Production of Monoclonal Antibody and Development of a New Immunoassay for Apramycin in Food.

Apramycin (APR) residue in food of animal origin can cause harmful effects on human health. In this study, a monoclonal antibody (mAb) was successfully produced using APR-BSA as immunogen, which was prepared by using the glutaraldehyde method. mAb 2A2 showed low cross-reactivity (<0.1%) with other aminoglycoside antibiotics, and its IC50 value was 0.35 ng/mL. On the basis of this mAb, a novel immunoassay in the format of an immunoaffinity test column (IATC) was developed. An immunoaffinity column filled with anti-APR antibody-Sepharose 4B gel was used as solid phase. APR in sample and HRP-APR conjugate compete with each other for the limited antibody on the column. The approach was able to give a naked-eye color signal for the detection of analyte. A blue color appears for negative results and no color for positive. The method was then successfully applied to the detection of APR in animal-origin food. To further evaluate the assay, direct competitive ELISA (dcELISA) based on the same antibody was developed for comparison in different aspects. Compared to the dcELISA, the detection time of IATC is shortened to 20 min, whereas a similar sensitivity for various samples was observed. The limits of detection (LOD) for raw milk, muscles, and livers are 3 ng/mL, 3 μg/kg, and 10 μg/kg, respectively.