Specific antibody directed to active hepatocyte growth factor activator and method for using the same
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EXAMPLE 1
Preparation of Inactive HGFA and Active HGFA
[0108] Inactive HGFA was prepared by using HGFA cDNA coding for the HGFA precursor described in Japanese Patent Laid-open Publication No. 6-153946 according to the method described in Japanese Patent Laid-open Publication No. 6-153966 to produce recombinant inactive HGFA. That is, the HGFA cDNA coding for the full length of the inactive HGFA precursor, 655 amino acids, was inserted into pcDNA3.1 (Invitrogen), which is an animal cell expression vector, downstream from a CMV promoter in a conventional manner.
[0109] The obtained expression vector was introduced into a CHO cell, which is an animal cell strain derived from a Chinese hamster ovary cell, by using a Transfectam (BioSepra) according to the attached instruction. Then, CHO cells expressing HGFA cDNA were selected by utilizing a property of a neomycin resistant gene existing on the introduced expression vector. That is, cells that could grow in an ERDF medium (Kyokuto Seiy...
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EXAMPLE 2
Preparation of ELISA Plate for Screening Active HGFA Specific Monoclonal Antibody
[0113] The active 36 kDa HGFA or active 98 kDa HGFA prepared in Example 1 was diluted with hydrogenphosphate buffered physiological saline (PBS(-)) to a final concentration of 1 μg / ml as an antigen for screening hybridoma, and 100 μl of the solution was added to each well of a 96-well plate and stored at 4° C. for 24 hours so that each antigen should be adsorbed on the 96-well plate. The solution was removed from this antigen-adsorbed plate, and then 250 μl of PBS(-) containing 5% bovine serum albumin (hereafter, abbreviated as “BSA”) was added to each well and left at 4° C. overnight (about 12 hours) or at 37° C. for 2 hours or longer to block the plate. Then, the plate was stored as an ELISA plate for screening active HGFA at 4° C.
[0114] Separately, the inactive HGFA prepared in Example 1 was diluted with PBS(-) to a final concentration of 1 μg / ml as an antigen for screening hybridoma. Then...
Example
EXAMPLE 3
Preparation of Active HGFA Specific Monoclonal Antibody
[0115] A solution containing 100 μg of the 36 kDa HGFA or 100 μg of the 98 kDa HGFA, which were prepared in Example 1, was subcutaneously and intraperitoneally administered to a Balb / c mouse with the same volume of complete Freund's adjuvant 6 times with 2-week intervals. After production of antibodies in the serum of the mouse was confirmed, a solution containing 100 μg of HGFA was administered into the caudal vein. Three days later, spleen was removed and spleen cells were fused with myeloma cells P3U1 by using polyethylene glycol 1500 according to “Monoclonal Antibody Experimental Manual” (Kodansha Scientific, 1987), introduced into wells of 96-well plate, added with HAT medium and cultured for 14 days.
[0116] Subsequently, hybridomas producing monoclonal antibodies specific to respective active HGFA in the medium were selected. That is, culture supernatant of a hybridoma subjected to selection was added to an ELIS...
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