High-Throughput Identification of Diagnostic Bovine Tuberculosis Antigens

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 AcroPrep^TM 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.