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Cell adhesion molecule 5 monoclonal antibody, preparation method and applications thereof

A monoclonal antibody and adhesion molecule technology, applied in biochemical equipment and methods, receptors/cell surface antigens/cell surface determinants, microbial-based methods, etc., can solve unstable test results, low activity, impact In-depth development of experimental research and other issues to achieve the effect of less antibody usage, high titer, and high expression

Active Publication Date: 2013-08-21
盛世菁华(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commercialized ICAM5 antibodies currently on the market are all polyclonal antibodies, which have low activity and unstable test results, which directly affect the in-depth development of experimental research.

Method used

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  • Cell adhesion molecule 5 monoclonal antibody, preparation method and applications thereof
  • Cell adhesion molecule 5 monoclonal antibody, preparation method and applications thereof
  • Cell adhesion molecule 5 monoclonal antibody, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0028] Embodiment 1 ICAM5 monoclonal antibody preparation

[0029] 1. Polypeptide antigen synthesis

[0030] From the human ICAM5 protein sequence, the second immunoglobulin-like region of cell adhesion molecule 1 (Ig2-ICAM-1-like, ICAM1-CD54) intermittently selects 6 amino acid sequences for peptide synthesis (amino acids 30-220). The peptide synthesis was completed by Abimate Biomedicine (Shanghai) Co., Ltd. The sequence of the 6 peptides is as follows:

[0031] RCARRT\LQARARGLIRT\FQRGLRWLARQL\VRASLTLTLLRTSLR\RNGTQRLQPRVAFV\ERRRSFAGEPPR, molecular weight 34KD, purity >90%.

[0032] The synthetic polypeptide sequence is shown as SEQ ID No.1:

[0033] RCARRTLQARARGLIRTFQRGLRWLARQLVRASLTLTLLRTSLRRNGTQRLQPRVAFVERRRSFAGEPPR

[0034] 2. Animal immunization

[0035] Three healthy female BALB / C mice aged 8-12 weeks were immunized with the synthesized polypeptide antigen. Take the rapid immunization method of mice.

[0036] Initial immunization: Antigen 50ug / body, plus Freund's...

Embodiment 2

[0112] Example 2 Application of ICAM5 Monoclonal Antibody in Immunofluorescence Experiment

[0113] Immunofluorescence Detection of ICAM5 on Cell Surface of Primary Cultured Neurons in Mouse Cerebral Cortex

[0114] Animal experiments were carried out in strict accordance with the relevant regulations on the protection of experimental animals. Fetal mouse brain tissue was collected after 16 days of pregnancy, and placed in dissecting separation solution (1 × Hanks balanced salt solution + 2.5mmol / L Hepes + 30mmol / L D-glucose + 2mmol / L CaCl) under an optical microscope 2 +0.5mmol / L MgSO 4 +2mmol / L NaHCO3+0.03%BSA+1% penicillin and streptomycin) to remove the dura mater and separate the cerebral cortex. After washing twice with the dissection separation solution, pipette gently to make the cortical tissue become a single suspended neuron cell. After filtering with a 200-mesh screen, centrifuge at 1500r / min (centrifugal radius: 10cm) for 3min, discard the supernatant, and add ...

Embodiment 3I

[0116] Example 3 Application of ICAM5 monoclonal antibody in Western blotting

[0117] Western blotting detection of ICAM5 protein in human brain tissue samples

[0118] Take the autopsy specimens of AIDS brain tissue frozen at -135 ℃, and extract tissue protein by high-salt lysis method. The specific steps are as follows: use small scissors to cut off tissue pieces about the size of mung bean grains, cut them as much as possible with scissors, and add 500 μl of ice-cold 1× PBS is homogenized with a homogenizer (note that the above operations are required to be on ice, and protease inhibitors should be added to PBS according to the concentration). After homogenization, pour the tissue homogenate into a clean Ep tube, Add ice-cold 2× high-salt lysate (1%NP40, 0.1%SDS, 0.04% sodium deoxycholate, 150mMNaCl, 50mMpH8.0Tris-HCl, 5mMEDTA, add 1:1000PMSF when used), place on ice for 20 minutes for complete lysis. After centrifugation at 4°C for 20 minutes, the supernatant was collec...

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Abstract

The present invention discloses cell adhesion molecule 5 (ICAM5) monoclonal antibody, a preparation method and applications thereof, wherein a preservation number of the hybridoma cell strain is CGMCC No.5798. The present invention provides the ICAM5 monoclonal antibody, which can be provided for ICAM5 antigen-based immunofluorescence and Western blotting, and other scientific researches, wherein test results show that the high and stable expression exists on the surface of human and mouse nerve cells. In addition, the antibody has characteristics of strong specificity and high titer, wherein an ELISA detection titer can be 1:400000, such that an ICAM5 antibody use concentration in a test is low, and an antibody use amount is low; and the ICAM5 antigen is positioned on the surface of nerve cell bodies and nerve fibers, such that structures of nerve cell bodies and nerve fibers can be clearly displayed with immunofluorescence staining.

Description

technical field [0001] The invention relates to a monoclonal antibody and a preparation method thereof, in particular to a cell anti-adhesion molecule 5 (ICAM5) monoclonal antibody and a preparation method thereof. Background technique [0002] Intercellular adhesion molecule 5 (Intercellularadhesionmolecule5, ICAM5) is a protein encoded by the human ICAM5 gene, which belongs to the ICAM family member of the immunoglobulin superfamily. All ICAM proteins are type I transmembrane glycoproteins with 2–9 immunoglobulin C2 domain-like domains that bind leukocyte integration-associated antigen (LFA-1). ICAM1 is expressed on the surface of non-neural cells in the brain, while ICAM5 is only expressed on the surface of nerve cells, and has two types of adhesion activities: homogeneous adhesion between neurons and heterogeneous adhesion between neurons and lymphocytes. ICAM5 can be expressed on the surface of nerve cell soma and protrusions, and exerts adhesion-dependent functions by...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/28C07K14/705G01N33/68C12R1/91
Inventor 陈德喜张玉林李宁石英宋凤丽丁谓乔录新侯庆生徐树莹
Owner 盛世菁华(北京)生物科技有限公司
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