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

A technology of monoclonal antibody and hybridoma cell line, applied in the field of medical bioengineering

Active Publication Date: 2012-10-17
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 antibodies against human SIRPα

[0038] 1. Antigen synthesis and coupling

[0039] The antigen peptide design refers to GenBank, GeneID: 140885, and the specific sequence is: RVTTVSESTKRENMDFSISISC (SEQ ID NO: 1), which was synthesized by Pocky Biotechnology (Shanghai) Co., Ltd. by a solid-phase peptide synthesis method using an automatic peptide synthesizer.

[0040] The coupling of peptides and keyhole limpet hemocyanin (KLH) uses the glutaraldehyde linkage described in "Molecular Cloning Experiment Guide" 2nd edition, written by Sambrook, translated by Jin Dongyan, Science Press, Beijing, 1999, page 856 law.

[0041] 2. Preparation and purification of monoclonal antibody 92CT57.39.3

[0042] 100μg of the polypeptide KLH conjugate purified in 1 above was diluted with PBS and mixed with equal volume of complete Freund’s adjuvant (CFA) to emulsify, and immunize 5 female BALB / c mice aged 5-6 weeks. Subcutaneous injections und...

Embodiment 2

[0046] Example 2: Identification and detection application of monoclonal antibodies against human SIRPα

[0047] 1. Type identification of monoclonal antibodies

[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 of IgG1 type, and the light chain was of kappa 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

IgG3

0.1084

0.1021

3.3212

IgM

0.0659

0.0621

3.3335

IgA

0.0631

0.0568

3.4426

Igκ

4

0.0611

3.4046

Igλ

0.0625

0.0536

2.9594

[0051] 2. Flow cytometry application of monoclonal antibodies

[0052] 1×10 6 Huh-7 / hSIRPα-4Y cells were resuspended i...

Embodiment 3

[0057] Example 3: Application of monoclonal antibodies against human SIRPα

[0058] Human monocyte line THP-1 cells 4×10 5 , Add 100ng / ml phorbol ethyl myristate (PMA) to the medium, change to normal medium after 24 hours, continue to cultivate for 48 hours, change to normal medium again, add corresponding antibody to 20μg / ml, 6 hours After the supernatant was collected, the concentration of TNF-α was determined (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 up to about 3 times that of the control.

[0059] Table 2: Measurement results of the concentration of TNFα in the supernatant of THP-1 cells stimulated by the monoclonal antibody 92CT57.39.3

[0060]

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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 α (SIRPα), a cell line secreting the monoclonal antibody, and a preparation method and application of the monoclonal antibody. Background technique [0002] Signal Regulator α (SIRPα) is a member of the immunoglobulin superfamily (IgSF). In MOLECULAR AND CELLULAR BIOLOGY in 1996 and NATURE in 1997, protein tyrosine phosphatase SIRPα in rats was first identified. 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 region 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 (ITIMs, whose characteristic...

Claims

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Application Information

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Patent Type & Authority Patents(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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