Author
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Brandon, David |
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ADAMS, LISA - Former ARS Employee |
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/21/2015 Publication Date: 3/30/2015 Publication URL: http://handle.nal.usda.gov/10113/60893 Citation: Brandon, D.L., Adams, L.M. 2015. Milk matrix effects on antibody binding analyzed by elisa and biolayer interferometry. Journal of Agricultural and Food Chemistry. 63(13):3593-3598. doi: 10.1021/acs.jafc5b01136. Interpretive Summary: Analytical methodology to detect biothreat and bacterial toxins in foods is important for food safety and food defense. Immunoassay, the use of antibodies in a test system, frequently provides rapid and sensitive methods, though test results are often influenced by components of the food sample. In order to understand some of the effects of milk on immunoassays, we used an optical sensor system called biolayer interferometry (BLI) to study the rate at which ricin toxin and a related protein bind to and dissociate from antibodies and to a protein which models the cellular system targeted by this potent toxin. We found that components of milk, including both low molecular weight components like sugars and high molecular weight components affect these binding reactions. Milk components accelerated the dissociation of complexes, explaining earlier observations that milk can inhibit ricin activity in a cellular assay system. Understanding toxin binding reactions could lead to better antibody-based analysis of foodborne contaminants and new strategies for food safety and defense. Technical Abstract: Biolayer interferometry (BLI) was employed to study the impact of the milk matrix on the binding of ricin to asialofetuin (ASF) and to antibodies. This optical sensing platform utilized ligands immobilized covalently or via biotin-streptavidin linkage, and the results were compared to those obtained by enzyme-linked immunosorbent assay (ELISA). In sandwich ELISA, the binding of ricin to ASF was dramatically decreased when galactose was present during the analyte or detection antibody binding step. Low concentrations of milk (1%, v/v) produced a similar reduction in ricin binding to ASF, but not to a high affinity monoclonal antibody (mAb), increasing the dissociation rate of ASF-ricin complexes up to 100-fold. The effect of milk on the binding of ricin to ASF was ascribable to dialyzable factors, and milk sugar can account for these effects. The use of high affinity mAbs in ELISA effectively limits the milk matrix effect on ricin analysis. |