Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor

Abstract

Salmonella Typhimurium is one of the leading causes of foodborne diseases worldwide. Biosensors and immunoassays utilizing monoclonal antibodies are widely used for the detection and subtyping of S. Typhimurium. However, due to insufficient information on the nature of binding with S. Typhimurium flagellin, the selection of appropriate antibodies for assay development is a cumbersome task. Hence, we aimed to compare the binding kinetics of a panel of monoclonal antibodies and their relative binding sites to flagellin antigen using a surface plasmon resonance biosensor. Initially, the flagellin was captured on the sensor surface through an immobilized anti-flagellin antibody. The interactions of different concentrations of monoclonal antibodies to flagellin were determined, and binding curves were fitted using 1:1 bio-interaction model to calculate the kinetic parameters. For epitope mapping, pairwise comparisons were completed to determine the binding inhibition of each paired combination of monoclonal antibodies. It was found that these monoclonal antibodies differed significantly (p < 0.05) in association rate, dissociation rate, and equilibrium dissociation constants. Of the five monoclonal antibodies, only two interfered with the binding of each other. Four distinct epitopes located within a 23 kDa domain of flagellin were identified. Findings from this study provide crucial information needed for the further development and optimization of biosensors and other immunoassays for the detection and subtyping of Salmonella.

Detection of Salmonella Typhimurium in Romaine Lettuce Using a Surface Plasmon Resonance Biosensor

“…A 500 kDa carboxymethyl dextran hydrogel surface sensor chip (SR7000 gold sensor slide) was purchased from Reichert Inc., NY, USA. Monoclonal antibodies (MAb 1E10 and MAb 1C8) specific to S. Typhimurium flagellin were produced in our laboratories as described in a previous study [29].…”

“…The flow rate of 20 µL/min and temperature of 25 °C were maintained throughout the immobilization process. Immobilization of MAb 1E10 on the sensor surface was performed as descried in our previous study [29]. In order to activate carboxyl groups on the surface of the sensor chip, fresh preparation of 40 mg EDC and 10 mg NHS dissolved in 1 mL of water was injected onto the sensor surface for 5 min.…”

“…The problem of nonspecific binding of polyclonal antibodies associated with S. Typhimurium in the SPR assay can be circumvented by using monoclonal antibodies specific to the targeted antigens. Monoclonal antibodies specific to flagellin of S. Typhimurium have been produced [28] and characterized in terms of their binding kinetics and epitope maps, as described in our previous work [29]. Use of the well characterized monoclonal antibodies either in direct or sandwich assays will render the SPR assay more specific and sensitive.…”

Surface Plasmon Resonance for Biomarker Detection: Advances in Non-invasive Cancer Diagnosis

“…Generally, SPR sensing can examine the interactions between biomolecules based on affinity binding analysis, including antibody-antigen (Hearty et al, 2018), ligand-receptor kinetics (Carroll et al, 2016; Teran and Nugent, 2019), enzyme-substrate reaction (Massumi Miyazaki et al, 2017), and epitope mapping (Bhandari et al, 2019). SPR is often used as a complementary method to analyze conformational changes study rather than as a primary technique.…”

Antibodies as Diagnostic Targets and as Reagents for Diagnostics

“…This Special Issue of Antibodies gives examples of current "state-of-the-art" uses of Abs as targets of diagnostics (infections and autoimmune diseases) [18][19][20][21][22] and as reagents for measuring analytical targets [23,24].…”