Xinxiang Institute of Engineering, College of Bioengineering, Xinxiang 453700, China.
Henan Institute of Science and Technology, College of Animal Science and Veterinary Medicine, Xinxiang 453003, China.
Molecules. 2022 Feb 27;27(5):1585. doi: 10.3390/molecules27051585.
: This study aimed to prepare monoclonal antibodies (mAbs) with high immunoreactivity, sensitivity, and specificity for the chelate (Cr(III)-EDTA) of trivalent chromium ion (Cr(III)) and ethylenediamine tetraacetic acid (EDTA). Further, the study established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting the total chromium content in food, feed, and environmental samples. : Hapten Cr(III)-iEDTA was synthesized by chelating Cr(III) with isothiocyanatebenzyl-EDTA (iEDTA). Immunogen Cr(III)-iEDTA-BSA formed by chelating Cr(III)-iEDTA with bovine serum albumin (BSA), and coating antigen Cr(III)-iEDTA-OVA formed by chelating Cr(III)-iEDTA with ovalbumin (OVA) were prepared using the isothiocyanate method and identified by ultraviolet spectra (UV) and inductively coupled plasma optical emission spectrometry (ICP-OES). Balb/c mice were immunized with the Cr(III)-iEDTA-BSA, and the anti Cr(III)-EDTA mAb cell lines were screened by cell fusion. The Cr(III)-EDTA mAbs were prepared by induced ascites in vivo, and their immunological characteristics were assessed. : The immunogen Cr(III)-iEDTA-BSA was successfully synthesized, and the molecular binding ratio of Cr(III) to BSA was 15.48:1. Three hybridoma cell lines 2A3, 2A11, and 3D9 were screened, among which 2A3 was the best cell line. The 2A3 secreted antibody was stable after six passages, the affinity constant (Ka) was 2.69 × 10 L/mol, its 50% inhibition concentration (IC50) of Cr(III)-EDTA was 8.64 μg/L, and it had no cross-reactivity (CR%) with other heavy metal ion chelates except for a slight CR with Fe(III)-EDTA (1.12%). An icELISA detection method for Cr(III)-EDTA was established, with a limit of detection (LOD) of 1.0 μg/L and a working range of 1.13 to 66.30 μg/L. The average spiked recovery intra-assay rates were 90% to 109.5%, while the average recovery inter-assay rates were 90.4% to 97.2%. The intra-and inter-assay coefficient of variations (CVs) were 11.5% to 12.6% and 11.1% to 12.7%, respectively. The preliminary application of the icELISA and the comparison with ICP-OES showed that the coincidence rate of the two methods was 100%, and the correlation coefficient was 0.987. : The study successfully established an icELISA method that meets the requirements for detecting the Cr(III)-EDTA chelate content in food, feed, and environmental samples, based on Cr(III)-EDTA mAb, and carried out its preliminary practical application.
本研究旨在制备对三价铬离子（Cr(III)）与乙二胺四乙酸（EDTA）的螯合物（Cr(III)-EDTA）具有高免疫反应性、灵敏度和特异性的单克隆抗体（mAbs）。此外，本研究建立了一种间接竞争酶联免疫吸附测定法（icELISA），用于检测食品、饲料和环境样品中的总铬含量。
通过将异硫氰酸苄基-EDTA（iEDTA）与 Cr(III)螯合，合成了半抗原 Cr(III)-iEDTA。通过将 Cr(III)-iEDTA 与牛血清白蛋白（BSA）螯合形成免疫原 Cr(III)-iEDTA-BSA，通过将 Cr(III)-iEDTA 与卵清蛋白（OVA）螯合形成包被抗原 Cr(III)-iEDTA-OVA，采用异硫氰酸法进行制备，并通过紫外光谱（UV）和电感耦合等离子体发射光谱法（ICP-OES）进行鉴定。用 Cr(III)-iEDTA-BSA 免疫 Balb/c 小鼠，通过细胞融合筛选出抗 Cr(III)-EDTA mAb 细胞系。通过体内诱导腹水制备 Cr(III)-EDTA mAbs，并评估其免疫学特性。
成功合成了免疫原 Cr(III)-iEDTA-BSA，Cr(III)与 BSA 的分子结合比为 15.48:1。筛选出 3 株杂交瘤细胞系 2A3、2A11 和 3D9，其中 2A3 是最佳细胞系。2A3 分泌的抗体在经过六次传代后仍保持稳定，亲和常数（Ka）为 2.69×10 L/mol，其 Cr(III)-EDTA 的 50%抑制浓度（IC50）为 8.64μg/L，除与 Fe(III)-EDTA 有轻微交叉反应（CR%）（1.12%）外，与其他重金属离子螯合物无交叉反应。建立了 Cr(III)-EDTA 的 icELISA 检测方法，检测限（LOD）为 1.0μg/L，工作范围为 1.13-66.30μg/L。平均批内回收率为 90%-109.5%，平均批间回收率为 90.4%-97.2%。批内和批间变异系数（CV）分别为 11.5%-12.6%和 11.1%-12.7%。icELISA 的初步应用及与 ICP-OES 的比较表明，两种方法的符合率为 100%，相关系数为 0.987。
本研究成功建立了基于 Cr(III)-EDTA mAb 的 icELISA 方法，用于检测食品、饲料和环境样品中的 Cr(III)-EDTA 螯合物含量，并进行了初步的实际应用。
