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Humanized antibody combined with tetanus toxin heavy chain C-terminal structural domain and application

An anti-tetanus and toxin technology, applied in the fields of immunology and microbiology, can solve the problems of easily causing allergic reactions, spreading infectious diseases, and high cost

Active Publication Date: 2020-11-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antitoxins derived from animals (horses) are heterologous proteins to the human body, which can easily cause allergic reactions, and there is also a risk of spreading zoonotic infectious diseases
Human tetanus immune globulin (TIG) obtained from immune healthy people is less likely to cause allergic reactions than equine antitoxin, but there are still limitations of small production scale, high cost and the risk of infectious disease transmission

Method used

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  • Humanized antibody combined with tetanus toxin heavy chain C-terminal structural domain and application
  • Humanized antibody combined with tetanus toxin heavy chain C-terminal structural domain and application
  • Humanized antibody combined with tetanus toxin heavy chain C-terminal structural domain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Screening and preparation of monoclonal antibodies

[0032] 1. Blood Sample Collection

[0033] After obtaining informed consent, the recombinant tetanus vaccine (the main component is TeNT-H, the C-terminal domain of recombinant tetanus toxin heavy chain C , GenBank: AF154828) 10 mL of blood sample 28 days after the second immunization of subjects in the clinical trial was used for follow-up experiments.

[0034] 2. FITC-labeled Tetanus Toxoid (TT)

[0035] Specific memory B cells need to be sorted by fluorescently labeled antigens, and the method of FITC-labeled tetanus toxoid (TT) is as follows:

[0036] 1) Fluorescein Isothiocyanate_FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg / mL.

[0037]2) Take 100 μL of 3.3 mg / mL TT solution, add pH 9.6 carbonate buffer to 400 μL.

[0038] 3) Add 8 μL FITC to the TT solution, and incubate at 4° C. in the dark for 3 hours.

[0039] 4) Use a 50kDa ultrafiltration tube to change the solution ...

Embodiment 2

[0123] Example 2.ELISA detection of antibody binding activity

[0124] 1. The day before the experiment, the 96-well enzyme-linked plate was coated with 1 μg / mL tetanus toxoid TT (purchased from China Food and Drug Control Institute) and tetanus toxin heavy chain C-terminal domain (TeNT-H C , GenBank: AF154828), 100 μL per well for coating. Put the coated ELISA plate in a humid box overnight at 4°C.

[0125] 2. Wash 5 times with a plate washer on the day of the experiment. Add 100 μL of blocking solution to each well and place at room temperature for 1 hour.

[0126] 3. Wash the plate 5 times. Add 150 μL of T9-6 monoclonal antibody with a concentration of 3.3 μg / mL to the first well, and add 100 μL of diluent to the remaining wells. Aspirate 50 μL from the first well and add to the second well, and so on, dilute the solution in a 1:3 gradient to a final volume of 100 μL in each well. Let stand at room temperature for 1 hour.

[0127] 4. Wash the plate 5 times. The HPR-l...

Embodiment 3

[0131] Example 3. Mice challenge protection experiment

[0132] To evaluate the neutralizing toxin effect of T9-6 in mice, the evaluation method is as follows:

[0133] 1. Mice: BALB / c, 10 in each group, half male and half male, 6-8 weeks.

[0134] 2. Sample preparation: boric acid buffer: 8.5g NaCl, 4.5g boric acid, 0.5g sodium tetraborate decahydrate, add water to 1L, 0.22μm filter sterilization, pH 7.4; dilute tetanus toxin with boric acid buffer, in LD in mice 50 It was 15.8ng / kg.

[0135] 3. T9-6 administration group: 10 μg antibody (diluted in 50 μL PBS) + 2LD 50Tetanus toxin (total system 0.5mL); control group: 50μL PBS+2LD 50 Tetanus toxin (total system 0.5mL), incubated at 37°C for 1 hour.

[0136] 4. Intraperitoneally inject the toxin / antibody mixture into mice, 0.5 mL per mouse.

[0137] 5. Observe for 10 days.

[0138] Results: All the mice in the control group died within 2 days, while the mice in the T9-6 administration group could die within 2LD 50 100% ...

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Abstract

The invention discloses a humanized antibody combined with a tetanus toxin heavy chain C-terminal structural domain. The antibody has a unique CDRs, and the affinity of the antibody with an antigen reaches 0.397 nM; the antibody can effectively neutralize tetanus toxin and can completely protect mice attacked by the tetanus toxin; and the antibody has a unique action site, the unique action site is different from the action site of an anti-tetanus toxin monoclonal antibody in the prior art, and it is suggests that the antibody has the potential of forming cocktail therapy with other neutralizing antibodies.

Description

technical field [0001] The invention discloses a monoclonal antibody, belonging to the technical fields of immunology and microbiology. Background technique [0002] Tetanus is a bacterial infection caused by Clostridium tetani (C.tetani). Clostridium tetani invades the human body through skin or mucous membrane wounds, grows and reproduces in a hypoxic environment, produces toxins that cause muscle spasms and autonomic nervous system dysfunction, and the mortality rate is extremely high without medical intervention. Clostridium tetani spores are ubiquitous in the environment and can infect wounds, minor abrasions and, in neonatal tetanus, the umbilical cord stump of newborns. Although rare in developed countries, tetanus remains a great diagnostic and treatment challenge in many low- and middle-income countries. The estimated number of deaths from neonatal tetanus in 2015 was 34,019. Tetanus occurring outside the neonatal period is not a notifiable disease in many countr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/85C12N5/10A61K39/40A61P31/04
CPCC07K16/1282C12N15/85A61P31/04C07K2317/24C07K2317/565C07K2317/56C07K2317/52C07K2317/92C07K2317/76C12N2800/107A61K2039/505A61K2039/507
Inventor 陈薇于长明张冠英于蕊李建民迟象阳范鹏飞房婷刘渝娇郝勐董韵竹宋小红陈旖刘树玲
Owner ACADEMY OF MILITARY MEDICAL SCI
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