Characterization of Flagellin Antigen Using a Panel of Monoclonal Antibodies and Their Applications for Detection of Salmonella Based on Surface Plasmon Resonance
Although Salmonella flagellin have been widely used in various immunoassays, it lacks information about its binding sites and binding kinetics with antibodies. Using inhibition assays in surface plasmon resonance (SPR) platform, we identified four distinct antibody binding sites on flagellin. We also compared the binding kinetics of flagellin with four monoclonal antibodies (MAb). SPR curves obtained due to interaction of different concentration of MAb with flagellin were fitted to 1:1 bio-interaction model to calculate binding kinetic parameters. Furthermore, SPR-based Salmonella enterica serovar Tyhimurium detection methods were developed, as there is no single method that is sufficiently faster, sensitive and selective to detect this most common foodborne pathogen. For the first method, S. Typhimurium inoculated romaine lettuce samples were enriched overnight, and flagellin was extracted. Three different assay formats were performed: direct assay-direct injection of flagellin, sequential two-step sandwich assay- flagellin injection followed by MAb injection, and pre-incubation one step sandwich assay-injection of incubated flagellin and MAb. It was found that buffered peptone water multiplied as low as 0.9 log cfu/mL of S. Typhimurium to the level that can be easily detected by SPR. This method was very specific to S. Typhimurium and had detection limit of 5.7 log cfu/mL. The second method was enrichment-free, where S. Typhimurium inoculated romaine lettuce samples were washed, followed by vacuum filtration of wash solution to capture bacteria on filter membrane, thereby to extract flagellin. Three assay formats were performed. In this method, antibody-coupled magnetic nanoparticles were used in sandwich assay types. Detection limit and detection time for this method were 5.5 log cfu/g of romaine lettuce and less than 4 hours, respectively. Results on binding sites and binding kinetics of flagellin will help to select MAbs for immunoassays that are aimed for detection and subtyping of Salmonella. Although enrichment-free detection method cannot discriminate viable and non-viable cells, it can quickly show the negative result for Salmonella negative samples. For samples with positive result, viability/non-viability can be determined by using alternate methods. In the future, these methods will be optimized to detect other Salmonella serotypes.
Devendra Prasad Bhandari,
"Characterization of Flagellin Antigen Using a Panel of Monoclonal Antibodies and Their Applications for Detection of Salmonella Based on Surface Plasmon Resonance"
ETD Collection for Tennessee State University.