ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen

A detection kit and marker technology, applied in the biological field, can solve the problems of high price and unfavorable promotion, and achieve the effects of strong specificity, simple and quick operation steps, and beneficial to clinical detection.

Active Publication Date: 2012-07-04
BEIJING C & N INT SCI TECH +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] Serum peptidomics, which has emerged in recent years, has been successfully applied to the diagnosis of several cancers, such as breast cancer, ovarian cancer, prostate cancer, etc., mainly using surface-enhanced laser desorption ionization (SELDI) technology and immunomagnetic bead technology , but the system has the disadvantages of being expensive and unfavorable for popularization

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  • ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting polypeptide marker antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation method of polypeptide marker antigen monoclonal antibody

[0036] 1) BALB / C mice were immunized with synthetic polypeptide 5 coupled to the carrier protein KLH as an immunogen; its full-length sequence is: Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp -Gln-Gly-His-Gly-His-Gln.

[0037] 2) After 2 weeks, the tail blood titer was detected, and when it reached above 1:1000, splenocytes from BALB / C mice were fused with SP2 / 0 mouse myeloma cells under the action of PEG;

[0038] 3) Screening by ELISA method, cloning and purifying hybridoma cells positive for secreting antibodies by limiting dilution method;

[0039]4) 10 strains of hybridoma cells targeting 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 detected ( 1:200000), titer (0.0005 μg / ml) and subtype ident...

Embodiment 2

[0040] The assembly of the ELISA detection kit of embodiment 2 polypeptide marker antigen

[0041] 1) 1.25 μg / ml horseradish peroxidase-labeled specific monoclonal antibody against polypeptide 5 of Example 1;

[0042] 2) Coating solution: 0.1M carbonate buffer solution with pH9.6;

[0043] 3) 5% skimmed milk powder: 5g / 100ml skimmed milk powder;

[0044] 4) TMB chromogenic solution: purchased from Beijing Kangwei Century Biotechnology Company;

[0045] 5) 2M sulfuric acid;

[0046] 6) 96-well enzyme-linked plate.

Embodiment 3

[0047] The ELISA detection method of embodiment 3 polypeptide marker antigen

[0048] Samples: 160 serums from clinically diagnosed liver cancer and 160 normal serums.

[0049] Detection process:

[0050] 1) Take 5 μL of clinical serum, add 95 μL of coating solution, place in a 96-well plate, and place overnight at 4°C; at the same time, add 100 μL of coating solution as a negative control;

[0051] 2) Discard the coating solution, wash 6 times with 200 μL of 0.01M, pH7.4 PBST buffer, and pat dry;

[0052] 3) Add 300 μL of 5% skimmed milk powder, and stand at 37°C for 2 hours to block;

[0053] 4) Discard the blocking solution, wash 6 times with 200 μL of 0.01M, pH7.4 PBST buffer, and pat dry;

[0054] 5) Add 100 μL of HRP-labeled polypeptide antibody and let stand at 37°C for 1 hour;

[0055] 6) Discard the antibody solution, wash 6 times with 200 μL of 0.01M, pH7.4 PBST buffer, and pat dry.

[0056] 7) Add 100 μL TMB color developing solution, and incubate at 37°C in th...

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Abstract

The invention discloses an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting a polypeptide marker antigen. The kit contains a monoclonal antibody of the polypeptide marker antigen; and the monoclonal antibody is secreted by a hybridoma cell strain CGMCC No.5269 of a polypeptide marker monoclonal antibody capable of resisting primary liver cancer. The invention also provides a method for detecting the polypeptide marker antigen. The monoclonal antibody provided by the invention has the advantage of strong specificity aiming at the polypeptide marker antigen; and the method for detecting the polypeptide marker antigen is simple, convenient and rapid in operation step and beneficial to clinical detection, and can be used for carrying out high-flux and low-cost detection of an enzyme linked detector.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an ELISA kit for detecting polypeptide marker antigens. Background technique [0002] With the maturity and development of liver cancer surgery, the treatment methods and means of liver cancer are becoming more and more abundant, and are no longer limited to a single surgical treatment. Liver resection and liver transplantation are still the main treatment methods for patients with liver cancer, but minimally invasive treatment, percutaneous hepatic arterial chemoembolization and other technical methods, as an important supplement to surgical treatment, have also been widely used. Surgical treatment opportunities, reducing or reducing tumor recurrence and metastasis have all played a certain role, prolonging the survival time of patients. Even so, HCC patients have insidious onset and no obvious symptoms, and most patients have lost the chance of radical surgery when they see a doct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCC07K16/18G01N33/577G01N33/57438C12Q1/00C07K16/303
Inventor 魏开华周晓明付海媛原剑杨保安侯利平孙云波黄亚娟郑俊杰甄蓓张拓王东茂
Owner BEIJING C & N INT SCI TECH
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