anti-alk2 antibody
An antibody and antigen technology, applied in the direction of antibodies, antibody medical components, anti-tumor drugs, etc., can solve unknown problems
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Embodiment 1
[0500] Preparation of rat anti-ALK2 antibodies (11E2, 15A6, 25C11 and 27D11: hereinafter abbreviated as A2-11E, A2-15A, A2-25C and A2-27D)
[0501] 1) Preparation of -1 antigen
[0502] As mALK2-Fc used as an antigen, mouse ALK2-His&Fc (Cat. #50297-M03H) manufactured by Sino Biological Inc. was used.
[0503] 1)-2 Immunization of animals
[0504] 1 mg / mL of mouse ALK2-His&Fc and TiterMax Gold (TiterMax, USA) were mixed in equal amounts to prepare an emulsion. To two 6-week-old Wister female rats, 100 μg of the antigen was subcutaneously administered together with the adjuvant twice. After 1 to 2 weeks, only 40 μg of the antigen solution was subcutaneously administered, and 3 days later, spleen cells and various lymph nodes were aseptically removed as antibody-forming cells, and used for subsequent cell fusion.
[0505] 1)-3 Cell fusion with myeloma cells
[0506] Adjust the ratio of the above-mentioned antibody-forming cells to the myeloma cell line (P3U1 cells) obtained f...
Embodiment 2
[0513] In vitro (invitro) evaluation of rat anti-ALK2 antibodies (A2-11E, A2-15A, A2-25C and A2-27D)
[0514] 2)-1 Antibody screening by flow cytometry
[0515] 2) Preparation of -1-1 mouse and human ALK2 expressing cells
[0516] HEK293A cells to reach 3 x 10 4 cells / cm 2 Inoculated into 100mm Petri dishes, with DMEM medium containing 15% FBS, at 37°C, 5% CO 2 cultured overnight under conditions. On the next day, pcDEF3 / mouse ALK2(WT)-EGFP, pcDEF3 / human ALK2(WT)-EGFP, pcDEF3 / human ALK2(R206H)-EGFP, and pcDEF3 were introduced into HEK293A cells using Lipofectamine 2000 (manufactured by Invitrogen), respectively, Further overnight incubation. Next day, adjust to 1×10 6 The cells / mL cell suspension was dispensed into 1.5mL microcentrifuge tubes every 100μL, centrifuged at 500g for 5 minutes, and the supernatant was removed. 100 µL of purified IgG diluted to 1 µg / mL was added to the cells, and the cells were left to stand at 4°C for 30 minutes. After the cells were washed...
Embodiment 3
[0536] Determination of the Nucleotide Sequence of the cDNA Encoding the Variable Region of Rat Anti-ALK2 Antibody (A2-11E, A2-15A, A2-25C, A2-27D)
[0537] 3)-1 Determination of the nucleotide sequence of the cDNA encoding the A2-11E variable region
[0538] 3)-1-1 Preparation of total RNA from the hybridoma producing A2-11E
[0539] To amplify the cDNA containing the A2-11E variable region, total RNA was prepared from the A2-11E-producing hybridoma using TRIzol Reagent (Ambion).
[0540] 3) Synthesis of 1-2 cDNA (5'-RACE-Ready cDNA)
[0541] Synthesis of cDNA (5'-RACE-Ready cDNA) was carried out using 1 µg of the total RNA prepared in Example 3)-1-1, and using SMARTer RACE cDNA Amplification Kit (Clontech Corporation).
[0542] 3)-1-3 Amplification and sequence determination of cDNA containing A2-11E heavy chain variable region by 5'-RACE PCR
[0543] As primers for amplifying the cDNA of the variable region of the A2-11E heavy chain gene by PCR, UPM (provided with Univer...
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