Anti-human apoa1 monoclonal antibody and its preparation method and application

A monoclonal antibody, cardiovascular and cerebrovascular disease technology, applied in biochemical equipment and methods, chemical instruments and methods, microbial-based methods, etc., can solve problems such as the inability to detect changes in HDL subtypes, and achieve risk prediction and Effects of clinical monitoring, improvement of sensitivity, and increase of detection rate

Active Publication Date: 2020-03-27
DIASYS DIAGNOSTIC SYST SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most detection methods are immunological methods based on ApoA1 polyclonal antibodies, which cannot detect changes in HDL subtypes

Method used

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  • Anti-human apoa1 monoclonal antibody and its preparation method and application
  • Anti-human apoa1 monoclonal antibody and its preparation method and application
  • Anti-human apoa1 monoclonal antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation and purification of ApoA1 monoclonal antibody D11-10

[0030] 1. Materials: fusion protein 6×His-ApoA1, 8-week-old female BALB / c mice

[0031] 2. Methods and Results

[0032] 2.1 Antigen preparation

[0033] 2.1.1 Construction of human ApoA1 recombinant protein expression plasmid

[0034] The gene sequence (Gene ID: 335) of ApoA1 was obtained by querying in GenBank, and polymerase chain reaction (PCR) primers were designed according to the sequence. The DNA fragment of human ApoA1 gene was amplified using human cDNA as a template. The PCR product was detected by 1.0% agarose gel electrophoresis and the corresponding fragment was recovered with a gel recovery kit. The size of the ApoA1 gene fragment amplified by PCR was 875bp. The PCR product was ligated into the expression vector pCold TM II (the 6×His tag contained in the vector is used for purification), transformed into TaKaRaDH5α host bacteria, picked a single clone for plasmid extraction an...

Embodiment 2

[0051] Example 2: Determination of the variable region sequence of ApoA1 monoclonal antibody D11-10

[0052] 1. Materials: Trizol (Invitrogen), primers were synthesized by Sangon Bioengineering Company, reverse transcription and PCR reagents were purchased from TaKaRa Company.

[0053] 2. Methods and results:

[0054] 2.1 Total RNA extraction and cDNA first-strand synthesis

[0055] The hybridoma cells in the logarithmic growth phase were collected, and total RNA was extracted according to the operating procedure of the Trizol manual. The qualitative and quantitative identification of total RNA was performed using a spectrophotometer and agarose gel electrophoresis.

[0056] According to TaKaRa PrimeScript TM II 1st Strand cDNA Synthesis Kit Instructions Synthesize cDNA.

[0057] 2.2 Gene fragment amplification and sequencing of ApoA1 monoclonal antibody D11-10 heavy chain variable region (VH) and light chain variable region (VL)

[0058] According to TaKaRa company's Taq...

Embodiment 3

[0062] Example 3: Clinical application of the detection method established based on ApoA1 monoclonal antibody D11-10 in the risk prediction and diagnosis of cardiovascular and cerebrovascular diseases

[0063] 1. Materials:

[0064] ApoA1 monoclonal antibody D11-10; serum samples from healthy people (no history of coronary heart disease) and patients with coronary heart disease (CHD).

[0065] 2. Methods and results:

[0066] 2.1 Preparation and assignment of mixed serum calibrator

[0067] Serum samples from 20 healthy individuals were used, and an equal volume of serum was taken from each case into centrifuge tubes (100 μL each), placed at 4°C for thorough mixing, then aliquoted in 200 μL / tube, and frozen in a -80°C refrigerator. One was taken out the next day, thawed at 4°C, and after equilibrating to room temperature, the ApoA1 immunoturbidimetric kit from Desai Company was used to assign values ​​to the mixed serum samples. The concentration of ApoA1 in the mixed serum...

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Abstract

The invention discloses an anti-human apolipoprotein A1 (ApoA1) murine monoclonal antibody D11-10. A heavy chain subtype of the ApoA1 monoclonal antibody D11-10 is IgG2b, a light chain subtype is kappa, and amino acid sequences of variable regions of a heavy chain and a light chain of the D11-10 are respectively shown in SEQ ID NO.3 and SEQ ID NO.4. Compared with a current ApoA1 detection method based on an ApoA1 polyclonal antibody, an ApoA1 detection method established based on the ApoA1 monoclonal antibody D11-10 disclosed by the invention can significantly improve a detection rate of a coronary heart disease (CHD), and especially has a significantly-improved detection rate for CHD patients with HDL-C in a normal range, so that the novel detection method established based on the ApoA1 monoclonal antibody D11-10 can better reflect correlation between a detection value of ApoA1 and cardiovascular and cerebrovascular diseases, and has important clinical significance for diagnosis, prevention, control and management of the cardiovascular and cerebrovascular diseases.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to an anti-human apolipoprotein A1 (ApoA1) mouse monoclonal antibody D11-10 and a hybridoma cell line secreting the monoclonal antibody, and using the monoclonal antibody in clinical detection of human ApoA1 protein content in peripheral blood to predict the risk of cardiovascular and cerebrovascular diseases and the diagnosis of the disease. Background technique [0002] Apolipoprotein A1 (ApoA1) is the main structural protein of high-density lipoprotein (HDL), and its physiological function is to help HDL capture free cholesterol. In addition, ApoA1 also stabilizes cycloprostaglandin, promotes vasodilation, inhibits platelet aggregation, and resists vascular plaque formation. [0003] Traditionally, the detection of HDL-C and ApoA1 protein is used clinically to reflect the level of HDL metabolism in vivo. The reduction of ApoA1 and HDL-C levels in serum / plasma ind...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/68G01N33/577C12R1/91
CPCC07K16/18C07K2317/56G01N33/577G01N33/6893G01N2333/775G01N2800/2871G01N2800/323G01N2800/324
Inventor 张杰张艳王荣芳钱震斌
Owner DIASYS DIAGNOSTIC SYST SHANGHAI
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