Production of a polyclonal antibody against acrylamide for immunochromatographic detection of acrylamide using strip tests

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.

Review of Research into the Determination of Acrylamide in Foods

“…Molecular imprinting technology (MIT) can chemically synthesize high-specific and Compared with the ELISA, the immunochromatographic strip (ICS) is a relatively mature integrated detection product, which is simpler to operate and can be completed without professional operators, meeting the real-time and fast-detection requirements of AA. Assaat et al have produced, purified, and characterized a polyclonal Ab against AA for ICS testing of AA [83]. Polyclonal anti-AA Ab was prepared by injecting N-acryloxysuccinimide conjugated BSA hapten into New Zealand white rabbits and further purified with protein A and conjugated with Au nanoparticles (AuNPs).…”

Enzyme-Induced Silver Deposition on Gold Nanorods for Naked-Eye and Smartphone Detection of Acrylamide in Food

“…Studies based on specific antibodies for the detection of acrylamide have been reported. , However, only a few antibodies can directly recognize acrylamide, and the insufficient epitope of low-molecular-weight acrylamide strongly limits the reproducible production of specific antibodies and their application for reproducible immunoassays. − As a consequence, acrylamide immunoassays are mainly based on antibodies against acrylamide derivatives. However, common derivatization reactions, which use 3-mercaptobenzoic acid or 4-mercaptobenzoic acid, result in low efficiency and relatively long reaction times (over 1 h) with the need for heating (over ∼50 °C).…”

“…10 Studies based on specific antibodies for the detection of acrylamide have been reported. 14,15 However, only a few antibodies can directly recognize acrylamide, and the insufficient epitope of low-molecular-weight acrylamide strongly limits the reproducible production of specific antibodies and their application for reproducible immunoassays. 16−18 As a consequence, acrylamide immunoassays are mainly based on antibodies against acrylamide derivatives.…”