Method of rapidly detecting concentration of heparin-combined protein in blood

A heparin-binding protein and blood technology, applied in the field of biomedical diagnosis, can solve problems such as differences in incubation time and different measurement results, and achieve the effect of high accuracy and simple operation

Inactive Publication Date: 2017-09-15
NINGBO ACCUTECH BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Different operations by different people, differences in the amount of sample added, differences in incubat

Method used

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  • Method of rapidly detecting concentration of heparin-combined protein in blood
  • Method of rapidly detecting concentration of heparin-combined protein in blood
  • Method of rapidly detecting concentration of heparin-combined protein in blood

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Preparation of anti-human heparin-binding protein monoclonal antibody

[0028] Recombinant human heparin-binding protein is commercially available. Anti-human heparin-binding protein monoclonal antibody was prepared according to the method of reference (Nature, 1975, 256:495-497). Specifically, complete Freund's adjuvant and human heparin-binding protein solution were mixed at a ratio of 1:1 to prepare an emulsion. Female Balb / c mice aged 6-8 weeks were immunized intraperitoneally (100 μl per mouse, containing 10 μg human heparin-binding protein). Two weeks later, the mice were boosted by intraperitoneal injection of 10 μg human heparin-binding protein with incomplete Freund's adjuvant. Two weeks later, blood was taken to determine the titer of antibodies. The mice were boosted by injecting 10 μg of human heparin-binding protein through the tail vein. Four days later, the mice were sacrificed, the spleen cells were isolated, and the isolated spleen cells we...

Embodiment 2

[0029]Example 2 Preparation of anti-human heparin binding protein antibody-latex particle complex

[0030] (1) Activation of latex particles

[0031] Add 100 μl of 10% carboxylated polystyrene latex particle solution into 1.0 mL of 50 mM MES buffer at pH 5.4, mix well, then add 20 μl of 10 mg / mL EDAC, and stir at 37°C Incubate for 0.5-1 hour, centrifuge at 13,000 rpm for 30 minutes, and carefully discard the supernatant. Suspend the pellet in 50 mM PBS buffer, pH 7.4, centrifuge at 13,000 rpm for 30 minutes, carefully discard the supernatant, repeat the washing twice, and finally resuspend the pellet in 1 mL of 50 mM PBS buffer, pH 7.4, to make the final solution The solids percentage of the polystyrene latex particles was 20 mg / ml.

[0032] (2) Selection of cross-linking buffer

[0033] Latex is activated according to step (1), and the latex after thawing is resuspended in MES, MOPOS or PBS damping fluid respectively, and the carboxylated polystyrene latex microsphere solu...

Embodiment 3

[0039] Example 3 Preparation of Heparin Binding Protein Detection Reagent

[0040] (1) Preparation of Reagent 1

[0041] Weigh 6.06g Tris (Tris), 9g NaCl, 40g PEG6000, 0.5mL Tween-20, 0.5gNaN 3 Dissolve in 0.8L deionized water, adjust the pH to 7.4 with dilute hydrochloric acid, and set the volume to 1L to obtain reagent 1. The content of each component in reagent 1 is 0.05mol / L Tris, 0.9% NaCl, 2% PEG6000, 0.05% Tween-20, 0.05% NaN 3 , pH8.0.

[0042] (2) Preparation of reagent 2

[0043] Dilute the antibody-latex particle complex prepared in Example 2 in a certain ratio with Tris-HCl buffer solution with a concentration of 50mM / L, so that the solid content of the floating antibody-latex particle complex is about 0.1-0.25%, adding Add bovine serum albumin BSA to a final concentration of 10 g / L, and add sodium azide to a final concentration of 0.5 g / L to prepare reagent 2.

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Abstract

The invention discloses a method of rapidly detecting concentration of heparin-combined proteins in blood. By means of a latex enhanced turbidimetric immunoassay method, an heparin-combined protein antibody is coated with latex particles to produce an antibody-latex particle composite; when the antibody-latex particle composite is specifically combined with the heparin-combined proteins in blood, the concentration of the heparin-combined proteins is measured by detecting the absorbance of a blood sample. The method is simple in operations, has high accuracy, allows accurate quantification and is suitable for detection of the heparin-combined proteins.

Description

technical field [0001] The invention belongs to the technical field of biomedical diagnosis, and in particular relates to a method for rapidly detecting the concentration of heparin-binding protein in blood and a corresponding kit. Background technique [0002] Heparin-binding protein is a glycoprotein with a molecular weight of 37 kDa, which is mainly synthesized in neutrophils. Structurally heparin-binding protein belongs to the superfamily of serine proteases and although it shares 45% sequence identity with human neutrophil elastase, it is inactive as a protease. The research on heparin-binding protein was initially due to its antibacterial activity, and later studies found that heparin-binding protein was involved in multiple inflammatory processes. Neutrophils are activated upon contact with endothelial cells, and the activated neutrophils release heparin-binding protein. Heparin-binding protein causes calcium-dependent cytoskeletal rearrangements in endothelial cell...

Claims

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Application Information

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IPC IPC(8): G01N33/536G01N33/577G01N33/535
CPCG01N33/535G01N33/536G01N33/577
Inventor 赖增祖叶欣杨奎东徐健
Owner NINGBO ACCUTECH BIOSCI LTD
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