new anti-dr5 antibody
A technology of antibodies and chimeric antibodies, applied in the direction of antibodies, antibody medical components, antibody mimics/stents, etc., can solve problems such as no correlation
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Embodiment 1
[0394] [Example 1] Production of mouse antibody B273
[0395] 1)-1 Production of human DR5 protein (human DR5 extracellular domain / human Fc fusion protein)
[0396] 1)-1-1 Production of human DR5 extracellular domain expression vector
[0397] A vector expressing human DR5 protein (isoform 2: NP_671716) was constructed by inserting a gene in which the extracellular domain of human DR5 was fused to the human IgG1 / Fc region downstream of the CMV promoter.
[0398] 1)-1-2 Production of human DR5 protein
[0399] Introduction of the expression vector into 293 FreeStyle cells and collection of the culture supernatant were performed by Invitrogen Corporation (now Life Technologies Japan Ltd.).
[0400] 1)-1-3 Purification of human DR5 protein
[0401] The culture supernatant obtained in b) above was purified using protein A affinity column chromatography. 5 L of the culture supernatant was applied to "HiTrap Protein AFF" (GE Healthcare Bio-Sciences Co., Ltd., catalog number 17-5...
Embodiment 2
[0428] [Example 2] Cloning of mouse antibody B273 gene and production of human chimeric antibody gene
[0429] 2)-1 Cloning and sequence determination of mouse antibody B273 cDNA
[0430] 2)-1-1 Determination of N-terminal amino acid sequences of heavy chain and light chain of mouse antibody B273
[0431]In order to determine the N-terminal amino acid sequences of the heavy and light chains of the mouse antibody B273, the mouse antibody B273 purified in Examples 1-8 was separated by SDS-PAGE. After separation, the protein in the gel was transferred from the gel to a PVDF membrane (pore size: 0.45 μm, manufactured by Invitrogen Corporation). The membrane was washed with wash buffer (25 mM NaCl, 10 mM sodium borate buffer, pH 8.0) and thereafter stained by immersion in dye solution (50% methanol, 20% acetic acid, 0.05% Coomassie brilliant blue) for 5 minutes, followed by 90% Methanol decolorizes. The band corresponding to the heavy chain (band with smaller mobility) and the b...
Embodiment 3
[0472] [Example 3] Measurement of human chimeric B273 (cB273) antibody activity (in vitro)
[0473] 3) Study on the selective binding properties of -1 cB273 antibody to the extracellular domain of human DR5
[0474] The binding properties of cB273 to human TRAIL R1-R4 and mouse TRAIL R2 extracellular domain proteins (manufactured by R&D Systems, Inc.) were investigated by the direct ELISA method described below. First, each of the ectodomain proteins of TRAIL R was diluted to 1 μg / ml with PBS, and the diluted solution was distributed in an immunoplate (manufactured by Nunc, Inc., #442404) at 50 μl / well, and the plate was incubated at 4 The protein was thus adsorbed to the plate by standing overnight at ℃. The next day, the liquid in each well was removed, and each well was washed once with PBS. Thereafter, in order to suppress non-specific adsorption of proteins, 200 μl / well of PBS containing 3% fetal bovine serum was dispensed, and the plate was allowed to stand at room tem...
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