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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

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the prevention or treatment is not proper, 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, tourism and rescue personnel.

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
Comparison scheme
Effect test

Embodiment 1

[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 shown in SEQ ID NO: 2, and the purity is greater than 99%.

[0041] 1.2 Obtaining of KLH-CRHC coupled peptide: Weigh 3 mg m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS) and dissolve it in 200 μL dimethylformamide (DMF), and take 1.5 mg KLH Dissolve in 0.5ml 0.05mol / L phosphate buffer (pH 7.0), add 70μL of prepared MBS solution to it, stir at room temperature for 30min; Buffer (pH 6.0) to elute, and collect 3.5mL of purified KLH / MBS; add 0.5mL of deionized water, 5mg of CRHC synthetic peptide dissolved in 100μL of DMF, quickly add 1mL of purified KLH / MBS, shake quickly and immediately wash with 2N Adjust the pH to 7.0-7.2 with NaOH, and place the mixed solution at 4°C overnight; finally add 3mL 0.1mol / LNH 4 HCO 3 , ly...

Embodiment 2

[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 shown in SEQ ID NO: 1, and the purity is greater than 99%.

[0066] 1.2 Obtaining of KLH-CRHN coupling peptide: Weigh 3 mg m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS) and dissolve it in 200 μL dimethylformamide (DMF), and take 1.5 mg KLH Dissolve in 0.5ml 0.05mol / L phosphate buffer (pH 7.0), add 70μL of prepared MBS solution to it, stir at room temperature for 30min; Buffer (pH 6.0) to elute, and collect 3.5mL of purified KLH / MBS; add 0.5mL of deionized water, 5mg of CRHN synthetic peptide dissolved in 100μL of DMF, quickly add 1mL of purified KLH / MBS, shake quickly and immediately wash with 2N Adjust the pH to 7.0-7.2 with NaOH, and place the mixed solution at 4°C overnight; finally add 3mL 0.1mol / LNH 4 HCO 3 ,...

Embodiment 3

[0088] Embodiment 3 The present invention detects the composition of the double antibody sandwich ELISA kit of CRH and optimal working concentration detection

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

[0090] The method for HRP-labeled monoclonal antibody C-D17 or N-D19 is as follows: First, weigh 10 mg of HRP dry powder, fully dissolve it in 5 mL double-distilled water, and add freshly prepared 0.2 mol / L NaIO 4 Solution 0.5mL, shake gently on a horizontal shaker at 4°C for 30min in the dark, add 10mg of monoclonal antibody C-D17 or N-D19 antibody that has been dialyzed into carbonate buffer, mix well, and fill the solution with molecular weight cut-off Dialyze it against 0.05mol / L pH9.6 carbonate buffer at 4°C in a 50kDa pre-treated dialysis bag, change the dialysate every 6h, and dialyze four times; after the dialysis, take out the dialysis bag Liquid in the bag, to which NaBH was added at a concentration of 5 mg / mL 4 100μL,...

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PUM

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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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