Development of an Immunoassay-Based Lateral Flow Dipstick for the Rapid Detection of Aflatoxin B1 in Pig Feed

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Abstract
The aim of this work was to develop an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. The test consisted of three main components: conjugate pad, membrane, and absorbent pad. The membrane was coated with two capture reagents, that is, aflatoxin B1−bovine serum albumin conjugate and rabbit anti-mouse antibodies. The detector reagent consisted of colloidal gold particles coated with affinity-purified monoclonal anti-aflatoxin B1 antibodies, which saturated the conjugate pad. A comparison of several extraction methods for the pig feed matrix is presented. A mixture of methanol/water (80:20, v/v) gave the best recoveries. After sample extraction and dilution, the dipstick was put in the sample solution at the conjugate pad side and developed for 10 min. Analyte present in the sample competed with the aflatoxin B1 immobilized on the membrane for binding to the limited amount of antibodies in the detector reagent. Thus, the line color intensity of an aflatoxin B1-positive dipstick is visually distinguishable from that of an aflatoxin B1-negative sample. The visual detection limit for aflatoxin B1 is 5 μg/kg. The major advantages of this one-step striptest are that results can be obtained within 10 min and that all reagents are immobilized on the lateral flow dipstick. Keywords: Aflatoxin B1; lateral flow; dipstick; rapid detection assay; pig feed