Preparation method for polypeptide marker Pep5 enzyme-linked immunosorbent assay (ELISA) kit

A technology for detection kits and markers, applied in the field of ELISA detection, can solve the problems of affecting the concentration and non-specificity of colorectal cancer, and achieve the effect of strong specific binding, beneficial to clinical detection, and simple and fast operation steps

Active Publication Date: 2014-09-10
BEIJING C & N INT SCI TECH +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Because endoderm-derived malignant tumors such as colorectal cancer, esophageal cancer, gastric cancer, liver cancer, and pancreatic cancer all have abnormal expression of CEA, and when CEA is released into the systemic circulation, it first enters the liver through the portal vein, and the liver has a degradative effect on CEA, which can affect Its concentration in peripheral blood, so CEA is not specific for the diagnosis of colorectal cancer

Method used

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  • Preparation method for polypeptide marker Pep5 enzyme-linked immunosorbent assay (ELISA) kit

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Embodiment 1

[0025] Example 1 Preparation method of polypeptide marker antigen monoclonal antibody

[0026] 1) BALB / C mice were immunized with synthetic polypeptide 5 coupled to the carrier protein KLH as an immune antigen; wherein, the full-length sequence of polypeptide 5 was: Asn-Leu-Gly-His-Gly-His-Lys-His-Glu- Arg-Asp-Gln-Gly-His-Gly-His-Gln.

[0027] 2) After 2 weeks, the tail blood titer was detected, and when it reached above 1:1000, spleen cells from BALB / C mice were fused with SP2 / 0 mouse myeloma cells under the action of PEG;

[0028] 3) Screen by ELISA method, clone and purify hybridoma cells that secrete positive antibodies by limited dilution method;

[0029] 4) 10 strains of hybridoma cells against synthetic peptide 5 were screened out, and one of them was unexpectedly found to be highly sensitive (1ng / well), the cell line was expanded, monoclonal antibody ascites was prepared and purified, and the titer of the monoclonal antibody was tested ( 1:200000), titer (0.0005μg / ml...

Embodiment 2

[0030] The research of embodiment 2 colon cancer ELISA detection kit chromogenic solution

[0031] 1) Hydrogen peroxide-TMB chromogenic solution: the volume concentration of hydrogen peroxide (H2O2) in substrate solution A is 0.3~2‰;

[0032] 2) Hydrogen peroxide-TMB color developing solution: the mass concentration of tetramethylbenzidine (TMB) in color developing solution B is 0.2~1‰;

[0033] 3) Hydrogen peroxide-TMB chromogenic solution: The buffer system in chromogenic solution A is acetate buffer system and citric acid buffer system.

Embodiment 3

[0034] The research of embodiment 3 colon cancer ELISA detection kit chromogenic solution

[0035] 1) Carbamide peroxide-TMB color developing solution: the volume concentration of carbamide peroxide in substrate solution A is 0.3~2‰;

[0036] 2) Carbamide peroxide-TMB color developing solution: the mass concentration of tetramethylbenzidine (TMB) in color developing solution B is 0.2~1‰;

[0037] 3) Carbamide peroxide-TMB chromogenic solution: DMF / DMSO ratio of organic phase in chromogenic solution B

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Abstract

The invention provides a polypeptide marker Pep5 enzyme-linked immunosorbent assay (ELISA) kit which contains a monoclonal antibody which is labeled by enzyme and secreted by monoclonal antibody hybridoma cell line with a China general microbiological culture collection center (CGMCC) No. 5269. The kit prepared by the monoclonal antibody secreted by the hybridoma cell line can be used for detecting colorectal adenocarcinoma, and is simple, convenient and rapid to operate, beneficial to high flux clinical detection and low in detection cost.

Description

technical field [0001] The invention relates to ELISA detection technology, in particular to a polypeptide marker Pep5 ELISA detection kit. Background technique [0002] Colon cancer is one of the common malignant tumors in humans, and its incidence rate accounts for the third of gastrointestinal tumors. In recent years, with the improvement of people's living standards and the change of diet structure, the incidence rate has been increasing year by year, seriously threatening human health. Commonly used diagnostic methods for colon cancer include fecal occult blood test, barium contrast, colonoscopy and serum tumor markers (tumor markers, TM). The sensitivity of the first two methods is low. Colonoscopy is an invasive test, and many patients Difficult to accept. TM detection plays an important role in cancer screening, diagnosis and follow-up after treatment, detection of recurrence and prognosis judgment. The prognosis of patients with early colorectal cancer is good, b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N21/78G01N21/31
Inventor 魏开华肖汉族孙云波傅海媛侯利平黄亚娟宋纯艳杨保安甄蓓徐淑芳张拓王东茂周晓明原剑
Owner BEIJING C & N INT SCI TECH
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