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ELISA kit for detecting human serum dlk1 protein and application thereof

A technology of kits and human serum, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of not being able to replace AFP, achieve reliable reference significance, improve sensitivity, high sensitivity and specificity

Active Publication Date: 2020-08-18
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, liver cancer markers have certain auxiliary significance in the diagnosis of primary liver cancer, especially AFP-negative cases, but they still cannot replace the status of AFP in the diagnosis of liver cancer

Method used

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  • ELISA kit for detecting human serum dlk1 protein and application thereof
  • ELISA kit for detecting human serum dlk1 protein and application thereof
  • ELISA kit for detecting human serum dlk1 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1. Establishment, screening and identification of a hybridoma cell line stably secreting anti-DLK1 monoclonal antibody

[0072] Step 1. Preparation of recombinant human DLK1 protein (immune antigen)

[0073] Using PCR technology, the BGH sequence was deleted from the 5' end of the self-built plasmid vector pcDNA3.1B-DLK1 (pcDNA3.1B sequence, and the T7 promoter, XhoⅠ, NotⅠ, EcoRⅤ and EcoRI restriction sites were added, and 3 flags were introduced. tag, the transformed vector is more conveniently applied to gene expression) to amplify the gene fragment encoding the human DLK1 secretory region sequence (DLK1solubleregion, 24-303aa), add the kappa signal peptide sequence to the front, and add the His 6tag tag sequence to the rear to construct The insert fragment kappa sp-DLK1soluble reg-His 6tag was correctly identified by sequence determination, and after being treated with restriction endonucleases, it was cloned into the expression vector pCPC(KpnI / NotI) (purc...

Embodiment 2

[0084] Example 2. In vitro preparation and purification of anti-DLK1 monoclonal antibody

[0085] The established 019 / 020 hybridoma cells were expanded and cultured in serum-free medium until the cell concentration reached 10 5 Stop adding liquid when cells / ml is above, and continue culturing until the cell culture medium turns yellow. The culture medium was collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane and stored at 4°C or -20°C for future use or directly used for the separation and purification of monoclonal antibodies in the next step.

[0086] Step 6. The hybridoma cells are amplified and cultured, the culture medium is collected, and the anti-human DLK1 monoclonal antibody is separated and purified by affinity chromatography.

[0087] In this example, the separation and purification of anti-DLK1 monoclonal antibody (019 / 020) adopts affinity chromatography. The purification steps are as follows: the...

Embodiment 3

[0088] Example 3, Preparation of human serum DLK1 protein enzyme-linked detection kit

[0089] 1. Solid phase carrier coated with antigen

[0090] The solid phase carrier can be selected from polystyrene or polyvinyl chloride, in the form of a 96-well plate or a microtiter plate.

[0091] Coat 100 ul / well (0.25 ug / ml) of the anti-human DLK1 monoclonal antibody (019 / 020) prepared in Example 2 into polystyrene or polyvinyl chloride reaction wells.

[0092] 2. Solution preparation

[0093] 2.1 Preparation of coating buffer

[0094] The coating solution uses 1*PBS. The coating solution is prepared by adjusting the pH to 7.3 with sodium hydroxide and then adjusting the volume to 1000ml with 8.18g sodium chloride, 0.2g potassium chloride, 3.58g 12 hydrate disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate .

[0095] 2.2 Preparation of enzyme conjugates (enzyme-labeled antigen)

[0096] There are two types of enzyme-labeled antigens, including:

[0097] Enzyme...

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Abstract

The invention discloses an ELISA kit for detecting human serum DLK1 protein and its application. The antigen of the ELISA kit of the present invention adopts recombinant human DLK1 antigen. The detection method of the present invention has high sensitivity and specificity, and can quickly detect the expression of DLK1 and liver cancer and hepatoblastoma. The ELISA kit of the present invention has broad application in the prevention and diagnosis of liver cancer and hepatoblastoma Application prospects.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an ELISA kit for detecting human serum DLK1 protein and its application. Background technique [0002] Hepatocellular carcinoma (HCC) has a high incidence in my country and is the fifth most common malignant tumor in the world. It lacks early preventive diagnosis and treatment measures, high degree of malignancy, rapid progression, poor prognosis, little curative effect of chemotherapy, surgery Treatment is difficult to cure. In my country, the morbidity and mortality of liver cancer ranks second among cancers. The occurrence of liver cancer is a multi-stage, multi-factor cumulative effect, as well as the result of the involvement and mutation of multiple genes, and its specific etiology and pathogenesis have not yet been clarified. The known possible external causes of primary liver cancer include viral hepatitis, liver cirrhosis, aflatoxin, drinking water pollution...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/574G01N33/577G01N33/535G01N33/543
CPCG01N33/6893G01N33/57484G01N33/57438G01N33/574G01N33/577G01N33/535G01N33/543G01N2333/4746G01N2800/085
Inventor 韩泽广徐晓翟杨杨
Owner SHANGHAI JIAOTONG UNIV
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