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

Active Publication Date: 2010-11-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing SIRPα antibodies do not have a good ability to perform western blot, flow cytometry and immunohistochemical detection at the same time.
And most of the existing functional SIRPα antibodies are blocking or inhibitory, but not activating

Method used

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  • Monoclonal antibody of anti-human SIRPalpha, cell strain, preparation method and application thereof
  • Monoclonal antibody of anti-human SIRPalpha, cell strain, preparation method and application thereof
  • Monoclonal antibody of anti-human SIRPalpha, cell strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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

negative control

positive control

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

negative control

positive control

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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Abstract

The invention belongs to the technical field of medical bioengineering, in particular to a monoclonal antibody of an anti-human signal regulating protein alpha (SIRPalpha), a preparation method and an application. The signal regulating protein alpha (SIRPalpha) is a member belonging to an immunoglobulin superfamily (IgSF), and tissues are mainly distributed in a medullary system (a macrophage cell, a dendritic cell and the like), therefore, the effect of the SIRPalpha in the immune adjustment of organism has been much accounted gradually. The invention provides the monoclonal antibody of the monoclonal antibody SIRPalpha, which is generated from a hybridoma cell strain with the preserved number of CGMCCNo.3801. The invention also provides the application of the monoclonal antibody in the preparation and detection of the SIRPalpha and in the preparation of drugs for stimulating macrophage TNF-alpha expression of a human body.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering. Specifically, the present invention relates to a monoclonal antibody against human signal regulatory protein alpha (SIRPα), a cell line secreting the monoclonal antibody, a preparation method and application of the monoclonal antibody. Background technique [0002] Signal regulatory protein alpha (SIRPα) is a member belonging to the immunoglobulin superfamily (IgSF). Rat protein tyrosine phosphatase SIRPα was first identified in MOLECULAR AND CELLULAR BIOLOGY in 1996 and NATURE in 1997. SIRPα is also called SHPS1, CD172α, P84, MYD1, BIT, PTPNS1, etc. It is a transmembrane protein mainly expressed in myeloid cells, GeneID: 140885. The extracellular domain of SIRPα contains three immunoglobulin superfamily (IgSF) domains and multiple glycosylation sites. Its cytoplasmic region is highly conserved among rats, mice and humans, with two immunoreceptor tyrosine inhibitory motifs (ITIM...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/577G01N15/10A61K39/395A61P37/02A61P43/00C12R1/91
Inventor 王红阳鄢和新唐亮刘琼
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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