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Vomiting toxin antigen-antibody immune complex-specific binding variable domain of heavy chain of heavy chain antibody 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 the problems of difficult immune analysis and difficult combination of two antibodies at the same time

Active Publication Date: 2016-10-12
NANCHANG UNIV
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  • 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

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  • Vomiting toxin antigen-antibody immune complex-specific binding variable domain of heavy chain of heavy chain antibody and application thereof
  • Vomiting toxin antigen-antibody immune complex-specific binding variable domain of heavy chain of heavy chain antibody and application thereof
  • Vomiting toxin antigen-antibody immune complex-specific binding variable domain of heavy chain of heavy chain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Affinity panning and identification of single-domain heavy chain antibodies that specifically bind to DON antigen-antibody immune complexes

[0027]Single-domain heavy chain antibodies against DON antigen-antibody immune complexes were panned from the camelid natural heavy chain antibody library by solid-phase affinity panning. Anti-DON mouse monoclonal antibody ascites was purified by affinity column to obtain anti-DON monoclonal antibody; anti-DON monoclonal antibody was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL, coated with microplate wells, 4°C Wrap overnight. The next day, after washing 15 times with PBST (10 mM PBS, 0.1% Tween-20 (v / v)), 1% gelatin was added to block at 37°C for 1 h; the blocking solution was aspirated, washed 5 times with PBST, and 100 μL of DON standard was added to the well. (20ng / mL), incubated at 37°C for 1 h to form DON antigen-antibody immune complexes; then washed 5 times with PBST, and added 100 μL of the c...

Embodiment 2

[0035] Example 2 Amplification of single-domain heavy chain antibody phagemids that specifically bind to DON antigen-antibody complexes

[0036] The phage displaying the positive single-domain heavy chain antibody was added to 20 mL of the culture inoculated with E.coli TG1, and incubated with shaking at 220 rpm at 30 °C for 6 h; the culture was transferred to another centrifuge tube, and centrifuged at 4 °C and 8000 rpm for 15 min. Transfer the supernatant to a fresh centrifuge tube, add 1 / 6 volume of PEG / NaCl, stand at 4 °C for 4 h, centrifuge at 4 °C and 8000 rpm for 10 min, discard the supernatant; resuspend the phage in 1 mL of PBS, add 1 / 6 volume of PEG / NaCl, after standing at 4 °C for 1 h, centrifuged at 10000 rpm for 10 min at 4 °C, discarding the supernatant, adding 500 μL PBS to resuspend, which is the phage amplification solution.

Embodiment 3

[0037] Example 3 Expression of single-domain heavy chain antibody of antibody immune complex that specifically binds to DON antigen in Escherichia coli.

[0038] The phagemid pHEN1 was partially digested with restriction enzymes 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, which was named pET25b-DON after sequencing verification.

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

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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 a DON 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 immunoassays are widely used in the analysis of macromolecular substances containing multiple antigenic epitopes such as microorganisms, viruses, and proteins due to their high sensitivity and simple steps. For small molecules, it is very difficult to develop a non-competitive mode based on double-antibody sandwich for the immunoassay of small molecules because the molecular weight of the small molecule is too sma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N15/13G01N33/68
CPCC07K16/14C07K2317/22C07K2317/565C07K2317/567C07K2317/569
Inventor 何庆华许杨
Owner NANCHANG UNIV
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