A kind of anti-human sirpα monoclonal antibody and its application
A monoclonal antibody and 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, and achieve novel sequences Effect
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Embodiment 1
[0041] Example 1 Obtaining specific anti-SIRPα mouse monoclonal antibody by fusion hybridoma technology
[0042] 1.1 Animal Immunization
[0043] Mice were immunized according to general methods 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MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNER)。 Use recombinant human SIRPα with his-tagged protein (produced by the company, the plasmid used is SRPA-ECD-His; the vector is pCMV-C-His; the amino acid sequence inserted between EcoR I and Xba I is ...
Embodiment 2
[0051] Example 2 In Vitro Assay for Determining the Functional Activity of SIRPα Monoclonal Antibodies
[0052] 2.1 Determination of antibody binding capacity based on capture ELISA
[0053] Goat anti-mouse IgG F(ab') in 1xPBS 2 A specific secondary antibody (Jackson ImmunoResearch Laboratories, Inc., Cat#115-005-072) was added to a final concentration of 2 μg / ml, and 100 μl / well was added to a 96-well microtiter plate at 37 degrees Celsius for 2 hours. After the coating, the plate was washed once with 1×PBST, and then 200 μl / well of PBST solution containing 5% nonfat dry milk was added and placed at 37 degrees Celsius to block for 2 hours. Wash the plate again, add 100 μl / well of diluted antibody solution and Benchmark (ie KWAR23 antibody, produced by the company, its heavy chain amino acid is
[0054] EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG...
Embodiment 3
[0076] Example 3 Binding activity of anti-SIRPα mouse monoclonal antibody
[0077] According to the assay method described in Example 2, the binding activity of the SIRPα antibody was assessed and summarized in Table 3 below.
[0078] The SIRPαBenchmark was used as a control. It can be seen from Table 3 that the anti-human SIRPα monoclonal antibody of the present invention has slightly better binding activity to human SIRPα antigen than Benchmark, and can cross-react with SIRPβ antigen at the same time.
[0079] Table 3 Binding activity of SIRPα antibody
[0080]
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