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Monoclonal antibody resisting human protein RN181, as well as hybridoma cell line 3953 and kit

A technology of hybridoma cell line and monoclonal antibody, which is applied in the direction of anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., and can solve the problem of expression decline

Inactive Publication Date: 2013-10-23
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our study found that the expression of human RN181 protein decreased in primary liver cancer cells

Method used

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  • Monoclonal antibody resisting human protein RN181, as well as hybridoma cell line 3953 and kit
  • Monoclonal antibody resisting human protein RN181, as well as hybridoma cell line 3953 and kit
  • Monoclonal antibody resisting human protein RN181, as well as hybridoma cell line 3953 and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Preparation of anti-RN181 monoclonal antibody

[0027] 1. Inoculate animals

[0028] The hybridoma cells with the preservation number CGMCC3953 in liquid nitrogen were recovered, cultured with 1640 medium containing 10% fetal bovine serum, and the hybridoma cells in the logarithmic growth phase were collected, and the hybridoma cells that had been sensitized with paraffin oil c mice were inoculated intraperitoneally with about 10 hybridoma cells in the logarithmic growth phase 6 indivual. After 10 days, ascitic fluid was collected and centrifuged at 12,000 r / min for 10 min to obtain supernatant, which was filtered with a 0.22 μm filter membrane. Purify the filtered ascites supernatant by Protein-G affinity chromatography: first equilibrate the column with 0.01mol / L PBS for 3 column volumes, dilute the supernatant with 0.01mol / L PBS 1:10, load the sample, 0.01mol / L After washing impurities with PBS, elute with 0.1mol / L glycine hydrochloride buffer (pH2.....

Embodiment 2

[0033] Example 2: Determination of the concentration of anti-RN181 monoclonal antibody

[0034] The anti-RN181 monoclonal antibody prepared by CGMCC3953 hybridoma cells was diluted according to the concentration of 4, 3, 2, 1, 0.5ug / ml, and the paraffin sections of liver cancer tissues and adjacent normal tissues of different pathological grades were tested by immunohistochemistry. The results found that : When the concentration of the monoclonal antibody is 2 μg / ml, the result is specific and clear, and can be well graded for specimens of different pathological grades, see Figure 2-5 .

Embodiment 3

[0035] Example 3: Composition of Immunohistochemical Diagnosis Kit for Primary Liver Cancer (120 pieces in size) and usage method

[0036] 1. The composition of the kit

[0037] 1) 3% H 2 o 2 6ml

[0038] 2) 2 μg / ml monoclonal antibody against human protein RN181 (prepared by CGMCC3953 hybridoma cells) 6ml

[0039] Biotin-labeled goat anti-mouse IgG antibody, streptomycin-labeled horseradish peroxidase (HRP), and DAB chromogenic buffer were purchased from Dako REAL TM Envision TM detection system, in which the volume of each component is 6ml.

[0040] 2. How to use the kit

[0041] The kits are used in the same way as follows:

[0042] 1) 3% H 2 o 2 Drop one drop per piece, incubate at room temperature for 10 minutes;

[0043] 2) Wash with 0.01M PBS (PH7.4) for 5min×3 times;

[0044] 3) Incubate with 2ug / ml monoclonal antibody against human protein RN181 for 1 hour at room temperature;

[0045] 4) Wash with 0.01M PBS (PH7.4) for 5min×3 times;

[0046] 5) One drop ...

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Abstract

The invention discloses a monoclonal antibody resisting human protein RN181, as well as a hybridoma cell line 3953 and a kit. The hybridoma cell line is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.3953. The monoclonal antibody resisting the human protein RN181 is excreted from the hybridoma cell line. A preferable implementation scheme is that: the monoclonal antibody resisting the human protein RN181 is prepared from the hybridoma cell line by a method of inoculating mouse peritoneal cavity and extracting ascites. The monoclonal antibody resisting human protein RN181 and a primary liver cancer immunohistochemistric diagnosis kit prepared by utilizing the monoclonal antibody can be used for distinguishing primary liver cancer tissues and non-tumor normal tissues, and grading the primary liver cancer tissues, so that diagnosis of the primary liver cancer is realized.

Description

technical field [0001] The invention relates to a monoclonal antibody against human protein RN181 and a kit thereof. Background technique [0002] Liver cancer is one of the most common deadly cancers in the world and ranks fourth in the incidence of various cancers. Most patients with liver cancer die within a year of diagnosis and have died in the past 20 years rate did not drop. Part of the reason is that the existing diagnostic methods are not very effective, and the discovery and diagnosis of liver cancer are relatively difficult, especially when primary liver cancer is mostly discovered at an advanced stage. Therefore, early detection of primary liver cancer can effectively improve the prognosis of this disease. However, the existing serum markers of liver cancer such as alpha-fetoprotein AFP and DCP lack sufficient detection specificity and sensitivity, so more specific detection targets need to be selected. [0003] Human protein RN181 is a newly discovered pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/574
Inventor 李明王穗海黄湘李月董慧敏
Owner SOUTHERN MEDICAL UNIVERSITY