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Monoclone antibody of ARD1 and application thereof

A monoclonal antibody and antibody technology, applied in the field of immunology, can solve the problems that cannot be used to detect ARD1

Inactive Publication Date: 2009-08-26
BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Abnova (Taiwan, China) provides a commercial anti-ARD1 monoclonal antibody (M01; 4B7-H4), but it cannot be used to detect ARD1 in mammalian cells or in immunohistochemistry

Method used

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  • Monoclone antibody of ARD1 and application thereof
  • Monoclone antibody of ARD1 and application thereof
  • Monoclone antibody of ARD1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 Preparation of anti-human ARD1 monoclonal antibody

[0060] 1. Preparation of ARD1 antigen

[0061] The 5' end sense primer 5'-ATTGGATCCTCGCCACCATGAACATCCGCAATGCG-3' (SEQ ID No.1) and the 3' end antisense primer 5'-AATGAATTCTAGGAGG CTGAGTCGGAG-3' (SEQ ID No.2) (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.), and BamH I and EcoR I restriction sites were introduced at both ends of the primers. The total RNA of human colon cancer cell LoVo (ATCC number: CCL-229) was extracted with Trizol (purchased from Invitrogen Company), reverse-transcribed into cDNA, and the ARD1 cDNA fragment was amplified by PCR using the above primers, and the product length was 712bp.

[0062] The PCR product and the pGEX-5X-3 vector (purchased from Amersham Biosciences) were digested with BamH I and EcoR I respectively and ligated. The ligated product was transformed into E. coli BL21 strain and a single clone was picked for enzyme digestion identification , a...

Embodiment 2

[0066] Embodiment 2 identifies the purity of 14D4 antibody or 10C12 antibody

[0067] The subtypes of 14D4 antibody or 10C12 antibody were identified by conventional ELISA method and confirmed to be IgG2a and IgG1 respectively. Therefore, Pro.A Sepharose-4B column was selected for purification, and its purity was identified by 12% SDS-PAGE electrophoresis. see results figure 1 , showing that the 14D4 antibody or 10C12 antibody has high purity and no bands.

Embodiment 3

[0068] Example 3 Determination of the titer of 14D4 antibody

[0069] The purified 14D4 antibody was dialyzed with PBS buffer, and the concentration of the antibody was measured at 280 nm by an ultraviolet spectrophotometer.

[0070] 1. Add 14D4 antibody at concentrations of 0, 0.18, 0.37, 0.625, 1.25, 2.5, and 5 μg / ml to the microtiter plate coated with GST-ARD1 protein. And the same concentration of 14D4 antibody was added to the GST protein-coated microtiter plate as a negative control. Incubate for 1 hour at room temperature.

[0071] 2. Wash the reaction wells 3 times with 0.05% Tween-20 / PBS, and 2 times with PBS.

[0072] 3. Add horseradish peroxidase-labeled goat anti-mouse IgG antibody and incubate at room temperature for 1 hour.

[0073] 4. Wash the reaction wells 3 times with 0.05% Tween-20 / PBS, and 2 times with PBS.

[0074] 5. Add horseradish peroxidase substrate OPD to the reaction well, after developing color at room temperature for 30 minutes, add stop solut...

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Abstract

The invention relates to a monoclone antibody of ARD1 and application thereof, in particular to the monoclone antibody aiming at ARD1 protein and excreted by a hybridoma of which the preserving number is CGMCC NO.2354 or CGMCC NO.2355, and a hybridoma cell line for excreting the antibody. The monoclone antibody has the characteristics of strong specificity and high sensitivity, and can be applied to immunological detections of immunohistochemistry, immunocytochemistry, immunoprecipitation, immunoblotting, enzyme-linked immunosorbent assay, and the like, thus the monoclone antibody can detect the ARD1 expression levels of tumors such as colon cancer, gastric cancer, esophageal cancer, and the like and various tumor cell lines, and is further applied to assistant diagnosis and correlative experimental investigations on the various tumors, and instructing clinical treatments.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a monoclonal antibody against ARD1 and a preparation method thereof, a hybridoma cell line secreting the antibody, and applications of the monoclonal antibody or its biologically active fragments. The invention also relates to a kit for detecting the ARD1 level of a sample by using the monoclonal antibody and an antitumor drug. Background technique [0002] The incidence of malignant tumors has continued to rise globally in recent years. Since the onset of malignant tumors is mostly insidious and there are no obvious symptoms in the early stage, the patients are often in the late stage when they see a doctor, and many patients with similar performances in clinical traditional pathology and TNM staging have great differences in prognosis, reflecting the occurrence and development of malignant tumors. complexity. At present, there is still a lack of ideal tumor markers that can be used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/18G01N33/577A61K39/395A61P35/00
Inventor 寿成超任婷婷姜北海吴健
Owner BEIJING CANCER HOSPITAL PEKING UNIV CANCER HOSPITAL
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