Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

文献类型： 外文期刊

第一作者： Zhang, Caixia

作者： Zhang, Caixia;Zhang, Weiqi;Tang, Xiaoqian;Zhang, Qi;Zhang, Wen;Li, Peiwu;Zhang, Caixia;Zhang, Weiqi;Tang, Xiaoqian;Zhang, Qi;Zhang, Wen;Li, Peiwu;Zhang, Caixia;Zhang, Weiqi;Tang, Xiaoqian;Zhang, Qi;Li, Peiwu;Tang, Xiaoqian;Zhang, Qi;Li, Peiwu;Tang, Xiaoqian;Zhang, Qi;Zhang, Wen;Li, Peiwu

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关键词： Amino acid residue; idiotypic nanobody; immunoassay; ochratoxin a; mycotoxin

期刊名称：TOXINS （ 影响因子：4.546； 五年影响因子：4.8 ）

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年卷期： 2020 年 12 卷 4 期

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收录情况： SCI

摘要： Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody's primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I-L), two of them in CDR2 (G-D, E-K), and three of them in CDR3 (Y-H, Y-W). Second, the affinity constant of NCA1 was tested as 1.20 x 10(8) L mol(-1), which is about 4 times lower than that of NCA2 (5.36 x 10(8) L mol(-1)). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL(-1), which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL(-1)). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL(-1) with a limit of detection (LOD) of 0.003 ng mL(-1). The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1-12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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