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Gene engineering monoclonal antibody combined with A-beta oligomer specificity

A technology of monoclonal antibodies and oligomers, which is applied in the direction of antibodies, drug combinations, and microbial-based methods, can solve the problems of three-dimensional epitopes that have not been explored clearly, and achieve inhibition of fibrosis aggregation, broad application prospects, Active effect

Active Publication Date: 2009-06-24
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The present invention solves the technical problem that existing antibodies bind to A-beta monomers, oligomers and filaments at the same time, and uses phage display technology to successfully screen out antibodies that specifically bind to A-beta oligomers but do not bind to A-beta oligomers. beta monomer and filament-bound monoclonal antibody
Its three-dimensional epitope has not been explored clearly so far

Method used

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  • Gene engineering monoclonal antibody combined with A-beta oligomer specificity
  • Gene engineering monoclonal antibody combined with A-beta oligomer specificity
  • Gene engineering monoclonal antibody combined with A-beta oligomer specificity

Examples

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Embodiment 1

[0049] The preparation of embodiment 1 A-beta oligomer

[0050] A-beta monomer (purchased from American Peptide Company, USA) was dissolved in HFIP (hexafluoroisopropanol, purchased from Sigma Company) to prepare a solution with a concentration of 1 mg / ml. Sonicate in a water bath at room temperature for 10 minutes, dispense into 1.5ml centrifuge tubes, place in a fume hood to allow HFIP to evaporate completely, and store at -20°C for later use.

[0051] After the above-treated A-beta was equilibrated at room temperature for 10 minutes, DMSO (dimethyl sulfoxide, purchased from Sigma) was added to fully dissolve A-beta to a final concentration of 1 mg / ml. A certain amount of A-beta was added to the PBS buffer at pH 7.4, so that the concentration of A-beta was 10 μM. After incubating the A-beta solution at 37° C. for 12 hours, centrifuge at 14,000 rpm for 20 minutes, and discard the bottom precipitate to obtain the supernatant containing A-beta oligomers. The formation of A-be...

Embodiment 2

[0052] Example 2 Screening of positive clones

[0053] The A-beta oligomer obtained in Example 1 was diluted to 10-100 μg / mL with coating buffer (PBS, pH=7.4), and 4 mL was added to an immunotube, and coated overnight at 4°C. Discard the supernatant and wash the tube 3 times quickly with PBS. The immunotube was filled with 3% BSA and sealed vertically for 2 hours at room temperature. Discard the supernatant and wash the tube 3 times quickly with PBS. The phage antibody library (purchased from the MRC center in the UK) was suspended in 4 mL of 3% BSA and added to the immunotube, incubated upside down at room temperature for 1 hour, and then incubated vertically for 1 hour. Wash 10 times with 0.1% Twenn-20 in PBS (20 times for the second round of selection and subsequent washes). After the PBS was blotted dry, 500 μL of trypsin-PBS solution was added to elute the phage, and incubated upside down at room temperature for 10 min. Add 250 μL of the eluted phage to 1.75 mL of Esc...

Embodiment 3

[0060] Example 3 Identification of Antibodies

[0061] Abeta monomers, oligomers, and fibrils (Abeta monomers were incubated at 37°C for more than 4 days and verified by atomic force microscopy) were spotted on 3 μL of NC (nitrocellulose) membranes. After the membrane was blocked with 5% BSA, scFv W20 was added to incubate for 1 hour, and the membrane was washed 3 times with PBS, 5 minutes each time. Add 1:5000 diluted Protein A (purchased from Santa Cruz, USA) and incubate for 1 hour, wash the membrane 3 times with PBST (Tween-20 concentration is 0.1%), 5 minutes each time, and develop color with DAB. Only clones showing spots in A-beta oligomers but not in A-beta monomers and fibrils were desired.

[0062] The above positive clones were sequenced and identified, and the clones that conformed to the basic structure of the antibody in the antibody library were complete single-chain genetically engineered antibodies. The sequencing primers are:

[0063] LMB3: 5'-CAG GAA ACA ...

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Abstract

The invention relates to the technical field of genetic engineering antibody and provides a monoclonal antibody. The variable region of heavy chain of the monoclonal antibody contains the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEO ID NO.3; the variable region of light chain of the monoclonal antibody contains the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The invention also specifically provides a humanization single-chain antibody generated by the recombinant strain of enterotoxigenic Escherichia coli with the accession number CGMCC No.2821 and the amino acid sequence of the humanization single-chain antibody is shown in SEQ ID NO.7. The antibody of the invention can be specifically bound with the A-beta oligomer, effectively inhibit the fibrosis aggregation of A-beta and obviously alleviate the toxic effect of the A-beta on cells. The invention also relates to a pharmaceutical composite containing the antibody. The antibody of the invention has strong activity, good specificity, easy preparation and wide prospect of experiment application and clinical application.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering antibodies, and specifically relates to a genetic engineering monoclonal antibody specifically combined with A-beta oligomers. The invention also relates to a preparation method of the monoclonal antibody and a pharmaceutical composition containing the monoclonal antibody. Background technique [0002] Studies have shown that Alzheimer's Disease (AD), commonly known as senile dementia, is composed of non-toxic β-amyloid monomer molecules (β-Amyloid, A-beta 40 / 42, hereinafter referred to as A-beta, also It can be written as Aβ) aggregates to form toxic oligomers in the elderly and is mainly characterized by memory loss and senile plaques in the brain. 1996, 9989-9990). Medical statistics show that 5-6% of the elderly over the age of 60 in my country and European and American countries suffer from Alzheimer's disease. The disease has been ranked as the fourth leading cause of death aft...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N1/21G01N33/577A61K39/395A61P25/28C12R1/19
Inventor 刘瑞田王小平
Owner TSINGHUA UNIV
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