Hybridoma cell strain, anti-CRH antibody C-D17 based on hybridoma cell strain, and application of anti-CRH antibody C-D17 to detection

A hybridoma cell line and hybridoma cell technology are applied in the field of anti-adrenocorticotropin-releasing hormone monoclonal antibodies, which can solve problems such as health threats to rescue workers in plateau work, and achieve high sensitivity, low material consumption, and good specificity. Effect

Inactive Publication Date: 2017-08-18
陈锋 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If the prevention or treatment is not proper, acute mountain sickness will further develop into high altitude cerebral

Method used

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  • Hybridoma cell strain, anti-CRH antibody C-D17 based on hybridoma cell strain, and application of anti-CRH antibody C-D17 to detection
  • Hybridoma cell strain, anti-CRH antibody C-D17 based on hybridoma cell strain, and application of anti-CRH antibody C-D17 to detection
  • Hybridoma cell strain, anti-CRH antibody C-D17 based on hybridoma cell strain, and application of anti-CRH antibody C-D17 to detection

Examples

Experimental program
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Example Embodiment

[0038] Example 1 Preparation and purification of the monoclonal antibody C-D17 of the present invention.

[0039] 1. Preparation of immunogen

[0040] 1.1 Preparation of CRHC synthetic peptide: Synthesize the C-terminal epitope of CRH, the sequence is the sequence shown in SEQ ID NO: 2, the purity is greater than 99%.

[0041] 1.2 Obtaining KLH-CRHC coupling peptide: Weigh 3 mg of meta-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS) and dissolve in 200 μL dimethylformamide (DMF), take 1.5 mg KLH Dissolve in 0.5ml 0.05mol / L phosphate buffer (pH 7.0), add 70μL of the prepared MBS solution to it, and stir for 30min at room temperature; the KLH / MBS mixed solution is passed through the PD-10 desalting column, 0.05mol / L phosphate Buffer (pH 6.0) eluted and collected 3.5mL purified KLH / MBS; add 0.5mL deionized water, 5mg CRHC synthetic peptide dissolved in 100μL DMF, quickly add 1mL purified KLH / MBS, shake quickly and immediately use 2N Adjust the pH to 7.0-7.2 with NaOH, and plac...

Example Embodiment

[0063] Example 2 Preparation and purification of the monoclonal antibody N-D19 of the present invention.

[0064] 1. Preparation of immunogen

[0065] 1.1 Preparation of CRHN synthetic peptide: Synthesize the N-terminal epitope of CRH, the sequence is the sequence shown in SEQ ID NO:1, and the purity is greater than 99%.

[0066] 1.2 Obtaining KLH-CRHN coupling peptide: Weigh 3mg of meta-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS) and dissolve in 200μL dimethylformamide (DMF), take 1.5mg KLH Dissolve in 0.5ml 0.05mol / L phosphate buffer (pH 7.0), add 70μL of the prepared MBS solution to it, and stir for 30min at room temperature; the KLH / MBS mixed solution is passed through the PD-10 desalting column, 0.05mol / L phosphate Buffer (pH 6.0) eluted and collected 3.5mL purified KLH / MBS; add 0.5mL deionized water, 5mg CRHN synthetic peptide dissolved in 100μL DMF, quickly add 1mL purified KLH / MBS, shake quickly and immediately use 2N Adjust the pH to 7.0-7.2 with NaOH, and place...

Example Embodiment

[0088] Example 3 The composition and optimal working concentration of the double antibody sandwich ELISA kit for detecting CRH of the present invention

[0089] 1. Preparation of HRP-labeled anti-human CRH monoclonal antibody C-D17 or N-D19

[0090] The method of HRP-labeled monoclonal antibody C-D17 or N-D19 is as follows: First, weigh 10 mg of dry HRP powder, dissolve it in 5 mL of double-distilled water, and add freshly prepared 0.2mol / L NaIO 4 0.5 mL of the solution, shake gently on a horizontal shaker at 4°C for 30 minutes, add 10 mg of the monoclonal antibody C-D17 or N-D19 antibody that has been dialyzed into carbonate buffer. After mixing, fill the solution with molecular weight cutoff In a 50kDa pre-treated dialysis bag, dialyze it against a 0.05mol / L carbonate buffer of pH 9.6 at 4°C, change the dialysate every 6h, and dialyze four times; after the dialysis, remove the dialysis Liquid in the bag, add NaBH with a concentration of 5mg / mL to it 4 100μL, 4°C, let stand for 4...

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Abstract

The invention relates to the field of biotechnology, and discloses an anti-CRH antibody C-D17 of a hybridoma cell strain, and application of the anti-CRH antibody C-D17 to detection. The hybridoma cell strain is preserved in China Center for Type Culture Collection on May 31, 2016 with the preservation number of CCTCC NO. C2015215, and is classified and named hybridoma cell C-D17. The monoclonal antibody C-D17 based on anti-cortin releasing hormone (CRH) generated by the hybridoma cell strain, and application of the monoclonal antibody to a kit for detecting acute altitude sickness are also provided. The anti-CRH monoclonal antibody C-D17 can recognize the C-end epitope of CRH, and has high specificity and high titer which can reach 1:4x10<6>. The double-antibody sandwich ELISA kit for detecting CRH has high specificity and sensitivity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a hybridoma cell strain, a monoclonal antibody against corticosteroid-releasing hormone (CRH), and a kit thereof. Background technique [0002] According to the report of the World Health Organization in 1996, the total number of people living above 2500m in the world is 140 million, and about 40 million people come to high mountains and plateau areas every year. And frequently-occurring diseases, various pathological reactions produced by the human body after being exposed to hypoxic environment rapidly. If not properly prevented or treated, acute mountain sickness will further develop into high altitude cerebral edema and pulmonary edema, which will pose a great threat to the health of altitude work, travel and rescue personnel. Therefore, how to realize the preventive diagnosis of people stationed in high altitude and early diagnosis of susceptible people i...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/26G01N33/74G01N33/577
CPCC07K16/26G01N33/74G01N2800/40
Inventor 陈锋刘英富李光宗霍景瑞侯世科樊毫军
Owner 陈锋
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