Antibody of mature brain-derived neurotrophic factor as well as application and diagnostic kit thereof

A technology of neurotrophic factors and antibodies, which is applied in application, disease diagnosis, anti-growth factor immunoglobulin, etc., can solve problems such as differences in processing methods and lack of specific antibodies, and achieve high specificity and sensitivity

Active Publication Date: 2021-04-13
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, due to differences in the processing methods of samples (such as serum and plasma), more importantly, there are two forms of BDNF, precursor BDNF (ProBDNF) and mature BDNF (mBDNF), and there is currently a lack of highly specific antibodies against mBDNF

Method used

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  • Antibody of mature brain-derived neurotrophic factor as well as application and diagnostic kit thereof
  • Antibody of mature brain-derived neurotrophic factor as well as application and diagnostic kit thereof
  • Antibody of mature brain-derived neurotrophic factor as well as application and diagnostic kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Screening of anti-mBDNF antibodies

[0049] The amino acid sequence (SEQ ID NO.11) of the antigen mBDNF used is as follows:

[0050] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR.

[0051] The above mBDNF protein was used as the antigen for immunization and the antigen 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 culture, during wh...

Embodiment 2

[0065] Purification of anti-mBDNF antibody

[0066] 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 concentration of the antibody was 0.862mg / ml, and the purity reached 98%.

Embodiment 3

[0068] Specific detection of anti-mBDNF antibody

[0069] Antigen sample: mBDNF (amino acid sequence shown in SEQ ID NO.11) or ProBDNF is detected by ELISA method:

[0070] 1) Coating: A 96-well plate was coated with 50 μl of mBDNF or proBDNF protein at a concentration of 2 μg / ml at 4° C. overnight.

[0071] 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.

[0072] 3) Add 50 μl of sample (the purified antibody of the above-mentioned Example 2), incubate at room temperature for 1 hour, wash the plate four times, and pat dry.

[0073] 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.

[0074] 5) 100 μl of TMB was reacted at room temperature for 10 minutes, and 50...

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PUM

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Abstract

The invention discloses an antibody of a mature brain-derived neurotrophic factor as well as application and a diagnostic kit thereof, and relates to the technical field of antibodies. The antibody of the mature brain-derived neurotrophic factor, disclosed by the invention, is provided with a heavy chain complementation determining region shown as SEQ ID NO.1-3 and a light chain complementation determining region shown as SEQ ID NO.4-6. The antibody can specifically recognize mature brain-derived neurotrophic factors, and has relatively high specificity and sensitivity.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to an antibody to mature brain-derived neurotrophic factor, its application and a diagnostic kit. Background technique [0002] As the most widely distributed neurotrophic factor (BDNF) in the central nervous system, the function and level of BDNF are closely related to various neurological diseases, such as Alzheimer's disease (Allen et al., 2011). Studies on samples from patients with Alzheimer's disease (AD) and its early symptoms of MCI have shown that the levels of BDNF in the hippocampus, cortex and other brain regions of patients are lower than those in normal human samples (Peng et al., 2005). Since BDNF can partially cross the blood-brain barrier, the level of BDNF in CSF is highly correlated with its peripheral level. In recent years, studies on AD and peripheral BDNF levels have gradually increased. The meta-analysis (Meta analysis) published by Qin et al. in 2017 sh...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13G01N33/68
CPCC07K16/22G01N33/74C07K2317/565C07K2317/34C07K2317/56G01N2333/4756G01N2800/044G01N2800/304G01N2800/302G01N2800/2835G01N2800/2821G01N2800/2871G01N2800/2878G01N2800/36
Inventor 郭炜
Owner RESEARCH INSTITUTE OF TSINGHUA UNIVERSITY IN SHENZHEN
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