Antibody against precursor brain-derived neurotrophic factor and application thereof

A technology of neurotrophic factors and antibodies, applied in application, anti-growth factor immunoglobulin, genetic engineering, etc., can solve the problem of lack of specific monoclonal antibodies

Active Publication Date: 2022-04-22
RESEARCH INSTITUTE OF TSINGHUA UNIVERSITY IN SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, BDNF exists in two forms: precursor BDNF (ProBDNF) and mature BDNF (mBDNF), and there is currently a lack of highly specific monoclonal antibodies against ProBDNF

Method used

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  • Antibody against precursor brain-derived neurotrophic factor and application thereof
  • Antibody against precursor brain-derived neurotrophic factor and application thereof
  • Antibody against precursor brain-derived neurotrophic factor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Anti-ProBDNF Antibody Screening

[0038] The amino acid sequence (SEQ ID NO.11) of the antigen ProBDNF used is as follows:

[0039] CDQKVRPNEENNKDA.

[0040] The polypeptide in the pro fragment of the above proBDNF protein is used as the antigen for immunization and the antigen for screening, and the proBDNF protein itself is also used as the antigen protein for screening. The immunized animals are female Balb / c mice aged 6-8 weeks, injected with antigen, and quickly immunized with a special adjuvant for about two weeks. After the animal was immunized, the antibody titer was detected. When the antibody titer was high enough, the animal was anesthetized with isoflurane and then sacrificed. The lymph node was taken, digested and fused with the myeloma. Use DMEM to culture sp2 / 0 myeloma cells. After the cells are in good condition and the rate of viable cells is over 80%, they are fused with lymphocytes through polyethylene glycol. Then use the HAT screening medium to c...

Embodiment 2

[0053] Purification of anti-ProBDNF antibody

[0054] The antibody gene sequence measured above was codon-optimized, synthesized and inserted into the pcDNA3.4 vector, and the Fc region used the original sequence of the antibody subtype IgG2b. The constructed expression vector was transferred into Escherichia coli for amplification and expression, and the expression plasmid was purified. The plasmid was electrotransfected into 293 cells. After 2 days of cultivation, the antibody protein in the supernatant was extracted, and the protein A affinity column was used for The antibody was purified and identified using HPLC-SEC and SDS-Page to determine its concentration and purity. After testing, the antibody concentration was 0.895 mg / ml, and the purity reached 94%.

Embodiment 3

[0056] Specific detection of anti-mBDNF antibody

[0057] Antigen sample: mBDNF or ProBDNF (amino acid sequence shown in SEQ ID NO.11)

[0058] Detection by ELISA method:

[0059] 1) Coating: A 96-well plate was coated with 50 μl of mBDNF or proBDNF protein at a concentration of 2 μg / ml overnight at 4° C., then washed three times with PBST (Tween 0.05%), and patted dry.

[0060] 2) Blocking: Add 200 μl of the prepared blocking solution (PBST solution containing 1% BSA) to a 96-well plate, block at room temperature for 1 hour, wash the plate four times, and pat dry.

[0061] 3) Add 50 μl of sample (purified antibody provided in Example 2), incubate at room temperature for 1 hour, wash the plate four times, and pat dry.

[0062] 4) Secondary antibody incubation: Secondary antibody (1:2000) was prepared with blocking solution, 100 μl of secondary antibody was added to each well, incubated at room temperature for 1 hour, washed four times, and patted dry.

[0063] 5) 100 μl of ...

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PUM

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Abstract

The invention discloses an antibody against precursor brain-derived neurotrophic factor and its application, and relates to the technical field of antibodies. The antibody of the precursor brain-derived neurotrophic factor disclosed in the present invention has the complementarity determining region of the heavy chain shown in SEQ ID NO.1-3 and the complementarity determining region of the light chain shown in SEQ ID NO.4-6. The antibody can specifically recognize the precursor brain-derived neurotrophic factor, is used for detecting the precursor brain-derived neurotrophic factor, and has high specificity and sensitivity.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to an antibody against precursor brain-derived neurotrophic factor and its application. Background technique [0002] As the most widely distributed neurotrophic factor in the central nervous system, the function and level of brain-derived neurotrophic factor (BDNF) are closely related to a variety of neurological diseases. BDNF can also be detected in peripheral blood. It is generally believed that BDNF in peripheral blood and The level of BDNF in the brain is correlated, so the detection of blood BDNF level is considered to be a diagnostic marker for certain neurological diseases (Hashimoto, 2010, 2014; Matsuoka et al., 2015). However, BDNF exists in two forms, precursor BDNF (ProBDNF) and mature BDNF (mBDNF), and currently there is a lack of highly specific monoclonal antibodies against ProBDNF. [0003] In view of this, the present invention is proposed. Contents of the in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/22C12N15/13G01N33/68
CPCC07K16/22G01N33/74C07K2317/565C07K2317/56C07K2317/34G01N2333/4756
Inventor 郭炜
Owner RESEARCH INSTITUTE OF TSINGHUA UNIVERSITY IN SHENZHEN
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