Anti-human SIRP alpha monoclonal antibody and application thereof
A monoclonal antibody, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, applications, etc., can solve the problems of limited specificity of anti-SIRPα antibodies, negative effects on T cell proliferation and recruitment, etc., and achieve novel sequences. Effect
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Embodiment 1
[0041] Embodiment 1 obtains the mouse monoclonal antibody of specific anti-SIRPα by fusion hybridoma technology
[0042] 1.1 Animal immunity
[0043] Mice were immunized according to the general method in the literature (E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998). 免疫原为C端含有人IgG1 Fc标签的重组人SIRPα(氨基酸Met 1-Arg 370)蛋白(公司自产,所用质粒为CD172-ECD-Fc;载体为pCP;插入氨基酸序列为MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATL RCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIR IGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARA TPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTA KVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAE NQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWL LVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGS NER). Use recombinant human SIRPα protein with his tag (produced by the company itself, the plasmid used is SRPA-ECD-His; the vector is pCMV-C-His; the amino acid sequence inserted between E...
Embodiment 2
[0051] Example 2 In vitro analysis method for measuring the functional activity of SIRPα monoclonal antibody
[0052] 2.1 Determination of antibody binding ability based on capture ELISA
[0053] Prepare goat anti-mouse IgG F(ab') in 1xPBS 2 Specific secondary antibody (Jackson ImmunoResearch Laboratories, Inc., Cat#115-005-072) was added to a 96-well microtiter plate at a final concentration of 2 μg / ml at 100 μl / well, and coated at 37 degrees Celsius for 2 hours. After coating, wash the plate once with 1xPBST, add 200 μl / well of PBST solution containing 5% skimmed milk powder and place at 37 degrees Celsius for 2 hours to block. Wash the plate again, add 100 μl / well of diluted antibody solution and Benchmark (that is, KWAR23 antibody, produced by the company, and its heavy chain amino acid is
[0054] EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIG RIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAY WGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQS...
Embodiment 3
[0076] Example 3 Binding Activity of Anti-SIRPα Mouse Monoclonal Antibody
[0077] According to the analysis method described in Example 2, the evaluation of the binding activity of the SIRPα antibody is summarized in Table 3 below.
[0078] Among them, SIRPα Benchmark was used as a control. It can be seen from Table 3 that the binding activity of the anti-human SIRPα monoclonal antibody of the present invention to human SIRPα antigen is slightly better than that of Benchmark, and can cross-react with SIRPβ antigen at the same time.
[0079] Table 3 Binding activity of SIRPα antibodies
[0080]
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