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Anti-DNP monoclonal antibody and preparation of hybridoma cell generating monoclonal antibody

A monoclonal antibody, myeloma cell technology, applied in the direction of anti-animal/human immunoglobulin, biological testing, biological material analysis, etc., to achieve the effect of fast response, high sensitivity and low cost

Active Publication Date: 2020-06-26
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

D-type natriuretic peptide (DNP), a new member of the natriuretic peptide family discovered in the past decade, appears in human plasma and atrial myocardium, and is increased in the plasma of patients with heart failure. The biological activity of DNP is still under study, but current research confirms that DNP may play an important role in the diagnosis and treatment of CHF

Method used

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  • Anti-DNP monoclonal antibody and preparation of hybridoma cell generating monoclonal antibody
  • Anti-DNP monoclonal antibody and preparation of hybridoma cell generating monoclonal antibody
  • Anti-DNP monoclonal antibody and preparation of hybridoma cell generating monoclonal antibody

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Experimental program
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Embodiment 1

[0032] The preparation of embodiment 1 hybridoma cell strain, comprises the following steps:

[0033] 1. Immunity

[0034] 1. Preparation of immunogen

[0035] (1) Coupling D-type natriuretic peptide (DNP) to carrier protein KLH by carbodiimide EDC method:

[0036] (a) Add 5 mg / 1250 μL of DNP polypeptide (equal molar number) to the EP tube of 5 mg / 500 μL of KLH solution, shake slowly in the shaker to form a mixture of polypeptide and carrier.

[0037] (b) Use the EDC solution that is currently prepared. After dissolving, quickly add 250 μL to the DNP-KLH mixture, place it in a shaker, and shake at a low speed of 100 rpm for 2 hours.

[0038] (c) Pretreatment of desalting column (10KD): centrifuge at 1000 rcf for 2 min, discard the bottom solution, and wash with 1×PBS for 3-4 times. Note: The column marks the level of the liquid in the tube after centrifugation.

[0039] (d) Desalting column: wash 3-4 times with PBS.

[0040] (e) Add the DNP-KLH coupled for 2 hours to a de...

Embodiment 2D

[0102] The preparation of embodiment 2DNP monoclonal antibody (mouse peritoneal induction method), comprises the following steps:

[0103] 1. Ascites preparation

[0104] (1) One week in advance, liquid paraffin was sterilized by filtration and injected into the peritoneal cavity of mice, 0.5mL / mouse, for backup;

[0105] (2) Tap the cells to suspend, then transfer to a 50mL sterile centrifuge tube; centrifuge at 1500rpm for 3min, discard the supernatant; add 10mL of 1640 medium for counting; centrifuge at 1500rpm for 3min, discard the supernatant; According to 1x10 6 Add 1640 culture medium at the amount of cells / mL.

[0106] (3) After 7 days, intraperitoneal injection of hybridoma cells, 1x10 6 pcs / only.

[0107] (4) Generally, after 7 days, ascites in mice is formed, and a 20G sterile needle is used to puncture the abdominal cavity of the mice, and the ascites is collected into a centrifuge tube;

[0108] (5) Centrifuge the ascites at 5000rpm / min for 3min, collect the ...

Embodiment 3

[0119] Embodiment 3 The method for monoclonal antibody specific identification

[0120] 1. The titer of the purified DNP monoclonal antibody was detected by ELISA, and the results were as follows: Figure 5 As shown, the titer of the purified DNP monoclonal antibody detected by ELISA reached 1:500,000.

[0121] 2. Western blot detection of the specificity of the purified DNP monoclonal antibody, from Figure 6 It can be seen that two clear bands appear between the positions of 35kDa-45kDa in lane 2. The upper chain is the heavy chain and the lower chain is the light chain.

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Abstract

The invention provides an anti-DNP monoclonal antibody and preparation of a hybridoma cell generating the monoclonal antibody, relates to the technical field of detection and particularly relates to ahybridoma cell line, a D-type natriuretic peptide monoclonal antibody based on the hybridoma cell line and a detection kit. The hybridoma cell line has a preservation number of CCTCC NO:C2019291. TheD-type natriuretic peptide monoclonal antibody based on the hybridoma cell line has a relatively high titer, can be used for detecting D-type natriuretic peptide by enzyme linked immunosorbent assay(ELISA) and has the characteristics of convenience in sample operation, low cost, rapid reaction, high sensitivity, strong specificity and the like.

Description

technical field [0001] The invention relates to the technical field of small molecule immunochemical analysis and detection, in particular to the preparation of an anti-DNP monoclonal antibody and a hybridoma cell line thereof. Background technique [0002] As endocrine hormones, the natriuretic peptide family mainly includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (C-type natriuretic peptide) and D-type natriuretic peptide (dendroaspis Natriuretic peptide (DNP), plays an important role in regulating blood volume, blood vessel elasticity, blood pressure, etc. Other natriuretic peptides include tree cobra natriuretic peptide, urodilatin, intestinal epithelial-derived peptide, guanylin and uroguanylin, etc., which have similar cardiovascular effects and participate in water balance and blood pressure regulation. Studies on natriuretic peptides (especially brain natriuretic peptides) have shown that plasma natriuretic pepti...

Claims

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Application Information

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IPC IPC(8): C07K16/26C07K17/10C07K14/58G01N33/74G01N33/577
CPCC07K16/26C07K17/10C07K14/582G01N33/74G01N33/577G01N2333/58
Inventor 张茂祥海那尔·乌拉孜巴依裴毓章文
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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