International Joint Research Laboratory for Biointerface and Biodetection, State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Shanxi Academy of Analytical Sciences, No. 17 Beiyuan Street, Taiyuan 030012, China.
Biosensors (Basel). 2021 Jul 13;11(7):235. doi: 10.3390/bios11070235.
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 HNO and HO 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.
猪肉中铜（Cu）残留浓度过高令人担忧，因此，本研究旨在开发一种高通量免疫分析法，用于检测食用猪肉组织中的此类残留。使用针对 Cu（II）-乙二胺四乙酸（EDTA）络合物的单克隆抗体（mAb）测量经 HNO 和 HO 消化后的猪肉样品中的 Cu 含量。使用 NaOH 将所得溶液中和至 pH 7，并使用 EDTA 将溶液中的游离金属离子螯合以进行免疫分析检测。建立了用于 Cu 离子分析的间接竞争酶联免疫吸附测定（ic-ELISA）方法。mAb 对 Cu（II）-EDTA 的半抑制浓度为 5.36ng/mL，线性检测范围在 1.30 和 27.0ng/mL 之间，检测限（LOD）为 0.43μg/kg，定量限（LOQ）为 1.42μg/kg。使用加标猪血清、肝脏和猪肉样品评估免疫分析的性能，回收率为 94.53-102.24%。重要的是，将所提出的免疫分析与电感耦合等离子体质谱法（ICP-MS）进行了比较，以评估其性能。三种类型样品（血清、猪肉和肝脏）的检测相关系数分别为 0.967、0.976 和 0.983。从当地市场采集了 30 份猪肉样品和 6 份猪肝样品，并用所提出的 ic-ELISA 进行了 Cu 检测。发现猪肉样品中的 Cu 含量为 37.31-85.36μg/kg，肝脏样品中的 Cu 含量为 1.04-1.9mg/kg。此外，我们还检测了补充三碱式氯化铜（TBCC）和硫酸铜（CS）（饲料中 60、110 和 210mg/kg）的猪的 Cu 含量。在 TBCC 和 CS 组中，猪肉组织中 Cu 的积累没有明显差异，而在 CS 组中，肝脏中的 Cu 积累在 210mg/kg 时明显增加，比 TBCC 组高两倍多。因此，所提出的免疫分析被发现对 Cu 的检测具有稳健性和敏感性，为其在食品和其他复杂样品中的检测提供了一种经济实用的工具。
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