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Hybridoma cells capable of secreting anti-h-fabp monoclonal antibody, monoclonal antibody, preparation method and application thereof

A monoclonal antibody and hybridoma cell technology, applied in the biological field, can solve the problems of unstable performance, long time-consuming ELISA detection, low concentration of H-FAPB, etc., and achieve high specificity, valuable treatment time, and high affinity.

Active Publication Date: 2019-12-27
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the concentration of H-FAPB in the blood of normal people is very low (generally less than <10ng / ml), although the concentration will increase after myocardial injury, it still cannot be detected by conventional chemical quantitative methods
Traditional enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), colloidal gold immunochromatography, etc. cannot accurately detect samples with very small amounts of H-FAPB content
Moreover, there are some defects in the above detection methods, which limit their large-scale application in clinical diagnosis.
For example, ELISA detection takes a long time, the operation steps are cumbersome, and the repeatability is poor
RIA requires specific instruments, and the operator will be exposed to radioactive materials, which will damage the operator's body. At the same time, the disposal of radioactive materials after use is also a serious problem.
At present, although there are many immunochromatographic products, there are generally problems such as low specificity and unstable performance.

Method used

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  • Hybridoma cells capable of secreting anti-h-fabp monoclonal antibody, monoclonal antibody, preparation method and application thereof
  • Hybridoma cells capable of secreting anti-h-fabp monoclonal antibody, monoclonal antibody, preparation method and application thereof
  • Hybridoma cells capable of secreting anti-h-fabp monoclonal antibody, monoclonal antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Establishment of hybridoma cell line and preparation of anti-H-FABP monoclonal antibody.

[0055] 1. Antigen immunity.

[0056] Mix the recombinant heart-type fatty acid binding protein (H-FABP) antigen (1.2mg / mL, produced by Shenzhen Feipeng Biological Co., Ltd., product name: K1-FABP) with complete Freund's adjuvant (SIGMA, F5881) in equal volumes , to obtain an oily emulsion. The emulsion was subcutaneously administered to the back site of BALB / c mice (Guangdong Provincial Medical Experimental Animal Center: No. 119, Poyang Road, Huangqi, Nanhai, Foshan City, Guangdong Province, 6-week-old female, 5) with a dose of 0.2 ml each. Fourteen days after the first immunization, intraperitoneal booster immunization (antigen mixed with incomplete Freund's adjuvant (SIGMA, F5506) in equal volumes), after booster immunization to four injections, tail blood was collected for titer detection, and the titer reached the fusion requirement.

[0057] Three days before the fusion, t...

Embodiment 2

[0078] Preparation of H-FABP detection test paper.

[0079] 1. Preparation of nitrocellulose membrane.

[0080] Coating buffer preparation: 0.01M PBS buffer solution containing 6% methanol with a pH of 7.2 is the coating buffer, filtered through a 0.22μ membrane, placed at 4°C for later use, and valid for one week. 1000mL 0.01M pH 7.2 PBS buffer solution of 6% methanol Recipe: NaCL 8g, KCL 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60mL, distilled deionized water to 1000mL.

[0081] Preparation of nitrocellulose membrane: Dilute the anti-heart fatty acid binding protein (H-FABP) monoclonal antibody 1F38 to 1 mg / mL-5 mg / mL with coating buffer, adjust the machine, and draw the line as T1 line, and the T1 line is For the detection line, the T1 line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad. Dilute the goat anti-mouse IgG antibody (manufactured by Shenzhen Feipeng Biological Co., Ltd., product name: go...

Embodiment 3

[0106] H-FABP detection kit

[0107] 1. H-FABP detection kit includes:

[0108] ①A pack of test strips (10 strips / pack)

[0109] ②One bottle of sample diluent (10mL / bottle)

[0110] Preparation of relevant solutions.

[0111] Sample diluent: The sample diluent is 8% NaCl solution. Preparation method: 80gNaCl, add distilled water to make up to 1000mL.

[0112] 2. Detection of H-FABP content by colloidal gold method

[0113] (1) Directly put 20 μL of collected venous whole blood into a plastic test tube containing 180 μL of diluent, mix well, take 120 μL of dissolved sample and add it to the sample hole of the test paper card, wait for 15 minutes and then observe the result.

[0114] (2) Judgment of results: When the test strip has a purple-red quality control line visible to the naked eye, there is no visible purple-red detection line (see Figure 4 ), the result was negative. When the test strip shows a purple-red quality control line visible to the naked eye, the first t...

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Abstract

The invention relates to a hybridoma capable of secreting an anti-H-FABP (Heart-type Fatty Acid-Binding Protein) monoclonal antibody, a monoclonal antibody, and a preparation method and application thereof. The hybridoma is divided into two strains which are reserved in the Wuhan University Center for Collection in the Wuchang Luojia Hill of Wuhan City, Hubei Province with the preservation number of CCTCC No: C2016220 and CCTCC No: C2016219. The secretion yield of the hybridoma is high, and the anti-H-FABP monoclonal antibody obtained by secretion has the advantages of high affinity, high specificity and the like and can be widely applied to the detection field of cardiovascular and cerebrovascular disease diagnosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell capable of secreting an anti-H-FABP monoclonal antibody, a monoclonal antibody and a preparation method and application thereof. Background technique [0002] Fatty acid binding protein (FABP) is a group of multi-source small molecular intracellular proteins with a molecular mass of about 12kDs ~ 15kDs, widely present in the myocardium, small intestine, liver, adipose tissue, brain, epidermis and other tissue cells of mammals. There are 9 different subtypes of FABP that have been discovered so far, the common ones are cardiac fatty acid binding protein (H-FABP), liver fatty acid binding protein (L-FABP), kidney fatty acid binding protein (K-FABP), bone Sarcofatty acid binding protein (S-FABP) and so on. [0003] Among them, H-FABP exists in a large amount in myocardial tissue, accounting for about 5% to 15% of all soluble proteins in the heart. It belongs to the fam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18C12N15/08G01N33/68G01N33/577C12R1/91
CPCC07K16/18C12N15/02G01N33/577G01N33/68G01N2333/46
Inventor 钟少霞刘德荣韩日才杨耿周孙康成
Owner FAPON BIOTECH INC
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