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Soluble fms-like tyrosine kinase-1 detection kit as well as preparation method and application thereof

The technology of tyrosine kinase and detection kit is applied in the field of soluble fms-like tyrosine kinase-1 detection kit and preparation thereof, and can solve the problems of poor detection sensitivity, narrow detection range and high detection cost

Pending Publication Date: 2022-03-29
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunosorbent assay is time-consuming and cumbersome to operate
In addition, these two detection methods have poor detection sensitivity, narrow detection range, and non-automatic testing
There is an electrochemiluminescence immunoassay method for the detection of sFlt-1 in foreign countries, but the detection cost of this method is high, which makes it unsuitable for pregnancy detection in areas with limited resources.

Method used

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  • Soluble fms-like tyrosine kinase-1 detection kit as well as preparation method and application thereof
  • Soluble fms-like tyrosine kinase-1 detection kit as well as preparation method and application thereof
  • Soluble fms-like tyrosine kinase-1 detection kit as well as preparation method and application thereof

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preparation example Construction

[0044] Some embodiments of the present invention provide a method for preparing a chemiluminescence-labeled protein, which includes the following steps:

[0045] (1) Protein activation: use a cross-linking agent to activate the protein;

[0046] (2) Activation of chemiluminescent substances: using dithiothreitol to activate chemiluminescent markers;

[0047] (3) Coupling: mixing and incubating the activated chemiluminescent marker in step (2) with the activated protein in step (1) to obtain a chemiluminescent marker-protein conjugate;

[0048] (4) Blocking: adding a blocking agent to the chemiluminescent marker-protein conjugate obtained in the step (3) for blocking to obtain the final product of chemiluminescent marker-protein coupling;

[0049] Wherein, the cross-linking agent in step (1) is selected from any one of SPDP, DTBP, DTSSP, AMAS, and the blocking agent in step (4) is MMTS.

[0050] In some of these embodiments, the crosslinking agent in step (1) of the above pre...

Embodiment 1

[0083] The preparation of embodiment 1sFlt-1 chemiluminescent immunoassay kit

[0084] (1) Preparation of tosylated magnetic particles coated with sFlt-1 monoclonal antibody:

[0085] Take a suspension containing 90 mg of tosylated magnetic particles (MagnosphereTM, product number: MS160) with a particle size of 0.9 μm to 1.8 μm, magnetically separate and remove the supernatant, and wash the magnetic particles with 0.01 M PBS buffer solution with a pH of 7.4. After removing the supernatant by magnetic separation, resuspend in 0.1M BBS buffer with pH 9.5, add 4mL of 3M ammonium sulfate solution with pH 9.5, and disperse evenly to obtain mixed solution 1, then add 3.6mg of soluble fms-like phenol Amino acid kinase-1 monoclonal antibody (Guangzhou Wanfu Biotechnology Co., Ltd., catalog number: sFlt-1-10#) added 5.4 μg of BS (PEG) 5 (PEGylated bis(sulfosuccinimidyl)suberic acid, ThermoFisher SCIENTIFIC) activated sFlt-1 monoclonal antibody, and then added the activated antibody t...

Embodiment 2

[0090] The detection method of embodiment 2sFlt-1 chemiluminescent immunoassay kit

[0091] The detection principle of the kit of the present invention is: the magnetic particles coated with soluble fms-like tyrosine kinase-1 (sFlt-1) capture antibody and the soluble fms-like tyrosine kinase-1 (sFlt-1) labeled with alkaline phosphatase ) The detection antibody combines with the placental growth factor in the sample, calibrator or quality control to form a "sandwich" complex. Under the action of an external magnetic field, the complex formed by the immune reaction is separated from other unbound substances. After the complex is washed, an enzymatic chemiluminescence substrate is added. The substrate is catalyzed and cleaved by the enzyme to form an unstable excited state intermediate. When the excited state intermediate returns to the ground state, photons are emitted to form a luminescent reaction, and the luminescence intensity of the reaction can be detected by a chemilumine...

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Abstract

The invention relates to a soluble fms-like tyrosine kinase-1 detection kit as well as a preparation method and application thereof, the inventor detects soluble fms-like tyrosine kinase-1 by adopting a double-antibody sandwich method combined with a magnetic particle chemiluminescence technology, and finds that an immune micro-magnetic particle preparation method optimized by the inventor is adopted. A soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit is formed by the prepared placenta growth factor capture antibody magnetic particles and an alkaline phosphatase labeled placenta growth factor detection antibody which is further prepared by the inventor through cross-linking agents DTBP and DTT and a sealing agent MMTS in a specific proportion. According to the present invention, the detection can be automatically completed by using the full-automatic chemiluminescence immunoassay analyzer as the detection tool, the detection performance is significantly improved, the detection sensitivity achieves 0.97 pg / mL, and the detection linear range is wide and can achieve 10-85000 pg / mL. Therefore, the method has a wide detection application prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a soluble fms-like tyrosine kinase-1 detection kit and its preparation method and application. Background technique [0002] VEGFR-1 (Flt-1) is a type of vascular endothelial growth factor receptor (VEGFR), and soluble fms-like tyrosine kinase-1 (sFlt-1) is its soluble form, which lacks transmembrane Region and tyrosine kinase activity, no signal transduction function, can act as VEGF antagonist. Soluble fms-like tyrosine kinase-1 (sFlt-1), a glycoprotein with tyrosine kinase activity, belongs to the type III receptor tyrosine kinase family and is a vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) A powerful inhibitor that binds to VEGF and PLGF to inactivate them. Under normal circumstances, the formation of placental vascular network is maintained by VEGF and its antagonistic factors (including sFlt-1). The expression of sFlt-1 in the peripheral blood o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/532G01N33/535G01N33/577G01N33/573G01N33/68G01N33/543G01N21/76
CPCG01N33/532G01N33/535G01N33/577G01N33/573G01N33/74G01N33/54326G01N21/76G01N2333/912G01N2333/71G01N2800/368
Inventor 秦静吴晗琪詹雯雯王保升
Owner GUANGZHOU WONDFO BIOTECH
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