Anti-gprc5d antibody and molecule containing same
An antibody, human antibody technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problem of undeveloped pharmaceutical products and so on
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Embodiment 1
[1132] Example 1. Preparation of rat anti-human GPRC5D antibody
[1133] 1)-1 Immunization using human GPRC5D expression vector
[1134] 1)-1-1 Construction of human GPRC5D expression vector (pcDNA3.1-DEST-hGPRC5D)
[1135] pcDNA3.1-DEST engineered as a destination vector was prepared from pcDNA3.1(+) using Gateway Vector Convention System (Thermo Fisher Scientific Inc.). The cDNA encoding human GPRC5D protein (NP_061124.1) was cloned into the pcDNA3.1-DEST vector using Gateway LR ClonaseEnzyme mixture (Life Technologies Corp.) to construct the human GPRC5D expression vector pcDNA3.1-DEST-hGPRC5D. For large-scale preparation of human GPRC5D expression vector, Endofree Plasmid Giga Kit (Qiagen N.V.) was used.
[1136] 1)-1-2 Rat Immunization
[1137]For immunization, WKY / Izm female rats (Japan SLC, Inc.) were used. First, both calves of each rat were pretreated with hyaluronidase (Sigma-Aldrich Corp.). Then, pcDNA3.1-DEST-hGPRC5D was intramuscularly injected into these sit...
Embodiment 2
[1171] Example 2. In Vitro Evaluation of Rat Anti-GPRC5D Antibodies (2A4, 2B1 and 7B4)
[1172] 2)-1 Binding activity of rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4) obtained by flow cytometry to human GPRC5D
[1173] The human multiple myeloma cell line KHM-1B cells (JCRB Cell Bank) expressing GPRC5D was adjusted to 5 × 10 with PBS containing 5% FBS 6 cells / mL, inoculate 100 μL / well into a 96-well U-bottom microplate, and centrifuge to remove the supernatant. Each of the rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4) adjusted to 0.32 ng / mL to 10 μg / mL in Example 1)-7 was added thereto in an amount of 100 μL / well, and the plate was incubated at 4 ℃ for 1 hour. Cells were washed twice with PBS containing 5% FBS. Then, PE goat anti-rat Ab (Becton, Dickinson and Company) diluted 100-fold with PBS containing 5% FBS was added thereto at a concentration of 100 μL / well, and the plate was left to stand at 4° C. for 1 hour. Cells were washed twice with PBS containing 5% FBS and the...
Embodiment 3
[1186] Example 3. Sequencing of cDNAs encoding the variable regions of rat anti-GPRC5D antibodies (2A4, 2B1 and 7B4)
[1187] 3)-1 Sequencing of cDNA encoding the variable region of 2A4
[1188] 3)-1-1 Preparation of total RNA from hybridoma producing 2A4
[1189] To amplify the cDNA encoding the variable region of 2A4, total RNA was prepared from 2A4-producing hybridomas using TRIzol reagent (Ambion / Thermo Fisher Scientific Inc.).
[1190] 3) Synthesis of -1-2 cDNA (5'-RACE-Ready cDNA)
[1191] cDNA (5'-RACE-Ready cDNA) was synthesized using approximately 1 µg of the total RNA prepared in Example 3)-1-1 and the SMARTer RACE cDNA Amplification Kit (Clontech Laboratories, Inc.).
[1192] 3)-1-3 The cDNA encoding the heavy chain variable region of 2A4 was amplified and sequenced by 5'-RACE PCR
[1193] The primers used for PCR amplification of the cDNA encoding the variable region of the heavy chain gene of 2A4 were UPM (Universal Primer A Mixture; attached to the SMARTer RAC...
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