Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method

An antibody-specific technology, applied in the fields of molecular biology and biology, can solve problems such as differences in the exposure of antibody-binding epitopes

Active Publication Date: 2013-02-20
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These evidences suggest that it is likely that different antibody-binding epitopes are exposed differently in different tissues and microenvironments

Method used

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  • Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method
  • Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method
  • Anti-human CD146 monoclonal antibodies, compositions containing anti-human CD146 monoclonal antibodies, and soluble CD146 detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Preparation and identification of monoclonal antibodies AA1-AA5 and AA7.

[0058] Application of hybridoma technology (Kohler and Milstein 1975; Yeh, Hellstrom et al.1979; Yeh, Hellstrom et al.1982) produced and screened to obtain antibodies AA1-AA6 and AA7. The brief description is as follows: the native CD146 protein (its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2) was isolated from human umbilical cord vein endothelial cells, according to (Yan, Lin et al. 2003) described monoclonal antibody AA98 antigen purification method purification, it is used as immunogen to carry out immunization to BALB / C mouse (Beijing Experimental Animal Center), each intraperitoneal injection 100 μ g albumen / mouse, once every two weeks, A total of three times. Before the splenocytes were collected, immunization was boosted once, and 100 μg protein / mouse was injected intraperitoneally. Three days after the booster immunizat...

Embodiment 2

[0082] Example 2: Epitope identification of monoclonal antibodies AA1-AA5 and AA7.

[0083] The antigenic epitopes of the six antibodies described in the present invention were identified by recombinantly expressing the five domain proteins of the extracellular region of human CD146 and immunoblotting.

[0084] like figure 2 As shown, domains 1-5 represent the five domains V-V-C2-C2-C2 of CD146 from extracellular to transmembrane region respectively. Overlapping sequences were designed to prevent possible loss of epitopes. Domain 1 (sequence is the amino acid at position 24-145 in the human CD146 sequence of SEQ ID NO: 1, as shown in SEQ ID NO: 9) is cloned on pET30a (Novagen), and the expression is His6-S tag fusion His6-S-D1 protein. Structural domain 2 (sequence is the amino acid of position 128-248 in the human CD146 sequence of SEQ ID NO: 1, as shown in SEQ ID NO: 10), structural domain 3 (sequence is the human CD146 sequence of SEQ ID NO: 1 The amino acid of the pos...

Embodiment 3

[0095] Example 3: Detection of human CD146 using monoclonal antibodies AA1-AA5 and AA7

[0096] The anti-human CD146 mouse monoclonal antibody of the present invention can detect human CD146 protein at molecular, cell and tissue levels.

[0097] In whole-cell western blot experiments, both AA1-AA5 and AA7 could recognize CD146 protein in both reduced and non-reduced states. The specific experimental method is as follows: collect human melanoma cells A375 (ATCC) highly expressing CD146, wash the cells twice with pre-cooled PBS, centrifuge at 800 rpm at 4°C for 5 minutes, and use lysate (Tris-HCl 50mM pH 8.0 , NaCl 150mM, EDTA 1mM, NP-40 1%, Glycerol 10%, PMSF 100μg / ml) to lyse the cells, centrifuge at 12000g at 4°C for 15 minutes, collect the supernatant, add DTT (final concentration 100mM) (dithiothreitol ) and DTT-free loading buffer (5x loading buffer: 0.313M Tris-HCl, pH 6.8, 10% SDS, 0.05% bromophenol blue, 50% glycerol), cook at 100°C. Whole-cell proteins were separated...

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Abstract

The present invention relates to a group of anti-human CD146 molecules developed by using a biological technology and an established high sensitivity sandwich ELISA method for soluble CD146 detection. The present invention provides anti-human CD146 mouse monoclonal antibodies (AA1, AA2, AA3, AA4, AA5 and AA7), and a method for specially detecting soluble CD146 by using antibody combinations and sandwich ELISA, wherein the antibodies can specifically recognize human source CD146 at a molecular level, a cellular level and a tissue level, and can be divided into two classes based on recognition of different epitopes. With an antibody AA1 and anti-human CD146 mouse monoclonal antibody AA98 combined sandwich ELISA method, nanogram-scale soluble CD146 in per ml of the solution can be detected. The antibodies and the detection method can become effective detection or diagnosis tools and methods in basic researches or clinical applications, and can further provide good carriers for targeted therapies of CD146-related diseases.

Description

[0001] This application is a divisional application of application number 2008100572607. technical field [0002] The invention belongs to the fields of molecular biology and biotechnology. Specifically, the present invention relates to a group of mouse monoclonal antibodies against human CD146, which can specifically recognize human CD146 protein at biochemical, cell and tissue levels. The present invention also relates to a method for detecting soluble CD146 by sandwich ELISA. In this detection method, the capture antibody is the mouse monoclonal antibody involved in the present invention, and the detection antibody is the mouse monoclonal antibody involved in the invention patent ZL 991075862 labeled with biotin. Cloned antibody AA98. Background technique [0003] CD146, also known as MUC18, Mel-CAM / MCAM, is a cell adhesion molecule belonging to the immunoglobulin superfamily. CD146 has five extracellular immunoglobulin-like domains (V-V-C2-C2-C2) (Holness and Simmons 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K16/24C12N5/20C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/395A61K49/16A61P35/00G01N33/68C12R1/91
Inventor 阎锡蕴张莹郑超固杨东玲冯静卢迪
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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