数字农科院2.0

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

作者： Zhang, CX; Zhang, WQ; Tang, XQ; Zhang, Q; Zhang, W; Li, PW

作者机构：

关键词： Amino Acid Residue; Idiotypic Nanobody; Immunoassay; Ochratoxin A; Mycotoxin

期刊名称： TOXINS

ISSN：

年卷期： 2020 年 12 卷 4 期

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收录情况： JCR（2021版）

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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