Monoclonal antibody of anti-human SIRPalpha, cell strain, preparation method and application thereof
A monoclonal antibody, hybridoma cell line technology, applied in the field of medical bioengineering
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Embodiment 1
[0037] Example 1: Preparation and purification of monoclonal antibody against human SIRPα
[0038] 1. Antigen Synthesis and Conjugation
[0039] Antigen peptide design refers to GenBank, GeneID: 140885, the specific sequence is: RVTTVSESTKRENMDFSISISC (SEQ ID NO: 1), synthesized by Baiqi Biotechnology (Shanghai) Co., Ltd. through the automatic peptide synthesizer by solid-phase peptide synthesis.
[0040] The coupling of polypeptide and keyhole limpet hemocyanin (KLH) adopts the glutaraldehyde connection described in "Molecular Cloning Experiment Guide" 2nd edition, written by Sam Brook, translated by Jin Dongyan, Science Press, Beijing, 1999, page 856 Law.
[0041] 2. Preparation and purification of monoclonal antibody 92CT57.39.3
[0042] Dilute 100 μg of the polypeptide KLH conjugate purified in 1 above with PBS, mix and emulsify with an equal volume of complete Freund’s adjuvant (CFA), and immunize 5 female BALB / c mice aged 5-6 weeks. For subcutaneous injections under t...
Embodiment 2
[0046] Example 2: Identification and detection application of monoclonal antibody against human SIRPα
[0047] 1. Type Identification of Monoclonal Antibody
[0048] The Ig class and subclass specific antibodies of mouse monoclonal antibodies were used for ELISA detection, and the identification results are shown in Table 1. The results showed that the heavy chain of the monoclonal antibody was IgG1 type, and the light chain was κ chain.
[0049] Table 1: Ig class and subclass identification results of monoclonal antibody 92CT57.39.3
[0050]
92CT57.39.3
IgG1
3.4102
0.0809
3.1634
IgG2a
0.0716
0.0581
3.186
IgG2b
0.0865
0.0611
3.1889
[0051]
92CT57.39.3
IgG3
0.1084
0.1021
3.3212
IgM
0.0659
0.0621
3.3335
IgA
0.0631
0.0568
3.4426
...
Embodiment 3
[0058] Embodiment 3: the application of the monoclonal antibody of anti-human SIRPα
[0059] Human mononuclear cell line THP-1 cells 4×10 5 , add 100ng / ml phorbol myristate (PMA) to the medium, change to ordinary medium after 24 hours, continue to cultivate for 48 hours, change to ordinary medium again, add corresponding antibody to 20μg / ml, 6 hours Afterwards, the supernatant was collected, and the concentration of TNF-α was determined therein (using Daktronics, DKW12-1720 kit). The results are shown in Table 2. The results show that the antibody can stimulate THP-1 cells to secrete TNF-α to about 3 times that of the control.
[0060] Table 2: The concentration determination results of TNFα in the supernatant of THP-1 cells stimulated by monoclonal antibody 92CT57.39.3
[0061]
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