Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Single domain heavy chain antibody specifically binding to vomitoxin antigen-antibody immune complex and application thereof

A single-domain heavy chain antibody and immune complex technology, applied in anti-fungal/algae/lichen immunoglobulin, application, immunoglobulin, etc., can solve problems such as difficult combination of two antibodies at the same time, difficulty in immune analysis, etc.

Active Publication Date: 2019-06-18
NANCHANG UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For small molecular substances, because the molecular weight of small molecular substances is too small, it is not easy to be combined by two antibodies at the same time, so it is very difficult to develop a non-competitive mode based on double antibody sandwich for immunoassay of small molecular substances

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Single domain heavy chain antibody specifically binding to vomitoxin antigen-antibody immune complex and application thereof
  • Single domain heavy chain antibody specifically binding to vomitoxin antigen-antibody immune complex and application thereof
  • Single domain heavy chain antibody specifically binding to vomitoxin antigen-antibody immune complex and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Affinity panning and identification of single domain heavy chain antibody specifically binding to DON antigen-antibody immune complex

[0027]The single-domain heavy chain antibody against DON antigen-antibody immune complex was screened from camel-derived natural heavy chain antibody library by solid-phase affinity panning. Purify the anti-DON mouse monoclonal antibody ascites with an affinity column to obtain the anti-DON monoclonal antibody; dilute the anti-DON monoclonal antibody to a final concentration of 50 μg / mL with PBS (pH 7.4), coat the wells of the microplate, and store at 4 °C Pack overnight. The next day, after washing 15 times with PBST (10 mM PBS, 0.1% Tween-20 (v / v)), add 1% gelatin to block at 37°C for 1 h; draw up the blocking solution, wash 5 times with PBST, and add 100 μL of DON to the well. Standard product (20ng / mL), incubated at 37°C for 1h to form DON antigen-antibody immune complex; then washed 5 times with PBST, and added 100μL cam...

Embodiment 2

[0035] Example 2 Amplification of Phagemids of Single Domain Heavy Chain Antibody Specifically Binding to DON Antigen-Antibody Complex

[0036] Add the phage displaying the positive single domain heavy chain antibody to 20mL culture inoculated with E.coli TG1, shake and culture at 220rpm at 30°C for 6h; transfer the culture to another centrifuge tube, centrifuge at 4°C and 8000rpm for 15min, Transfer the supernatant to a fresh centrifuge tube, add 1 / 6 volume of PEG / NaCl, let stand at 4°C for 4h, centrifuge at 4°C, 8000rpm for 10min, discard the supernatant; resuspend the phage in 1mL PBS, add 1 / 6 After standing still at 4°C for 1 hour, centrifuge at 10,000 rpm for 10 minutes at 4°C, discard the supernatant, and add 500 μL PBS to resuspend, which is the phage amplification solution.

Embodiment 3

[0037] Example 3 Expression of a single domain heavy chain antibody specifically binding to the DON antigen antibody immune complex in Escherichia coli.

[0038] Phagemid pHEN1 was incompletely digested with restriction endonuclease NotI / NcoI, and the target fragment was recovered from agarose gel.

[0039] The obtained single-domain heavy chain antibody double-enzyme-digested gene fragment was cloned into the expression vector pET-25b, and after sequencing verification, it was named pET25b-DON.

[0040] The recombinant plasmid pET25b-DON was transformed into Escherichia coli Rosetta and induced to express. Pick a single colony and inoculate it into 5mL liquid LB / Amp medium, and culture it on a shaker at 37°C and 200rpm for 10h; inoculate the above culture solution with 1% inoculum size into 50mL liquid LB medium, shake it at 37°C and 200rpm After culturing to an OD of 0.5, a final concentration of 0.05 mM IPTG was added to induce culture at 30° C. and 180 rpm on a shaking ta...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology, and specifically relates to vomiting toxin antigen-antibody immunocomplex-specific binding variable domain of heavy chain of heavy chain antibody (VHH) preparation and application thereof. The amino acid sequence is shown in SEQIDNO. 1. The variable domain of heavy chain of heavy chain antibody can specifically bind a vomiting toxin antigen-antibody immunocomplex, can replace the traditional antibody, and can be applied to a non-competitive immunology analysis of DON. The amino acid sequence provided by the invention can be used as a precursor, and is modified by a random or orientated mutation technique to obtain a mutant with better characters, and the mutant is used to develop proteins or peptides in the field of industry and food safety further.

Description

technical field [0001] The present invention relates to single-domain heavy chain antibody technology (also known as nanobody technology) and genetic engineering antibody technology, belonging to the field of biotechnology, in particular to a single-domain heavy chain that can specifically bind to vomitoxin antigen-antibody complex (Immunocomplex) Antibody (Variable domain of heavy chain of heavy chain antibody, VHH) preparation and application. Background technique [0002] Immunoassay methods can be divided into competitive and non-competitive forms. Non-competitive immunoassay is widely used in the analysis of macromolecular substances containing multiple antigenic epitopes, such as microorganisms, viruses, and proteins, due to its high sensitivity and simple steps. For small molecular substances, because the molecular weight of small molecular substances is too small, it is not easy to be combined by two antibodies at the same time, so it is very difficult to develop a ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/14C12N15/13G01N33/68
CPCC07K16/14C07K2317/22C07K2317/565C07K2317/567C07K2317/569
Inventor 何庆华许杨
Owner NANCHANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com