Use of Cloneable Peptide–MBP Fusion Protein as a Mimetic Coating Antigen in the Standardized Immunoassay for Mycotoxin Ochratoxin A

2014 ◽  
Vol 62 (35) ◽  
pp. 8830-8836 ◽  
Author(s):  
Yang Xu ◽  
Zhenyun He ◽  
Qinghua He ◽  
Yulou Qiu ◽  
Bo Chen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ochratoxin A ◽  
Coating Antigen

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

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 273
Author(s):  
Caixia Zhang ◽  
Weiqi Zhang ◽  
Xiaoqian Tang ◽  
Qi Zhang ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Limit Of Detection ◽  
Basic Knowledge ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Assay Sensitivity ◽  
Coating Antigen ◽  
Relative Standard

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.

scholarly journals Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 4044 ◽  
Author(s):  
Zhichang Sun ◽  
Xuerou Wang ◽  
Qi Chen ◽  
Yonghuan Yun ◽  
Zongwen Tang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
Solvent Tolerance ◽  
Enzyme Linked Immunosorbent Assay ◽  
One Step

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

One-step detection of ochratoxin A in cereal by dot immunoassay using a nanobody-alkaline phosphatase fusion protein

Food Control ◽  
2018 ◽  
Vol 92 ◽  
pp. 430-436 ◽  
Author(s):  
Zongwen Tang ◽  
Xuerou Wang ◽  
Jingwen Lv ◽  
Xiangrong Hu ◽  
Xing Liu
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Step Detection ◽  
One Step ◽  
Dot Immunoassay

scholarly journals Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay

RSC Advances ◽  
2020 ◽  
Vol 10 (56) ◽  
pp. 33700-33705
Author(s):  
Qi Chen ◽  
Yuanyuan Wang ◽  
Fujing Mao ◽  
Benchao Su ◽  
Kunlu Bao ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Visual Detection ◽  
Low Molecular Weight ◽  
Low Molecular Weight Compounds ◽  
Diagnostic Reagent ◽  
Dot Immunoassay

The horseradish peroxidase-nanobody fusion protein was developed as a promising immunological diagnostic reagent for toxic low-molecular-weight compounds in food.

Sign in / Sign up

Export Citation Format

Share Document