Development of an Immunoassay for the Detection of Copper Residues in Pork Tissues

The presence of high concentrations of copper (Cu) residues in pork is highly concerning and therefore, this study was designed to develop a high-throughput immunoassay for the detection of such residues in edible pork tissues. The Cu content in the pork samples after digestion with HNO₃ and H₂O₂ was measured using a monoclonal antibody (mAb) against a Cu (II)-ethylenediaminetetraacetic acid (EDTA) complex. The resulting solution was neutralized using NaOH at pH 7 and the free metal ions in the solution were chelated with EDTA for the immunoassay detection. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed for Cu ion analysis. The half maximal inhibitory concentration of the mAb against Cu (II)-EDTA was 5.36 ng/mL, the linear detection range varied between 1.30 and 27.0 ng/mL, the limit of detection (LOD) was 0.43 μg/kg, and the limit of quantification (LOQ) was 1.42 μg/kg. The performances of the immunoassay were evaluated using fortified pig serum, liver, and pork samples and had a recovery rate of 94.53-102.24%. Importantly, the proposed immunoassay was compared with inductively coupled plasma mass spectroscopy (ICP-MS) to measure its performance. The detection correlation coefficients of the three types of samples (serum, pork, and liver) were 0.967, 0.976, and 0.983, respectively. Thirty pork samples and six pig liver samples were collected from local markets and Cu was detected with the proposed ic-ELISA. The Cu content was found to be 37.31~85.36 μg/kg in pork samples and 1.04-1.9 mg/kg in liver samples. Furthermore, we detected the Cu content in pigs with feed supplemented with tribasic copper chloride (TBCC) and copper sulfate (CS) (60, 110, and 210 mg/kg in feed). There was no significant difference in Cu accumulation in pork tissues between the TBCC and CS groups, while a remarkable Cu accumulation was found for the CS group in liver at 210 mg/kg, representing more than a two-fold higher level than seen in the TBCC group. Therefore, the proposed immunoassay was found to be robust and sensitive for the detection of Cu, providing a cost effective and practical tool for its detection in food and other complicated samples.