Kit for clinically detecting active urokinase receptor in plasma of new coronal pneumonia patient

A technology of urokinase receptor and kit, which is applied in the field of medical detection and can solve the problem of low expression

Inactive Publication Date: 2021-01-05
FUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Urokinase-type plasminogen activator receptor (uPAR) is a multi-domain cell surface receptor including DI, DII, and DIII, and a highly glycosylated surface membrane protein widely expressed on activated s...

Method used

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  • Kit for clinically detecting active urokinase receptor in plasma of new coronal pneumonia patient
  • Kit for clinically detecting active urokinase receptor in plasma of new coronal pneumonia patient
  • Kit for clinically detecting active urokinase receptor in plasma of new coronal pneumonia patient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of an ELISA detection kit pre-coated with a fusion protein that can bind to active suPAR

[0022] (1) Prepare washing solution / sample diluent

[0023] The washing solution and sample diluent contained 0.05% Tween 20 in TBS buffer, and the formula of TBS was 20 mM Tris-HCl, 150 mM NaCl, 2.7 mM KCl.

[0024] (2) Configure the coating solution

[0025] The coating solution is 50 mM bicarbonate buffer solution (pH 9.6), specifically 1.59 gNaCO in 1L buffer solution 3 ,, 2.93 g NaHCO 3 .

[0026] (3) Prepare blocking solution

[0027] The blocking solution was prepared from TBST, pH 7.4 buffer solution containing 4% sucrose and 3% BSA.

[0028] (4) Preparation of substrate chromogenic solution

[0029] The substrate chromogenic solution is 0.1 M Tris-HCl, 0.1 M NaCl, 5 mM MgCl and 1 mg / ml PNPP.

[0030] (5) Recombinant expression and purification of suPAR standard

[0031] The recombinant suPAR was expressed in Drosophila embryonic cell S2 and p...

Embodiment 2

[0038] Example 2 Using an ELISA detection kit to detect the level of active soluble urokinase receptor (suPAR) in COVID-19 patients, asymptomatic infected persons or healthy individuals

[0039] 1. Add 100 μl of standard substances of different concentrations to the microplate, and then add the diluted plasma sample of the patient to be tested (diluted 5 times with the sample diluent) to the other wells of the microtiter plate. Each plasma sample has 2 replicates. The 96-well plate was sealed with sealing tape to prevent liquid evaporation, and incubated at 37°C for 2h. Uncover the sealing tape, dry the liquid in the wells of the microplate reader, and absorb the washing solution with a row gun, wash each well 5 times, after the last wash, pat the microplate dry on absorbent paper and ensure that the wells are completely dry. No air bubbles remained.

[0040] 2. Add the rabbit-derived anti-active suPAR polyclonal antibody diluted 200-800 times with the sample diluent to the E...

Embodiment 3

[0044] Example 3 Active suPAR levels in plasma of COVID-19 patients (common type, severe type and critical type)

[0045] The COVID-19 patients in Example 2 were classified in more detail into common type, severe type and critical type, and then the levels of active suPAR in these three groups were analyzed, and it was found that the level of suPAR in the blood of patients with severe disease The higher the result, see image 3 . The average plasma concentrations of normal, severe and critical patients were 4.57 ng / ml, 5.97 ng / ml and 6.68 ng / ml, respectively.

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Abstract

The invention belongs to the field of biotechnology application, and relates to a novel prediction technology for human severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) infection, inparticular to a method for determining the content of an active urokinase receptor in a human body through enzyme-linked immunosorbent assay to serve as a prognosis biomarker of a COVID-19 patient. Bydetecting the level of an active urokinase receptor in a COVID-19 patient, an asymptomatic infector or a healthy human body, it is found that the level of active suPAR in the COVID-19 patient and theasymptomatic infector is far higher than that of active suPAR in the healthy human body, and the level of active suPAR in the asymptomatic infector is slightly higher than that of active suPAR in theCOVID-19 patient. The result obtained by the invention shows that the detection of the level of the suPAR in the body of the COVID-19 patient can be used as a prognosis biomarker of the patient, anda certain value is provided for early triage of the COVID-19 patient.

Description

technical field [0001] The invention relates to the field of medical detection, in particular to an ELISA detection kit for human severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) infection and the severity of symptoms of new coronary pneumonia. Background technique [0002] Since its discovery in early 2020, the rapid spread of novel coronavirus pneumonia around the world is a major challenge facing the world. As of August 31, 2020, the global pandemic caused by SARS-CoV-2 has infected more than 25.2 million people in more than 180 countries and regions around the world and caused 840,000 deaths. While there are many available methods to detect the virus and combat the disease in the current pandemic, none of these available diagnostic or predictive methods have their limitations. Therefore, the development of new assays or biomarkers will facilitate the presumptive diagnosis or prognosis of individuals with a high index of clinically suspected infection. ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/68G01N33/543G01N2800/26G01N2800/12
Inventor 黄明东江龙光袁彩李林林王蕊
Owner FUZHOU UNIV
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