High-Throughput Identification of Diagnostic Bovine Tuberculosis Antigens
High-throughput filter plate protein purification helps to determine the antigens with highest reactivity and sensitivity for use in diagnostic TB testing
December 22, 2021
Bovine tuberculosis (TB) is a chronic infectious disease caused by three bacteria that form part of the Mycobacterium group: M. bovis, M. avium, and M. tuberculosis. Bovine TB caused by M. bovis, can be transmitted from livestock to humans and other animals, making it a very serious health concern. Once the most prevalent infectious disease of cattle in the United States, in the first part of the 20th-century, bovine TB caused more deaths amongst farm animals than all other infectious diseases combined.
Steps towards the eradication of Tuberculosis
The National Tuberculosis Eradication Program was set up in 1917 to eliminate TB within the US and is today administered by the U.S. Department of Agriculture(1). While today the US experiences only small and irregular outbreaks of bovine TB, the disease is extremely serious where it does arise, due to its ability to infect people with potentially life-threatening consequences. Early detection and diagnosis are key to limiting both the risk to human and animal health as well as mitigating the economic loss to farmers.
Current diagnostic tests must improve if we are to achieve full eradication of TB
The current frontline diagnostic test for bovine TB is the tuberculin skin test which detects the cellular immune response to administered purified protein derivative (PPD). However, this test has a known high false-positive rate due to the immune response to PPD proteins that are conserved across non-tuberculosis causing mycobacteria. Serodiagnostic tests are available but are effective chiefly in the advanced stages of infection, limiting their use for early detection. If the US Department of Agriculture’s goal of eliminating TB is to be realized there is clearly a need for improvement in our technology for the early detection of bovine TB.
In her 2020 Ph.D. thesis(2) at the University of Ottawa Department of Medicine, Nadia Assal described a screening process to identify novel M. bovis antigens that could be used for diagnostic purposes. As the complete genomes of several M. bovis isolates have already been sequenced and are available in the public domain, the author was able to take a whole-genome approach, utilizing bioinformatics to identify genes encoding cell surface proteins in silico. This approach provided a candidate list of 77 extracellular and outer membrane proteins for further study.
A novel high-throughput approach identifies new antigens with diagnostic potential
A high-throughput system was then developed to express these antigen candidates, involving the PCR amplification, cloning, and expression of the selected proteins. The number and volume of proteins involved in the study necessitated the development of a high-throughput purification process for which the author selected AcroPrepTM Advance 96-well filter plates. The 96-well format of the AcroPrep filter plates enables the processing of multiple proteins in parallel, and the filter membrane can recover the target protein at a high level of quality and purity suitable for use in the creation of a microarray. This microarray spotted with high purity recombinant proteins was then used for high-throughput screening. By exposing the microarrays to sera from M. bovis-infected animals plus uninfected negative controls, it was possible to rapidly screen the proteins and identify those that exhibited an immunological reaction.
The screening process identified 13 antigens that were able to repeatably distinguish between the infected and uninfected animals. Out of these 13 antigens, six showed high levels of reactivity and sensitivity, of which four were novel and had not been previously described. These four were taken forward into further evaluation using dot blot and ELISA assays in which two of the antigens exhibited a strong ability to discriminate between infected and control sera. The author suggests that these two novels, high-sensitivity antigens should be considered for inclusion in the existing serodiagnostic test to enhance the early detection of outbreaks caused by M. bovis.
At Pall we manufacture a wide range of high-quality filtration products that are used in critical applications by researchers across the globe. You can learn more about the AcroPrep Advance Filter Plates used in this study, along with other laboratory research solutions, for protein purification and analysis in our Life Science Research section.
- U.S. Department of Agriculture. Animal and Plant Health Inspection Service. 2020. National Tuberculosis Eradication Program [online] Available at: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/cattle-disease-information/national-tuberculosis-eradication-program [Accessed 28 October 2021]
- Nadia Assal. High Throughput Discovery of Novel Diagnostic Antigens for Mycobacterium bovis Using a Whole Genome Approach. [Ph.D. Thesis]. Ottawa, Canada: University of Ottawa; 2020