Anti-epha4 antibody
An antibody and variable technology, applied in the direction of antibody, anti-enzyme immunoglobulin, recombinant DNA technology, etc., can solve the problem of no antibody report
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example 1
[0438] Example 1: Preparation of anti-mouse EphA4 monoclonal antibody
[0439] Preparation of mouse anti-mouse EphA4 monoclonal antibody
[0440] In order to prepare a monoclonal antibody that binds to mouse EphA4 (GenBank accession number NP_031962.2, SEQ ID NO: 1), the extracellular region (20 to 547) of mouse EphA4 (SEQ ID NO: 1) was prepared by the following steps. ID NO: 2) A protein fused with secreted alkaline phosphatase (SEAP) and a histidine tag (hereinafter referred to as "mouse EphA4 extracellular region-SEAP-His protein", SEQ ID NO: 43).
[0441] First, using total RNA derived from mouse brain, the DNA sequence encoding the signal sequence (SEQ ID NO: 42) and extracellular region (SEQ ID NO: 2) of mouse EphA4 was amplified by RT-PCR, and cloned to the SalI / NotI sites of the pENTR1A vector (Invitrogen / Life Technologies) with the DNA sequence encoding SEAP and histidine tag. Secondly, the signal sequence, extracellular region, SEAP, and histidine tag DNA seque...
example 2
[0460] Example 2: Binding affinity of anti-EphA4 monoclonal antibodies against mouse and human EphA4
[0461] The binding affinity of antibody A obtained in Example 1 to mouse and human EphA4 was determined by surface plasmon resonance (SPR method) using Biacore A100 (GE Healthcare). First, mouse IgG will be used 1 Rat anti-mouse IgG prepared by immunizing the antibody against rats by common methods 1 Antibodies were immobilized on the sensor chip CM5. rat anti-mouse IgG 1 Antibody immobilization on the sensor chip CM5 was achieved by using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was carried out by the amine coupling method, and ethanolamine was used for blocking (both the sensor chip and the immobilization reagent were manufactured by GE Healthcare). Use fixation buffer (10 mM sodium acetate, pH 4.5), dilute to 1 to 2 μg / mL, and immobilize on the sensor chip according to the operation instructions attached to Bi...
example 3
[0463] Example 3: Mouse EphA4-Mouse Ligand Binding Inhibitory Activity of Anti-EphA4 Monoclonal Antibodies
[0464] For antibody A obtained in Example 1, the evaluation of the binding inhibitory activity of mouse EphA4 to the mouse ligand was carried out according to the following procedure. Anti-alkaline phosphatase antibody (Thermo Fisher Scientific) was coated on the wells of a 96-well culture plate (Nunc). After overnight incubation at 4°C, the wells were blocked with 1% Block Ace (DS Pharma Biomedical) for 1 hour at room temperature. After washing 3 times with 0.05% Tween 20 / PBS (Thermo Fisher Scientific), the mouse EphA4 extracellular region-SEAP-His protein (final concentration 10 nM) obtained in the method of Example 1 was added to the well, and incubated at room temperature 1 hour. After washing 3 times, ligands and serially diluted antibody A (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, and 1000 nM) were added to the wells as KYL peptide (KYLP...
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