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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 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 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.
Caixia Zhang; Weiqi Zhang; Xiaoqian Tang; Qi Zhang; Wen Zhang; Peiwu Li. Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals. Toxins 2020, 12, 273 .
AMA StyleCaixia Zhang, Weiqi Zhang, Xiaoqian Tang, Qi Zhang, Wen Zhang, Peiwu Li. Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals. Toxins. 2020; 12 (4):273.
Chicago/Turabian StyleCaixia Zhang; Weiqi Zhang; Xiaoqian Tang; Qi Zhang; Wen Zhang; Peiwu Li. 2020. "Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals." Toxins 12, no. 4: 273.
Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC50 value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027-0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products.
Caixia Zhang; Qi Zhang; Xiaoqian Tang; Wen Zhang; Peiwu Li. Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals. Toxins 2019, 11, 280 .
AMA StyleCaixia Zhang, Qi Zhang, Xiaoqian Tang, Wen Zhang, Peiwu Li. Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals. Toxins. 2019; 11 (5):280.
Chicago/Turabian StyleCaixia Zhang; Qi Zhang; Xiaoqian Tang; Wen Zhang; Peiwu Li. 2019. "Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals." Toxins 11, no. 5: 280.
Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 monoclonal antibodies were induced by low or high doses of aflatoxin B1-BSA, and 34 monoclonal antibodies were induced by low or high doses of aflatoxin M1-BSA. The specificities of the antibodies induced, whether by aflatoxin B1 conjugate or aflatoxin M1 conjugate, indicated that the low dose of the immunogen induced a narrow spectrum of antibody specificity, while the high dose of the immunogen showed an advantage to form a broad spectrum of antibody specificity. Therefore, this report provides important information for the development of new antibodies against small molecules like aflatoxins.
Peiwu Li; Jing Wu; Li Zhang; Zhiyong Fan; Tingting Yu; Feng Jiang; Xiaoqian Tang; Zhaowei Zhang; Wen Zhang; Qi Zhang. Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin. Toxins 2017, 9, 172 .
AMA StylePeiwu Li, Jing Wu, Li Zhang, Zhiyong Fan, Tingting Yu, Feng Jiang, Xiaoqian Tang, Zhaowei Zhang, Wen Zhang, Qi Zhang. Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin. Toxins. 2017; 9 (5):172.
Chicago/Turabian StylePeiwu Li; Jing Wu; Li Zhang; Zhiyong Fan; Tingting Yu; Feng Jiang; Xiaoqian Tang; Zhaowei Zhang; Wen Zhang; Qi Zhang. 2017. "Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin." Toxins 9, no. 5: 172.
An on-site, ultra-sensitive, and quantitative sensing method was developed based on quantum dot nanobeads (QDNBs) and a test strip for the determination of total aflatoxins (AFTs) in rice and peanuts. The monoclonal antibody against AFT (mAbAFT) was homemade and labeled with QDNB. After the pre-coating of the AFT antigen on the test line (T line), the competitive immunoreactions were conducted between AFT and AFT antigen on the T line with QDNBs-mAbAFT. Under optimal conditions, this approach allowed a rapid response towards AFT with a considerable sensitivity of 1.4 pg/mL and 2.9 pg/mL in rice and peanut matrices, respectively. The put-in and put-out durations were within 10 min. The recoveries for AFT in rice and peanut sample matrices were recorded from 86.25% to 118.0%, with relative deviations (RSD) below 12%. The assay was further validated via the comparison between this QDNB strip and the conventional HPLC method using spiked samples. Thus, the design provided a potential alternative for on-site, ultra-sensitive, and quantitative sensing of AFT that could also be expanded to other chemical contaminants for food safety.
Suiyan Ouyang; Zhaowei Zhang; Ting He; Peiwu Li; Qi Zhang; Xiaomei Chen; Du Wang; Hui Li; Xiaoqian Tang; Wen Zhang. An On-Site, Ultra-Sensitive, Quantitative Sensing Method for the Determination of Total Aflatoxin in Peanut and Rice Based on Quantum Dot Nanobeads Strip. Toxins 2017, 9, 137 .
AMA StyleSuiyan Ouyang, Zhaowei Zhang, Ting He, Peiwu Li, Qi Zhang, Xiaomei Chen, Du Wang, Hui Li, Xiaoqian Tang, Wen Zhang. An On-Site, Ultra-Sensitive, Quantitative Sensing Method for the Determination of Total Aflatoxin in Peanut and Rice Based on Quantum Dot Nanobeads Strip. Toxins. 2017; 9 (4):137.
Chicago/Turabian StyleSuiyan Ouyang; Zhaowei Zhang; Ting He; Peiwu Li; Qi Zhang; Xiaomei Chen; Du Wang; Hui Li; Xiaoqian Tang; Wen Zhang. 2017. "An On-Site, Ultra-Sensitive, Quantitative Sensing Method for the Determination of Total Aflatoxin in Peanut and Rice Based on Quantum Dot Nanobeads Strip." Toxins 9, no. 4: 137.