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Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146

An antibody and human body 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: 2008-08-20
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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  • Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146
  • Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146
  • Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation and identification of monoclonal antibodies AA1-AA5 and AA7.

[0055] 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. Briefly described as follows: Isolate natural CD146 protein (its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2) from human umbilical cord vein endothelial cells, according to (Yan, Lin et al.2003 ) to purify the monoclonal antibody AA98 antigen, and use it as an immunogen to immunize BALB / C mice (Beijing Experimental Animal Center), inject 100 μg protein / mouse intraperitoneally, once every two weeks, for 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 immunization, the spleen was taken, and the splenocytes were suspended ...

Embodiment 2

[0079] Example 2: Epitope identification of monoclonal antibodies AA1-AA5 and AA7.

[0080] 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.

[0081] As shown in Figure 2, domains 1-5 respectively represent the five domains V-V-C2-C2-C2 of CD146 from the extracellular to the transmembrane region. 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 what is expressed is His6-Stag 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...

Embodiment 3

[0092] Example 3: Detection of human CD146 using monoclonal antibodies AA1-AA5 and AA7

[0093] The anti-human CD146 mouse monoclonal antibody of the present invention can detect human CD146 protein at molecular, cell and tissue levels.

[0094] 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 pH8. 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) (dithiothreose) Alcohol) and DTT-free loading buffer (5x loading buffer: 0.313M Tris-HCl, pH6.8, 10% SDS, 0.05% bromophenol blue, 50% glycerol), cook at 100°C. Whole-cell proteins were sepa...

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Abstract

The invention relates to a group of anti-human CD146 molecules which are developed by utilizing the biological technology and an established high-sensitive sandwich ELISA method for detecting the solubility CD146. The invention includes anti-human CD146 mouse monoclonal antibodies of AA1, AA2, AA3, AA4, AA5 and AA7, and the method of utilizing the combination of the antibodies and the sandwich ELISA specificity for detecting the solubility CD146. The group of antibodies can identify the human source CD146 on the molecular, the cellular and the tissue levels, which can be divided into two categories based on the identified different epitopes. The sandwich ELISA method which is combined by the antibody AA1 and another strain of anti-human CD146 mouse monoclonal antibody AA98 can detect the solubility CD146 at each milliliter nano-gram level. The antibodies and the detection means can become the effective detection or diagnosis tools and method in the basic research or the clinical application and provide good carriers for the targeted therapy of the CD146-related diseases at the same time.

Description

technical field [0001] 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 [0002] 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 1994) and is highly glycosylated, mediating calcium ion-independent intercellular adhesion. ...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K16/28C12N5/18C12N15/13C12N15/63A61K39/395A61P35/00G01N33/577
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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