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Immunomicrosphere chromatography quick HIT antibody detection test paper

An immune microsphere and antibody detection technology, applied in biological testing, measuring devices, analytical materials, etc., can solve the problems of long test report time, complicated clinical operation, complicated operation, etc., to ensure the validity and quality control, and test results. Clear and accurate results with improved sensitivity

Inactive Publication Date: 2018-07-10
PRO MED BEIJING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the HIT antibody detection reagents used in western countries are mainly ELISA method, which is very popular in foreign clinical screening, but due to the need for professional testing personnel, the operation is complicated, time-consuming, and the positive predictive value is not high
flow cytometry HIT Kit and SRA radioimmunoassay products have complicated clinical operations, long test report time, and low clinical popularity. After clinical practice, HemosIL's immunoturbidimetric method HIT-Ab (PF4-H) has a good laboratory Applicable value, but this method is a large-scale coagulation instrument detection item, not suitable for bedside POC, and a more convenient and rapid method is needed for clinical detection. STic Expert from Stago and PIFAPlussPF4 from Akers Biosciences TM It can be used for clinical rapid POCT detection, but the HIT antibody has not been quantitatively detected, and the sensitivity of clinical detection is low

Method used

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  • Immunomicrosphere chromatography quick HIT antibody detection test paper
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Experimental program
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Effect test

Embodiment 1

[0049] Such as figure 1 Shown, a kind of immune microsphere chromatography rapid HIT antibody detection test paper preparation method comprises the steps:

[0050] S1. Specific steps for preparation of heparin / PF4 complex:

[0051] 40ul of heparin (1000U / ml), dissolved in PBS buffer of pH 7.4, mixed with 94ul of 1.4mg / ml of platelet factor 4, added 56ul of pure water, incubated at room temperature for 30min and turned into a yellow complex solution, then added 4ul 50% sucrose solution and 4ul of 25% trehalose solution were mixed, and 2ul of 1M TAPS buffer solution of pH 9.0 was added for use.

Embodiment 2

[0053] S2. Preparation of protein A particles

[0054] Specific steps: take 100ml of 10% 0.3um fluorescent microspheres, wash 3 times with PH4.5 50mM MES solution, add 1ml of freshly prepared 10mg / ml EDAC solution (prepared with PH4.5 50mM MES), and incubate at room temperature 30min, then centrifuged at 2000rpm for 5min, then washed with 50mM MES solution of pH 4.5, incubated overnight at room temperature with 1ml of 0.65mg / ml recombinant protein A (PBS), incubated with 10% BSA, 100×Tris-EDTA at room temperature for 1 Hours, centrifuged and washed 3 times with 1ml PBS, stored in 4% sucrose, 0.05% sodium azide solution.

Embodiment 3

[0056] S3, preparation of reaction film

[0057] The compound was sprayed onto a Millipore Hi-Flowmylar backed nitrocellulose membrane (25 mm wide, SHF0900425) at 0.075 μl / mm, spraying at a constant velocity of 45 mm / s to form a test line. At the same time, the reference reagent containing 0.25 mg / mL recombinant protein A was mixed with 1% sucrose / 0.5% trehalose / 10 mM TAPS pH 9.0, and the quality control line was sprayed at 0.075 μl / mm at 45 mm / sec. The films were then baked at 56 °C for one week.

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Abstract

The invention relates to the technical field of clinical heparin-induced thrombocytopenia (HIT) detection, in particular to immunomicrosphere chromatography heparin / platelet factor 4-antibody detection test paper. A method includes the following steps that an antibody compound is prepared; a test paper strip carrier membrane is prepared, protein A latex particles are prepared, a reaction pad is prepared, a sample pad is marked, a separation membrane is assembled, a test paper strip is assembled, a clinical sample is detected, and the dried test paper strip is placed in an aluminum foil bag. The HIT antibody lateral flow immunodetection method has high cost effectiveness, and the level of HIT antibodies in body fluids of patients is quantitatively detected and evaluated. The method is usedfor a POCT detection system and screening or detection of titer changes of the HIT antibodies in the body fluids of the patients to determine a detection method sensitive to HIT so that the HIT antibody detection technology can be a clinical conventional detection project.

Description

technical field [0001] The invention relates to the technical field of heparin-induced thrombocytopenia detection, in particular to a method for preparing a test paper for rapid detection of HIT antibody by immune microsphere chromatography. Background technique [0002] HIT is an antibody-mediated syndrome with high morbidity and mortality. In the past, it was often considered a rare disease because many doctors did not have enough understanding and it was difficult to diagnose it. And there is no better treatment than heparin. The incidence of HIT is mainly related to factors such as heparin use time, heparin type, route of use, patient type, gender and race. [0003] The pathogenesis of HIT is not very clear. It is generally believed that when heparin-like compounds appear in the blood circulation, PF4 (platelet factor 4) binds to it with high affinity to form a heparin-PF4 (H-PF4) complex (heparin loses active). After the H-PF4 complex is formed, its conformation chan...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/558
Inventor 郝存范秋苹潘志红张宁高巍巍陈美艳南洋田茹汪廷枫
Owner PRO MED BEIJING TECH
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