Aflatoxin-enzyme-linked immunosorbent assay device based on nano-antibody and antigen analog peptide

An aflatoxin and nanobody technology, which is applied in the detection field to achieve the effects of easy production, reduced labor intensity, and reduced washing steps

Inactive Publication Date: 2018-11-13
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the improvement of antibodies and antigens used in immunoassays at the same time, that is, the use of genetically engineered antibodies and antigen-mimicking peptide pairs to establish a mycotoxin immunoassay method

Method used

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  • Aflatoxin-enzyme-linked immunosorbent assay device based on nano-antibody and antigen analog peptide
  • Aflatoxin-enzyme-linked immunosorbent assay device based on nano-antibody and antigen analog peptide
  • Aflatoxin-enzyme-linked immunosorbent assay device based on nano-antibody and antigen analog peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of aflatoxin nanobody Nb28 immunomagnetic beads

[0041] In this study, according to the amino acid sequence of aflatoxin nanobody Nb28 provided in the literature, as shown in SEQ ID NO.1 (He T, Wang Y, Li P, et al. Nanobody-based enzyme immunoassay foraflatoxin in agro-products with high tolerance to cosolvent methanol[J].Analytical chemistry,2014,86(17):8873-8880.), the corresponding DNA sequence was obtained by reverse translation of DNAMAN software, and the rare codons of Escherichia coli were replaced, and the optimized DNA sequence was SEQ ID Shown in NO.2. After gene synthesis, PCR amplification, enzyme digestion, and pET-BAD plasmid connection, the pET-BAD plasmid construction method refers to (Zhao F, Wang H, Han X, et al. Development and comparative study of chemosynthesized antigen andmimotope-based immunoassays for class- specific analysis of O,O-dimethylorganophosphorus pesticides[J].Scientific reports,2016,6:37640.), construction of...

Embodiment 2

[0055] Example 2, Screening of Nb28 Antigen Mimetic Peptides

[0056] The present invention takes the prepared nanobody immunomagnetic beads Nb28-SA-MB as the target, and performs three rounds of panning on the phage random dodecapeptide library to enrich the phage antigen mimic peptide corresponding to Nb28. The specific process is as follows:

[0057] (1) Dilute 20 μL Nb28-SA-MB into 1 mL 1×PBS, add 50 μL phage display random dodecapeptide library (2.0×10 13 pfu / mL), shake and mix well and incubate at 37°C for 40min;

[0058] (2) After incubation, place the centrifuge tube on a magnetic stand for magnetic separation, absorb the supernatant and discard it, then add 1 mL of PBST to wash 3 times (add PBST to shake and mix well during washing, and place it on the magnetic stand after centrifugation for a short time for magnetic separation). separation);

[0059] (3) Add 1mL eluent (0.2M Glycine-HCl, 1mg / mL BSA, pH 2.2), vortex to mix and incubate at 37°C for 10min;

[0060] ...

Embodiment 3

[0080] Example 3, Preparation of Enzyme-labeled Antigen Mimic Peptide

[0081] The present invention selects the Nb28 antigen mimetic peptide ME17 with the best hydrophilicity to carry out polypeptide synthesis. For the subsequent coupling with HRP, a cysteine ​​(Cysteine) is added to the C-terminus of the polypeptide to introduce a sulfhydryl group (-SH ). According to the presence or absence of the connecting arm (Gly-Gly-Gly-Ser) between the antigen-mimicking peptide and cysteine, two kinds of antigen-mimicking peptides were designed and synthesized, respectively ME17-13AA without the connecting arm, the amino acid sequence is as follows: The amino acid sequences shown in SEQ ID NO.7 (Y-S-W-H-E-W-Y-I-P-Q-L-S-C) and ME17-17AA with linker arms shown in SEQ ID NO.8 (Y-S-W-H-E-W-Y-I-P-Q-L-S-G-G-G-G-S-C) were chemically synthesized by Nanjing Peptide Biotechnology Co., Ltd. The synthesis steps of antigen mimetic peptide and HRP are as follows:

[0082] (1) Weigh 10mg HRP into ...

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Abstract

The invention relates to the technical field of detection, in particular to an aflatoxin-enzyme-linked immunosorbent assay device based on a nano-antibody and antigen analog peptide. An aflatoxin antigen mimic peptide uses immune prepared by an aflatoxin nano-antibody Nb28 as a target and is obtained through screening of a phage display technology, its amino acid sequence is shown as SEQ ID NO.3,SE QID NO.4, SEQ ID NO.5 or SEQ ID NO.6. For the first time, the nano-antibody Nb28 is applied to the antigen mimic peptide corresponding to the nano-antibody and meanwhile is used for aflatoxin-enzyme-linked immunosorbent assay (ELISA), and quick aflatoxin detection can be achieved. The genes and amino acid sequences of genetically engineered antibodies and antigen-mimicking peptides used in themethod can be determined and are easy to be produce, popularize and apply.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to an aflatoxin magnetic bead-ELISA detection method based on a nanobody and an antigen mimic peptide. Background technique [0002] Aflatoxins are mainly toxic secondary metabolites produced by Aspergillus flavus and Aspergillus paraciticus, and more than 20 kinds have been isolated, including AFB 1 、AFB 2 、AFG 1 、AFG 2 、AFB 2a , AFGM, AFH 1 、AFM 1 、AFM 2 、AFP 1 , AFQ and toxic alcohol. where AFB 1 The most toxic effect, in 1993, the World Health Organization (WHO) will AFB 1 and aflatoxin mixtures are listed as Class I carcinogens. Aflatoxin pollution is very widespread in the world, especially in hot and humid environments. For example, the Yangtze River Basin in my country has become a highly polluted area of ​​aflatoxin due to its climate characteristics. In view of the characteristics of mycotoxins such as wide pollution range and strong toxicity, it is extremely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08G01N33/535G01N33/543
CPCC07K7/08G01N33/535G01N33/54326G01N2333/38
Inventor 赵凤春杨正友申强朱常香
Owner SHANDONG AGRICULTURAL UNIVERSITY
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