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Kit for high-specificity detection of heparin binding protein and application thereof

A heparin-binding protein and kit technology, applied in the field of immunodetection, can solve the problems of low accuracy, complicated operation process, long experiment time, etc., and achieve the effects of high specificity, good stability and avoiding interference.

Pending Publication Date: 2021-12-10
北京森美希克玛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme-linked immunoassay method can accurately detect the concentration of heparin-binding protein in the sample, but the actual operation takes a long time and the operation process is relatively complicated
Latex turbidimetry and immunofluorescence chromatography can be quickly detected, but the accuracy is low and cannot provide a reliable basis for clinical judgment
The existing magnetic particle chemiluminescence method uses a biotin-streptavidin amplification system. Although the specificity and sensitivity have been improved, the interference of endogenous biotin cannot be avoided, so that the detection results cannot be guaranteed.

Method used

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  • Kit for high-specificity detection of heparin binding protein and application thereof
  • Kit for high-specificity detection of heparin binding protein and application thereof
  • Kit for high-specificity detection of heparin binding protein and application thereof

Examples

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preparation example Construction

[0039] Preferably, the HBP-binding antibody is prepared by the following steps: mixing and labeling the HBP antibody and fluorescein isothiocyanate at a ratio of 1 mg: (100-200 g);

[0040] Add 1~10M NH to the mixed product4 Cl solution to NH 4 Cl final concentration is 50mmol / L, to stop reaction;

[0041] The product after terminating the reaction is dialyzed in the buffer solution, and after the dialyzing, it is diluted to the required concentration with a diluent to obtain the HBP-binding antibody.

[0042] Preferably, the kit further includes: at least one of a buffer, a diluent, a calibration product, a quality control product and a cleaning solution.

[0043] Preferably, the diluent contains 1.0wt% BSA, 0.1wt% Proclin-300 and 0.1mol / L (phosphate concentration), pH7.4 phosphate buffer.

[0044] Preferably, the calibration product and / or the quality control product are prepared by mixing heparin-binding protein and matrix fluid.

[0045] Preferably, the heparin-binding ...

Embodiment 1

[0053] A kit for detecting heparin-binding protein, which includes the following reagents.

[0054] (1) Magnetic particle reagent

[0055] Preparation method: Take 10 mg of magnetic particles, magnetically separate for 5-10 minutes, discard the supernatant, add 0.5M MES solution to wash, repeat three times, and wash the magnetic particles and the antibody of fluorescein isothiocyanate according to the mass ratio of 100 Mix evenly in 0.5M MES solution at a ratio of :1, add 100 μL of EDC solution, react for 18-20 hours, then resuspend with magnetic particle protection solution and dilute to 1.5 mg / ml, and equilibrate for 22-24 hours at 2-8°C , and then stored at 2-8°C for later use.

[0056] The above magnetic particle protection solution contains 1wt% bovine serum, 0.2wt% casein, 0.2wt% Proclin-300, 0.1mol / L, phosphate buffer solution with pH 7.6.

[0057] (2) HBP binding antibody

[0058] Preparation method: Take 2mg of HBP monoclonal antibody, slowly dissolve it in the ant...

Embodiment 2

[0085] A kit and method for detecting heparin-binding protein are provided, which are roughly the same as in Example 1, except that the luminescent marker of the HBP-labeled antibody is alkaline phosphatase (ALP), and the chemiluminescent substrate corresponding to it is The substance is (adamantane)-1,2-dioxetane (AMPPD); the solution contains: 0.5wt% AMPPD, tris(0.05mol / L, pH range 7.00-9.50), 0.2 mol / L sodium sulfate, 0.1mol / L sodium chloride, 0.2wt% Proclin-300 and 2wt% luminescence enhancer.

[0086] The above-mentioned alkaline phosphatase-labeled HBP-labeled antibody preparation method is as follows:

[0087] Weigh 5 mg of alkaline phosphatase and add it to 1 ml of antibody solution (2 mg / ml) to dissolve it, put it into a dialysis bag, and dialyze it with 0.01 mol / L PBS at pH 7.2 for 18 hours at 4°C, change the solution 3 times during this period, and add 2.5% 20 μL of glutaraldehyde, stirred at room temperature for 2 hours, dialyzed with 0.01mol / L pH7.2 PBS at 4°C for...

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Abstract

The invention discloses a kit for high-specificity detection of heparin binding protein and application thereof, and relates to the technical field of immunodetection. The kit comprises an HBP binding antibody connected with fluorescein isothiocyanate, an HBP labeled antibody labeled with a luminous marker and a solid-phase carrier with the surface coated with a fluorescein isothiocyanate antibody, a sample is detected through a fluorescein isothiocyanate and anti-fluorescein isothiocyanate system, interference of endogenous biotin is well avoided, the specificity is high, the stability is good, and the kit has more advantages in detection limit.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a kit for highly specific detection of heparin-binding protein and its application. Background technique [0002] Infectious diseases are a serious threat to health, and sepsis and some serious infectious diseases have become the main causes of morbidity and death. Sepsis refers to the systemic inflammatory response syndrome (systemic inf1animatory response syndrome, SIRS) caused by infection, clinically confirmed infection or highly suspicious infection, once it occurs, the disease develops rapidly, systemic coagulation disorder and immune inflammatory response imbalance, Inducing organ failure and leading to death, how to take reasonable countermeasures early in the prevention and treatment of infectious diseases is the key to success. early identification method. [0003] Heparin-binding protein (HBP) is a protein present in neutrophils, which was discovered and isolated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/577G01N33/543G01N33/533G01N21/76
CPCG01N33/6893G01N33/582G01N33/581G01N33/577G01N33/54326G01N33/533G01N21/76G01N2333/47G01N2800/26
Inventor 肖文峰庞启锋胡国茂
Owner 北京森美希克玛生物科技有限公司
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