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Preparation and application of anti-PCSK9 antibody and PCSK9 detection kit

A kit and antibody technology, applied in the field of clinical detection, can solve problems such as high price and increased clinical diagnosis and treatment costs

Active Publication Date: 2018-08-21
成都金洛克锶生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are commercialized human PCSK9 detection kits for scientific research (such as R&D company, DYC900) on the market in foreign countries, there is no research data or proof for its use in clinical biological sample detection, and the price is expensive. If it is applied to clinical detection, it will greatly increase Increased clinical diagnosis and treatment costs

Method used

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  • Preparation and application of anti-PCSK9 antibody and PCSK9 detection kit
  • Preparation and application of anti-PCSK9 antibody and PCSK9 detection kit
  • Preparation and application of anti-PCSK9 antibody and PCSK9 detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of anti-human PCSK9 monoclonal antibody

[0038] 1. Preparation of anti-human PCSK9 monoclonal antibody expression plasmid

[0039] 1.1 Preparation of antibody variable region sequences

[0040]Using PCR amplification technology, the variable region sequence of the antibody was obtained from the hybridoma cell line secreting the anti-human PCSK9 antibody. The PCR reaction system was as follows (total volume: 50 μl): 5 μl of 10×Buffer, 2 μl and 1 μl of dNTP (see Table 1 for primer sequences), cDNA as template, 0.2 μl of rTaq enzyme, and make up to 50 μl with double distilled water. Reaction conditions: pre-denaturation at 95°C for 3 minutes, 33 cycles at 95°C for 15s, 52°C for 30s, and 72°C for 30s, and finally 72°C for 15 minutes. The obtained heavy and light chain variable region gene fragments were named 5H and 6K, respectively.

[0041] Table 1 Antibody variable region amplification primers

[0042]

[0043] 1.2 Construction of antibody e...

Embodiment 2

[0069] Example 2 Binding Affinity of Monoclonal Antibody to Human PCSK9

[0070] The binding affinity of the monoclonal antibody of the present invention to human PCSK9 is determined by biomembrane interference technology. The specific measurement process is as follows: First, the human PCSK9 protein was biotin-labeled, specifically human PCSK9 and NHS-LCLC-biotin (Thermo-Fisher Company) were mixed at a molar ratio of 1:5, and after incubation at room temperature for 1 h, PD- 10 A desalting column (GE Company) was used to remove the remaining NHS-LCLC-biotin to obtain biotinylated human PCSK9. Then in the protein molecular interaction instrument OtectQK e (Pall Company) it is immobilized on SA Biosensor sensor (Pall Company), the antibody of different concentration (600nM to 0.82nM) is set, with Sample Diluent buffer as blank control; Adopt Otect QKe The StandardKinetics Assays (0.6HZ, averaged by 5) detect the binding kinetics of human PCSK9 and monoclonal antibodies, record...

Embodiment 3

[0073] Example 3 ELISA method for quantitative detection of human PCSK9

[0074] 1. Establishment of antibody-sandwich ELISA method

[0075] The anti-human PCSK9 monoclonal antibody described in Examples 1-2 was used to establish an antibody-sandwich ELISA method for quantitative detection of human PCSK9. Specifically, firstly, the anti-human PCSK9 monoclonal antibody was immobilized on a microtiter plate as a capture reagent, and the immobilization method was to dilute the anti-human PCSK9 antibody to 2 μg / ml with carbonate buffer (pH9.6), and 100 μl Add / well to a microtiter plate and incubate at 4°C for 16-20h; then wash three times with PBS solution containing 0.05% Tween-20, and block with 3% BSA in PBS for 2h at 37°C; finally immobilized The microtiter plate was sealed and stored at -20°C for future use. The process of detecting the content of human PCSK9 is as follows: equilibrate the above-mentioned microtiter plate with pre-immobilized capture reagent to room tempera...

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Abstract

The invention discloses preparation methods and application of a monoclonal antibody against human proprotein convertase subtilisin / kexin type 9 (PCSK9) and a human PCSK9 detecting kit containing theantibody. The invention discloses an amino acid sequence of the anti-PCSK9 monoclonal antibody and a coding gene thereof, and a host cell containing a vector and a preparation method thereof. Based onthe antibody, the invention provides an antibody-sandwich ELISA method and kit for human PCSK9 used as an antigen; and the method and kit are used for detecting the levels of PCSK9 in biological samples and can be used as indexes for clinical medication guidance and diagnosis / prognosis.

Description

[0001] manual technical field [0002] The invention relates to the field of diagnostic reagents, in particular to the use of antibodies and kits containing antibodies for clinical detection. Background technique [0003] Proprotein convertase subtilisin 9 (PCSK9) is the ninth member of the kexin-like proprotein convertase subtilisin family, consisting of 692 amino acid residues, divided into signal peptide, prodomain, catalytic domain, carboxyl Terminal domain; mainly expressed in liver, kidney and small intestine. The synthesis of PCSK9 occurs in the endoplasmic reticulum, and then it is transported to the Golgi apparatus through the endoplasmic reticulum, and secreted into the blood after a series of modifications in the Golgi apparatus. At present, it is believed that only PCSK9 secreted by the liver can be secreted into the blood, and bind to the low-density lipoprotein receptor (LDLR) on the surface of liver cells, mediate the degradation of LDLR, and regulate plasma ...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13G01N33/577G01N33/573
CPCC07K16/40C07K2317/565C07K2317/92G01N33/573G01N33/577G01N2333/96433G01N2800/04G01N2800/324
Inventor 张晟付伟李生伟何刚代燕平程琳高小平
Owner 成都金洛克锶生物技术有限公司
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