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Heparin-binding protein assay kit as well as preparation method and assay method thereof

A heparin-binding protein and kit technology, applied in the field of immunoassay, can solve the problems of lack of high-tech, the sensitivity and specificity of detection results are not ideal and so on

Pending Publication Date: 2020-11-13
HANGZHOU JOINSTAR BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Heparin-binding protein is superior to procalcitonin and C-reactive protein in the diagnosis of infectious diseases, especially for the diagnosis and prognosis of sepsis. In recent years, the number of manufacturers of in vitro diagnostic reagents has increased significantly, but the products are all in Most of them are ordinary varieties without high technology, which leads to the unsatisfactory sensitivity and specificity of the test results

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  • Heparin-binding protein assay kit as well as preparation method and assay method thereof
  • Heparin-binding protein assay kit as well as preparation method and assay method thereof
  • Heparin-binding protein assay kit as well as preparation method and assay method thereof

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Embodiment 1

[0089] Embodiment 1 A kind of heparin-binding protein assay kit

[0090] A heparin-binding protein assay kit, comprising magnetic particles, HBP antibody 1, enzyme-labeled HBP antibody 2, HBP calibrator, concentrated washing solution and substrate.

[0091] Magnetic particles: avidin-labeled magnetic particles with a particle size of 0.9 μm to 1.1 μm;

[0092] HBP antibody 1: biotin-labeled mouse anti-human HBP monoclonal antibody 1;

[0093] Enzyme-labeled HBP antibody 2: horseradish peroxidase-labeled modified mouse anti-human HBP monoclonal antibody 2;

[0094] HBP calibrator: recombinant human HBP;

[0095] Concentrated washing solution: 2mol / L, pH7.5 phosphate buffer solution with mass fraction of 2% Tween-20 and 1% Procline300.

[0096] Substrate: luminol at a concentration of 1 mmol / L.

Embodiment 2

[0097] Embodiment 2 Heparin-binding protein assay kit preparation method

[0098] Step 1, preparation of avidin-labeled magnetic particles:

[0099] Activate the magnetic particles with EDC and NHS, mix them with avidin, rotate at room temperature for 2 hours, and block with blocking solution for 1 hour. The blocking solution is 1mol / L glycine, and perform magnetic separation. After removing the blocking solution, add pH 7.4, 0.02mol / L phosphate buffer containing 0.5% Tween-20, 1% BSA, 1% EDTA, 0.1% Procline300, 4% PEG for storage, and store at 2°C to 8°C for later use;

[0100] Step 2, prepare biotin-labeled mouse anti-human HBP monoclonal antibody 1:

[0101] Biotin was dissolved in phosphate buffer, the phosphate buffer was 0.02mol / L anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate solution, mixed with HBP antibody 1 and rotated at room temperature for 1 hour; the mixed solution was passed through Chromatography column to collect biotin-labeled HBP antib...

Embodiment 3

[0110] Embodiment 3 heparin-binding protein assay kit assay method

[0111]Step 1: Equilibrate the HBP calibrator, magnetic particle suspension, biotin-labeled antibody, enzyme-labeled antibody, concentrated washing solution and substrate to room temperature (20°C to 25°C);

[0112] Step 2: Dilute the concentrated lotion 10 times with purified water to become a washing working solution for later use;

[0113] Step 3: Add 50 μL of the HBP calibrator and 50 μL of the sample to be tested into 50 μL of the magnetic particle suspension in turn, add 50 μL of the biotin-labeled antibody and 50 μL of the enzyme-labeled antibody, and shake at 20°C to 25°C Incubate for 18 minutes and wash 5 times with the washing working solution; the concentration of the magnetic particles is 1 mg / ml, the concentration of the biotin-labeled antibody is 5 ug / ml, and the concentration of the enzyme-labeled antibody is 5 ug / ml;

[0114] Step 4: Add 50 μL of water and mix well;

[0115] Step 5: add 100 μ...

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Abstract

The invention discloses a heparin-binding protein assay kit applying a magnetic particle chemiluminescence enzyme immunoassay method. The heparin-binding protein assay kit comprises avidin-labeled magnetic particles, a biotin-labeled HBP antibody 1, a horseradish peroxidase-labeled HBP antibody 2, an HBP calibrator, a concentrated washing liquid and a substrate. The invention also discloses a preparation method and a determination method of the kit. The novel immunoassay kit disclosed by the invention is used for quantitatively assaying HBP in human body fluid in vitro, overcomes personal errors of a traditional enzyme immunoassay (EIA) method in the aspect of operation, can quantitatively and objectively reflect the existence of HBP, and provides a more powerful experimental diagnosis basis for clinical diagnosis, curative effect observation and prognosis judgment. The heparin binding protein determination method provided by the invention is high in specificity, good in repeatability,high in detection sensitivity, wide in linear range, high in automation degree and simple, convenient and rapid to operate; compared with an EIA method, the method has the advantages of rapidness, simplicity, convenience, accuracy and small interference.

Description

technical field [0001] The application relates to the technical field of immunoassay, in particular to a heparin-binding protein assay kit using magnetic particle chemiluminescent enzyme immunoassay and a heparin-binding protein assay method. Background technique [0002] Infectious diseases are a class of diseases that seriously threaten human health. Sepsis is one of the infectious diseases with a high mortality rate. Early diagnosis and treatment play an important role in improving the curative effect and prognosis of sepsis. Therefore, the biggest clinical challenge now is how to diagnose sepsis patients as early as possible. In recent years, heparin-binding protein (HBP) has been confirmed as one of the early diagnostic markers of sepsis. [0003] Sepsis refers to systemic inflammatory response syndrome (SIRS) caused by infection, clinically confirmed infection or highly suspected infection. Although sepsis is caused by infection, once it occurs, its occurrence and de...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/535G01N21/76
CPCG01N33/6893G01N33/54326G01N33/535G01N21/76G01N2800/26
Inventor 王涛姜珊陈莹菲洪龙斌龚庆姚静岚
Owner HANGZHOU JOINSTAR BIOTECH
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