Characterization of Campylobacter Flagellin Using a Panel of Monoclonal Antibodies and Their Application in Direct Detection Using Surface Plasmon Resonance
Campylobacter bacteria is one of the main causes of gastroenteritis worldwide. Flagellin is a major antigen of the Campylobacter flagellar filament and plays vital role in immunogenicity and pathogenicity. Limited studies have been conducted based on flagellin immunoassays; therefore, there are insufficient information on flagellin antibody binding pattern and binding kinetics. The binding patterns of two different groups (Group I and Group V) of monoclonal antibodies (MAbs) were studied to identify the common and strain specific binding pattern against flagellin between Campylobacter strains and species using western blot approaches. The MAb binding patterns on trypsin-digested flagellin was studied for the possibility of structural relatedness between flagellin among Campylobacter species. Comparison between the binding kinetics and binding affinity of flagellin antigen to its intact anti-flagellin antibody placed on the sensor surface was measured by Surface Plasmon Resonance (SPR). The obtained SPR data were fitted to simple 1:1 Langmuir interaction to measure the binding kinetics parameters. Pair-wise epitope mapping was tested initially with individual Group V MAbs and with PAbs in a separate experiment on the immobilized sensor against their specific anti-flagellin antibodies followed by sequent interactions with flagellin and their respective anti-flagellin antibodies. Finally, for SPR direct detection of Campylobacter, different concentration of C. jejuni antigen was injected on the sensor surface immobilized with one of the MAbs (5A6). Collectively, the results from western blot indicated that Group V MAbs showed two binding bands at 65 and 42 kD in C. jejuni strains but showed only single band at 65 kD in C. coli strains along with similar but unique banding patterns among Campylobacter strains. The binding affinities ranged from 4.34 nM to 98.4 nM were obtained from five different MAbs tested against three different strains of C. jejuni antigen. No epitope pair was obtained from MAbs and PAbs against flagellin of C. jejuni species The sensitivity of 7.7 × 104 CFU/ml was obtained from SPR direct assay. This study has contributed significant information in understanding the diversity of flagellin among Campylobacter strains and species. Findings from this study also suggest the use of alternative antibodies for affinity measurements and epitope mapping.
Shreya Singh Hamal,
"Characterization of Campylobacter Flagellin Using a Panel of Monoclonal Antibodies and Their Application in Direct Detection Using Surface Plasmon Resonance"
ETD Collection for Tennessee State University.