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Heparin binding protein detection kit, preparation method and application

A technology of heparin-binding protein and detection kit, which is applied in the field of immunoassay, can solve the problems of no heparin-binding protein detection kit, time-consuming exploration, few antigen detection methods, etc., achieve small inter-batch difference, evaluate prognosis effect, good thermal stability

Pending Publication Date: 2022-01-18
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it requires time-consuming exploration in raw material screening and process parameter selection. In addition, because the application of heparin-binding protein in inflammation detection, disease diagnosis, and guidance of antibiotic use has just emerged, there are relatively few studies on the detection method of this antigen. Therefore, there is currently no heparin-binding protein detection kit based on chemiluminescence

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  • Heparin binding protein detection kit, preparation method and application
  • Heparin binding protein detection kit, preparation method and application
  • Heparin binding protein detection kit, preparation method and application

Examples

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preparation example Construction

[0061] The present invention also provides a preparation method of the HBP detection kit according to the , wherein the preparation method comprises:

[0062] Coating step of magnetic microspheres: coating the first antibody strain on the magnetic microspheres;

[0063] The step of labeling the tracer label: labeling the tracer label on the second antibody strain;

[0064] Prepare calibrator steps.

[0065] The present invention finds that in the coating step of the magnetic beads, the coupling time of the first antibody strain and the magnetic microspheres has little effect on the detection signal and the coincidence rate of the sample, and the coupling time can be 0.5 to 1.5 hours. In order to save time, A preferred coupling time is 0.5 hours.

[0066] As one of the embodiments of the present invention, the coupling is carried out in 2-(N-morpholino)ethanesulfonic acid (MES) buffer in the step of coating the magnetic microspheres with the first strain of antibody. Compared...

Embodiment 1

[0095] This embodiment provides a method for preparing a HBP detection kit and a method for detecting HBP in human blood.

[0096] Preparation 1: The first antibody-coated magnetic microspheres

[0097] (1) Measure 10mg of magnetic microspheres (average particle size 1.5μm, purchased from Bangs Laboratories, solid content 2.54%), and suspend in 1mL of 0.1M MES buffer, magnetize for 5-10min, discard After the supernatant, repeat the above cleaning steps 3 to 5 times, add 1 mL of the above buffer (0.1M MES buffer), and vortex to mix.

[0098] (2) Add 150 μg of the first HBP monoclonal antibody (the sequence of the amino acid site of HBP recognized by the first antibody is the sequence shown in SEQ ID NO: 4), so that the mass ratio of the magnetic microspheres to the antibody is 100:1.5, vortex and mix well, and incubate at 37°C for 30min.

[0099] (3) Add 10 μL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethyldiimine hydrochloride (EDC), vortex and incubate at 37° C. for 1.5 h.

...

Embodiment 2

[0126] Panel screening of antibodies used in HBP detection kits

[0127] There are four kinds of HBP monoclonal antibodies in the process of preparation 1 and preparation 2 in this example: the sequence of the amino acid site recognized by antibody 1 is the sequence shown in SEQ ID NO: 1, and the sequence of the amino acid site recognized by antibody 2 is as shown in SEQ ID For the sequence shown in NO:2, the sequence of the amino acid site recognized by antibody 3 is the sequence shown in SEQ ID NO:3, and the sequence of the amino acid site recognized by antibody 4 is the sequence shown in SEQ ID NO:4.

[0128] Coat antibodies 1, 2, 3 and 4 on magnetic microspheres to form antibody 1-magnetic microsphere suspension, antibody 2-magnetic microsphere suspension, antibody 3-magnetic microsphere suspension and antibody 4-Magnetic microsphere suspension, antibody 1 and antibody 4 are respectively labeled on acridinium ester to obtain antibody 1-acridine ester and antibody 4-acridin...

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Abstract

The invention relates to a heparin binding protein detection kit, a preparation method and application thereof. The kit provided by the invention comprises a first antibody coated on magnetic microspheres and a second antibody marked with a tracing marker. The specific binding sites of the heparin binding protein identified by the first antibody and the second antibody are different. The kit has better sensitivity and specificity, and a detection result can be quickly obtained.

Description

technical field [0001] The invention belongs to the field of immunoassay, and in particular relates to a heparin-binding protein detection kit, preparation method and application. Background technique [0002] Heparin-binding protein (HBP): It is a granular protein derived from neutrophils, and its structure consists of a single-chain protein of 222 amino acids, containing 8 cysteine ​​residues, at 100, 114 or 145 There are glycosylation sites on aspartic acid residues. It was first isolated and identified in 1984 by Shafer et al. HBP itself has a strong positive charge, which leads to its remarkable bactericidal ability and heparin binding ability. At the same time, the strong positive charge of HBP will also produce chemotactic activity, which can be used to activate monocytes and macrophages, and induce vascular permeability and tissue edema. Studies have shown that high concentrations of HBP can lead to severe vascular infiltration, causing hypotension and even shock....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/68G01N33/54326G01N2800/26G01N2800/52
Inventor 王萧慧张瑞石晓强徐建新李福刚
Owner SHANGHAI UPPER BIO TECH PHARMA