A kind of neutralizing antibody against tetanus toxin and its application

A tetanus toxin and anti-tetanus technology, applied in the field of genetic engineering, can solve the problems of exogenous virus contamination and high price of HTIG, and achieve the effects of low dosage, high affinity activity, and strong neutralizing activity

Active Publication Date: 2019-05-24
CHANGCHUN BCHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides an anti-tetanus toxin neutralizing antibody and its application to solve the problems of high price and exogenous virus contamination of existing HTIG

Method used

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  • A kind of neutralizing antibody against tetanus toxin and its application
  • A kind of neutralizing antibody against tetanus toxin and its application
  • A kind of neutralizing antibody against tetanus toxin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Sorting B cells by flow cytometry and screening and purification of anti-tetanus toxin antibodies

[0088] 1. PBMC isolation and plasma cell sorting

[0089] 15 ml of venous blood was collected from volunteers who had been vaccinated against tetanus and developed protective antibodies, and placed in anticoagulant tubes containing heparin. The 15ml blood sample was separated from PBMC (peripheral bloodmononuclear cell, peripheral blood mononuclear cell) with Ficoll (polysucrose). After PBMC counting, BD FACSria flow cytometer was used to sort single B cells from PBMCs, and the B cells with good morphology were placed in a 96-well PCR (Polymerase Chain Reaction, polymerase chain reaction) plate, so that each well contained one B cells. The 96-well PCR plate with B cells was placed in a -80°C refrigerator and stored for later use.

[0090] 2. Isolation of antibody variable region genes

[0091] Add 0.5 μM constant region primers of heavy chain and light chain...

Embodiment 2

[0103] Embodiment 2 ELISA detects the neutralizing activity of antibody TRN1015

[0104] 1. Experimental process

[0105] 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. Each well contained an overnight coating with a volume of 100 μ and the temperature was 4 ° C, and blocked with the blocking solution for 2 hours at room temperature. Serially dilute the expressed and purified antibody TRN1015 as the primary antibody and incubate at 37°C for 2h, use HRP / anti-His-tag (1:2000 dilution) as the secondary antibody and incubate at 37°C for 1h, add the substrate chromogenic solution and keep away from light at room temperature Stand for 5 minutes, stop the reaction with 2M sulfuric acid, and detect and analyze with a wavelength of 450nm.

[0106] 2. Results

[0107] The result is as image 3 As shown, after 10,000 dilutions of the expressed and purified antibody (the antibody concen...

Embodiment 3

[0108] Example 3 Kinetic Analysis of Affinity Activity of Antibody TRN1015

[0109] 1. Capture method test

[0110] The antibody affinity test adopts the capture method, the buffer is HBS-EP, and the CM5 chip is first coupled with proteinA. Then dilute the capture antibody so that its concentration is 1ug / ml, and the binding time is 50s. The analyte tetanin was sequentially passed through the chip at 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 3M MgCl2 to return to the state of the original uncaptured antibody, and the regeneration time was 30s. The obtained signal curve was analyzed with BiaCore X-100System software to obtain the detection graph of the affinity activity between the neutralizing antibody and tetanus toxin provided in the embodiment of the present invention.

[0111] 4. Results

[0112] The result is as Figure 4 As s...

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Abstract

Provided are a neutralizing antibody against tetanus toxins, and a use thereof. The provided neutralizing antibody against tetanus toxins is capable of specifically binding to tetanus toxins, and thus has strong neutralizing activity and a high affinity, and can be used in a low amount. The provided neutralizing antibody against tetanus toxin can be used for prevention and treatment of tetanus toxin infections and for isolated antibodies of clostridium tetani-mediated diseases or disease conditions, and can also be used to diagnose and/or monitor tetanus toxin infections and isolated antibodies of clostridium tetani infections. Furthermore, the neutralizing antibody against tetanus toxin is free from foreign virus contaminations and can be widely applied to various populations.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-tetanus toxin neutralizing antibody and its application. Background technique [0002] Tetanus toxin is a secreted protein produced by Clostridium tetani under anaerobic conditions to inhibit the release of neurotransmitters. Tetanus toxin can cause hyperreflexia and striated muscle spasm. On the basis of muscle tonic contraction (muscle rigidity, stiffness), paroxysmal strong spasm can be fatal in severe patients, and the case fatality rate can be as high as 50%. [0003] For the prevention and treatment of tetanus toxin, horse serum TAT (Tatanus Antitoxin, tetanus antitoxin) is usually used for treatment. However, due to differences in human body constitution and different acceptance levels of horse serum TAT, many people may experience adverse reactions such as allergic reactions, anaphylactic shock or serum sickness. Therefore, horse serum TAT is not sui...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13A61K39/40A61P31/04A61P21/02G01N33/577G01N33/569
CPCA61K2039/505A61P21/02A61P31/04C07K16/1282C07K2317/565C07K2317/76G01N33/56911G01N33/577
Inventor 廖化新王月明袁晓辉郑伟宏
Owner CHANGCHUN BCHT BIOTECH
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