Preparation for aflatoxin B1-cationized bovine serum albumin based on Mannich-type reaction

Aflatoxin B1 (AFB1), which is commonly found in agricultural commodities, is one of the most potent carcinogenic mycotoxins. To ensure food safety, rapid and low-cost immunological methods have been applied to detect AFB1 worldwide. A key step in these immunological methods is coupling AFB1 to carrier proteins; AFB1 is usually deviated to AFB1-oxime and coupled to carrier proteins to form the AFB1-oxime–protein conjugate. In the current research, AFB1 was directly coupled with cationized bovine serum albumin (cBSA) using a method based on Mannich-type principles. The coupling effects were investigated with different initial molar ratios of AFB1 to cBSA. The conjugate molar ratio was 6.4:1 when the initial molar ratio was 40:1. The cationized proteins and their conjugates were identified by UV–Vis and FT-IR spectra, which showed the characteristic bands of the ethylendiamine group and AFB1, respectively. After BALB/c mice were immunized with AFB1–cBSA, a quicker immunological response and a similar sensitivity of antisera against AFB1 were observed, compared with immunization by AFB1-oxime–BSA. This suggests that the Mannich-type reaction might be an alternative method of preparation for AFB1–protein.