Preparation and utilization of an immunoadsorption column for aflatoxin B1 composed of nanobodies and pre-crosslinked agarose microspheres.

Recent studies on immunoaffinity chromatography (IAC) columns for aflatoxin B1 (AFB1) have not provided a comprehensive preparation procedure or explored their capacity, reusability, and stability. In this study, we outline a preparation process for an AFB1 immune affinity column filler (AFB1-IAC) for extracting AFB1 from food sources like rice, peanuts, and soybeans. Using commercially available IAC is often limited due to high costs, low capacity, slow flow rates, and poor reusability. To overcome these issues, we developed succinic anhydride (SA)-modified agarose pre-crosslinked microspheres (SA-A) activated with 1,1'‑carbonyldiimidazole (CDI). The activated microspheres were coupled with the anti-AFB1 nanobody Nb02, creating an immunoaffinity packing called CDI-SA-A-Nb02 (D1-IAC). After verification, the results indicated that the column capacity of D1-IAC was about 4.67 times that of several commercial IAC brands. Additionally, the maximum flow rate at a pressure of 0.04 MPa was 25 % greater than specific commercial IAC, and the recovery rate remained above 80 % after being reused five times. This method is considered effective for extracting and detecting AFB1 in food products. The preparation process for D1-IAC shows great consistency, and the column's performance is better than that of available immunoaffinity columns. This study explores using an IAC to extract and purify AFB1, which is then quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).