Abstract
Objective:To produce, purify, and characterize a polyclonal antibody against acrylamide (anti-AA) for an application to immunochromatographic strip tests for AA.Materials and Methods:Polyclonal anti-AA was prepared by injecting N-acryloxysuccinimideconjugated bovine serum albumin hapten-antigen into New Zealand white rabbits. The antibody was purified using protein A, characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and conjugated with gold nanoparticles (AuNP). The conjugated antibody was then characterized using UV–Vis and FTIR spectroscopy and transmission electron microscopy (TEM). Immunochromatographic strip tests were performed using sample pads, conjugated pads, test zones, control zones, and absorbent pads. Strip tests were finally validated using standard AA solutions followed by the application of various concentrations of coffee samplesResults:Using SDS-PAGE, the purified anti-AA antibody was resolved at 50 and 25 kDa, indicating the presence of heavy and light chains, respectively. The conjugation of anti-AA with AuNP was confirmed using wavelength shifts in UV–Vis and FTIR spectra, and TEM analyses revealed increased diameters of AuNPs after conjugation. The immunochromatographic strip test was sensitive to 1 mgml-1 standard AA. Various concentrations of coffee samples resulted in red color differences in the test zone. High and low coffee concentrations produced thick and thin red lines, respectively.Conclusion:Purified anti-AA can be conjugated with AuNP to produce strip tests for detecting AA in coffee samples. The present immunochromatographic strip tests quantitatively showed increasing intensities of red lines with increasing AA concentrations.
Highlights
Acrylamide (AA) is generally present in low concentrations in food samples and is difficult to detect
Various methods have been developed to detect AA in food samples and these are commonly based on chromatographic techniques, including liquid chromatography [4], gas chromatography [5], and capillary electrophoresis [6]
N-acryloxysuccinimide (NAS), bovine serum albumin (BSA), phosphate buffer saline (PBS), polyethylene glycol (PEG) 2000, 3,3ʹ, 5,5ʹTetramethylbenzidine (TMB), Tween 20, goat antirabbit IgG-HRP, Freund’s complete and incomplete adjuvants and protein A antibody purification kits, and other chemicals were supplied from Sigma Aldrich
Summary
Acrylamide (AA) is generally present in low concentrations in food samples and is difficult to detect. Various methods have been developed to detect AA in food samples and these are commonly based on chromatographic techniques, including liquid chromatography [4], gas chromatography [5], and capillary electrophoresis [6]. Production of a polyclonal antibody against acrylamide for immunochromatographic detection of acrylamide using strip tests. Res., 6(3): 366–375, September 2019 and competitive indirect ELISA [8] were reported All of these methods require sophisticated laboratory instruments that need to be operated by experts. We developed a rapid test kit for detecting AA in food using principles of immunochromatography. The detection of food contaminant is important to increase the health quality of humans. Easy and applicable sensors are required for food quality and in industrial process or environmental issues related
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