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Tetanus toxin-resistant neutralizing antibody as well as preparation and application thereof

A tetanus toxin and anti-tetanus technology, applied in the fields of cellular immunology and genetic engineering, can solve the problems of being in the laboratory research stage, expensive, and foreign virus contamination.

Active Publication Date: 2018-07-24
ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty in the source of human blood, the high price, and the risk of foreign virus contamination, its industrial production and clinical application have been greatly restricted.
[0007] At present, there have been research reports abroad on the transformation of mouse-derived monoclonal antibodies to prepare humanized genetically engineered tetanus antitoxin, etc., but they are still in the stage of laboratory research.

Method used

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  • Tetanus toxin-resistant neutralizing antibody as well as preparation and application thereof
  • Tetanus toxin-resistant neutralizing antibody as well as preparation and application thereof
  • Tetanus toxin-resistant neutralizing antibody as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Sorting of B cells by flow cytometry and screening and purification of anti-tetanus toxin antibodies

[0068] 1. PBMC isolation and plasma cell sorting

[0069] 100ml of venous blood was collected from volunteers who had been injected with tetanus vaccine and developed protective antibodies into anticoagulant tubes containing heparin. Use Ficoll to separate 100ml blood samples from mononuclear cells (PBMCs); after cell counting, use BDFACSria flow cytometer to sort from PBMCs, and place single cells with good morphology in a 96-well PCR plate, so that each well contains a memory B cells were stored in a -80°C refrigerator for later use.

[0070] 2. Isolation of antibody variable region genes

[0071] A 96-well plate containing a single B cell was added with 0.5 μM constant region primers of heavy and light chains of each subtype and Superscript III reverse transcriptase, and incubated at 37°C for 1 hour; PCR amplification was carried out under the following ...

Embodiment 2

[0083] Example 2 ELISA detection of neutralizing activity of TRN0012 antibody

[0084] The standard tetanus toxin was used as the antigen, and the antigen was diluted 10 times with the coating solution to coat the 96-well ELISA plate, 100 μl per well was coated overnight at 4°C, and blocked with the blocking solution for 2 hours at room temperature. After serial dilution of the expressed and purified antibody TRN0012, it was used as the primary antibody and incubated at room temperature for 2 hours, and was incubated with HRP / anti-His-tag (1:2000 dilution) as the secondary antibody at room temperature for 1 hour. After adding the substrate chromogenic solution, place it in the dark at room temperature for 5 minutes. , the reaction was terminated with 2M sodium sulfate, and the detection analysis was performed at a wavelength of 450 nm.

[0085] The result is as figure 2 As shown, after 50,000 dilutions of the expressed and purified antibody (the antibody concentration is app...

Embodiment 3

[0086] Example 3 Kinetic Analysis of Affinity Activity of Antibody TRN0012

[0087] Using the CM5 chip and human antibody capture kit, the anti-human FC secondary antibody was coupled first, then the anti-tetanus toxin monoclonal antibody was captured, and finally the tetanus toxin protein of different concentrations was used as the analyte. The tetanus standard toxin was diluted with HBS-EP buffer as the analyte, and the analyte flowed through the chip in sequence with gradually increasing concentrations, and the signal curves were respectively obtained. Each concentration was regarded as one cycle, and after completing one cycle, the chip was regenerated with 3 mol / L magnesium chloride to return to the original state of unbound antigen. BiaCore X-100System software was used to analyze the affinity and kinetics of monoclonal antibody binding to tetanus standard toxin (antigen).

[0088] The result is as image 3 As shown, the neutralizing antibody TRN0012 has an affinity of...

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PUM

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Abstract

The invention discloses a tetanus toxin-resistant neutralizing antibody as well as preparation and application thereof. The antibody can be used for preventing, treating and diagnosing tetanus infection and / or treating one or more symptoms mediated by clostridium tetani infection. The invention further provides a method for producing an antibody capable of combining with tetanus toxin immunologically specifically.

Description

technical field [0001] The present invention belongs to the fields of cellular immunology and genetic engineering, and relates to an anti-tetanus toxin neutralizing antibody and its preparation and application. Background technique [0002] Tetanus (Tetanus) disease is an acute disease characterized by general muscle tonic spasm caused by Clostridium tetani (Clostridium tetani) invading the body wound, multiplying in anaerobic environment, producing neurotropic exotoxin. Severe patients may die due to laryngospasm or secondary severe lung infection; newborns and severe patients, the case fatality rate can be as high as 20-50%; about 1 million people die from tetanus every year in the world, most of which occur in underdeveloped countries or region. The clinical characteristics of tetanus can be divided into four categories: partial tetanus, head tetanus, generalized tetanus, and neonatal tetanus. [0003] Clostridium tetani is a strict anaerobic bacterium. Its spores can r...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/63A61K39/40A61P31/04G01N33/577G01N33/569
CPCA61K2039/505C07K16/1282C07K2317/565C12N15/63G01N33/56911G01N33/577
Inventor 廖化新王月明袁晓辉
Owner ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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