculty from Wayne State University has discovered a new technology that will quickly and easily detect active antibodies to Mycobacterium tuberculosis (TB) infection. Their work, “Discovery of novel transketolase epitopes and development of IgG-based serodiagnosis of tuberculosis,” is published in a recent issue of Spectrum MicrobiologyA journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, MD, professor in the Center for Molecular Medicine and Genetics at the College of Medicine. Samavati is joined by Jaya Talreja, PhD, and Changia Peng, research scientists in the Department of Internal Medicine at Wayne State.
Tuberculosis remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest WHO report, tuberculosis is the 13th leading cause of death and the second infectious killer after COVID-19. Latent tuberculosis infection (LTBI) is a reservoir for TB bacteria and people with active TB can develop it. One-third of the world’s population has TB, and on average, 5 to 10% of people with LTBI will develop active TB disease over the course of their lives, usually within the first five years after initial infection.
The gold standard tests for determining whether an active TB infection is the smear test and the culture test. However, these methods require sputum collection, which is expensive, time-consuming, requires trained personnel, and lacks sensitivity. Current conventional tests that differentiate LTBI from uninfected controls—such as tuberculin skin tests (TST) and/or interferongamma release assay (IGRA)—do not differentiate between active TB infection and latent TB. Despite advances in rapid molecular techniques for tuberculosis diagnosis, there is an unmet need for an inexpensive simple point-of-care (POC), a rapid, non-sputum-based test.
Samavati’s research group has worked for more than 15 years to develop a technology to detect antibodies in various respiratory diseases. Her lab developed a new non-sputum-based technique and discovered several new immune epitopes that differentially bind to immunoglobulin (IgG) in people with tuberculosis. Epitope-specific IgG levels in the somme can distinguish active TB from subjects with LTBI, healthy infections and other respiratory diseases. This technique can be used as a simple, non-sputum-based serologic POC-TB test, which is highly specific and sensitive to active LTBI-resistant tuberculosis.
“Previously, we developed a T7 phage antigen display platform and after immunological screening of large batches of serum samples, we identified 10 clones that bind differentially to serum antibodies (IgG) of active TB patients that differentiate TB from other respiratory diseases,” Samavati said. “One of these high-performance clones had homology to the bacterium tuberculosis transketolase (TKT) enzyme which is an essential enzyme required for the intracellular growth of the bacterium in the host. We hypothesized that the abundance of serum IgG against the specific novel antigen that we named TKTµ might differentiate between active TB and LTBI and other granulomatous lung diseases other than tuberculosis such as sarcoidosis.We have developed a novel direct peptide ELISA assay to determine IgG levels in serum samples against TKTµ.We designed two additional nested M.tb TKT-peptide homologs with a potential antigen matching of M.transketolase determinant (M-TKT1 f M-TKT3) and thus three peptide ELISA (TKTμ, M.tb TKT1 and M.tb TKT3) were standardized for serodiagnosis of tuberculosis. “
After developing and standardizing a direct peptide ELISA for three peptides, the research team tested 292 people, and the results of the TKT-peptide ELISA showed that TB patients had significantly higher levels of TKT-specific antibodies than patients who were healthy and with LTBI. Presumably, increased levels of TKT-specific antibodies are associated with the growth of M.tb bacteria in active TB patients. TKT plays a key role in the transition from the dormant to proliferative stage and TKT-specific IgG may reveal differences between active TB and LTBI. Thus, IgG-based serodiagnosis of tuberculosis using TKT-peptide ELISA is promising.
Currently, commercially available serotyped tuberculosis tests show poor sensitivity and specificity. ELISA results obtained using TKT peptides discovered by the Wayne State team yielded high resolution and sensitivity. Their results show that IgG antibodies against transketolase can discriminate active TB disease.
“Our TKT peptide ELISA assay requires chemically synthesized TKT peptides to encapsulate wells in an ELISA plate, less than 100 μL of serum sample from a patient, detection reagents and an ELISA plate reader,” Samavati said. “We are very excited about our technology and the fact that with a simple test we can distinguish active TB from LTBI and other respiratory diseases. We believe that our method and the TKT peptide ELISA can fit the requirements of WHO and Centers for Disease Control and Prevention as a POC screening method.”
The research team has applied for a patent on its technology and is actively seeking out companies interested in investing.
This research was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health, with grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.