CN112816702A - Kit for detecting content of soluble endothelial factor in human body fluid - Google Patents
Kit for detecting content of soluble endothelial factor in human body fluid Download PDFInfo
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- CN112816702A CN112816702A CN202011609713.XA CN202011609713A CN112816702A CN 112816702 A CN112816702 A CN 112816702A CN 202011609713 A CN202011609713 A CN 202011609713A CN 112816702 A CN112816702 A CN 112816702A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
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Abstract
The invention provides a kit for detecting the content of soluble endothelial factor in human body fluid. The invention combines the chemiluminescence technology with the immunomagnetic particles, provides a reaction system close to homogeneous phase, adopts a one-step reaction mode, greatly improves the detection sensitivity and the precision, enlarges the detection range, greatly shortens the reaction time, and shortens the time from the beginning of sample adding to the detection result, which is less than 20 min; and a plurality of samples can be simultaneously measured on a full-automatic chemiluminescence apparatus, high-flux rapid measurement of the soluble endothelial factor (sENG) is realized, the accuracy is high, the specificity is strong, and the accuracy and the detection efficiency are greatly improved.
Description
Technical Field
The invention belongs to the field of detection kits, and particularly relates to a soluble endothelial factor detection kit.
Background
Endoglin (ENG) is a membrane glycoprotein involved in the formation of transmembrane receptors of the transforming growth factor-beta (TGF- β) receptor complex on endothelial and monocyte membranes. During embryogenesis, the placenta, and especially the syncytiotrophoblast, is the primary source of ENG. The physiological role of ENG is mainly to promote angiogenesis and endothelial cell differentiation, as well as to regulate vascular tone by eNOS. The extracellular portion of ENG produces soluble endothelial factor (sENG) under the action of proteolytic enzymes, and sENG has a limiting effect on TGF- β and eNOS. TGF-beta has anti-inflammatory and pro-angiogenic effects, and sENG can clear blood TGF-beta from causing vascular endothelial dysfunction such as vasoconstriction, adhesion molecule overexpression and T cell depletion, which are usually characteristic of Preeclampsia (PE). Similarly, increased levels of sENG can be found in the blood of PE patients before clinical symptoms appear, and, in relation to the severity of the disease, gradually decrease after delivery. Therefore, the sENG can be used as a reliable marker for predicting the development of severe early-onset PE by pregnant and lying-in women.
Disclosure of Invention
The invention aims to provide a kit for measuring the content of soluble endothelial factor (sENG) in a human body fluid sample (including but not limited to serum/plasma/cerebrospinal fluid/ascites/amniotic fluid and the like) by adopting a magnetic particle chemiluminescence method.
According to one aspect of the present invention, the kit for detecting the content of the soluble endoglin in the human body fluid comprises a calibrator, a quality control material, an anti-reagent, a magnetic particle reagent and a luminescent substrate.
According to an aspect of the present invention, in the kit for detecting a content of soluble endoglin in a human body fluid, the anti-reagent is an anti-soluble endoglin labeled antibody labeled with alkaline phosphatase.
According to an aspect of the present invention, in the kit for detecting a content of soluble endoglin in human body fluid, the magnetic particle reagent is a magnetic particle-anti-soluble endoglin coated antibody conjugate.
According to an aspect of the present invention, in the kit for detecting the content of soluble endoglin in human body fluid, the chemiluminescent substrate is AMPPD.
According to an aspect of the present invention, in the above-mentioned kit for detecting the content of soluble endoglin in human body fluid, the sample source of the human body fluid is one or more of serum, plasma, cerebrospinal fluid, ascites or amniotic fluid.
The technical principle is as follows: the anti-soluble endoglin (sENG) antibody coated magnetic beads are combined with an Alkaline Phosphatase (AP) labeled soluble endoglin (sENG) partner antibody and placental growth factor in the sample, calibrator or quality control to form a "sandwich" complex. Separating the complex formed by immunoreaction from other unbound substances under the action of an external magnetic field, washing the complex, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction. Within the detection range, the luminous intensity is in direct proportion to the content of the placenta growth factor in the sample, and the concentration of the placenta growth factor in the sample can be calculated by using improved four-parameter Logistic equation fitting.
The invention combines the chemiluminescence technology with the immunomagnetic particles, provides a reaction system close to homogeneous phase, adopts a one-step reaction mode, greatly improves the detection sensitivity and the precision, enlarges the detection range, greatly shortens the reaction time, and shortens the time from the beginning of sample adding to the detection result, which is less than 20 min; and a plurality of samples can be simultaneously measured on a full-automatic chemiluminescence apparatus, high-flux rapid measurement of the soluble endothelial factor (sENG) is realized, the accuracy is high, the specificity is strong, and the accuracy and the detection efficiency are greatly improved.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a standard graph in an embodiment of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the "guide to molecular biology experiments" (Aus. works), or according to conditions recommended by the manufacturer.
Example 1 kit for detecting content of soluble endoglin in human body fluid
The embodiment provides a kit for detecting the content of soluble endothelial factor in human body fluid, wherein reagents in reagent holes are pre-packaged into reagent strips, aluminum-plastic composite materials are adopted for membrane sealing, and the reagents comprise a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate (AMPPD) and other reagents.
Example 2 soluble endoglin assay
(I) sample-magnetic bead incubation
Transfer 100 μ L of plasma sample to reaction well, then transfer 100 μ L of magnetic particle-anti-soluble endoglin-coated antibody-containing conjugate in reagent well to reaction well where sample is located, and maintain reaction well at constant temperature of 37 ℃.
(II) magnetic bead transfer washing
At the end of the previous incubation step, the magnetic separation module of the instrument adsorbs and enriches the magnetic particles in the reaction wells, and then the waste liquid without the magnetic particles is aspirated. And then, adding the cleaning solution in the reagent hole into the reaction hole, closing the magnetic separation module, blowing, beating and mixing the reaction hole, standing for 30 seconds, starting the magnetic separation module, adsorbing the magnetic particles, and sucking the cleaning solution by the liquid transfer module to finish one-time cleaning.
(III) detection of antibody binding
(IV) magnetic bead transfer washing
(V) Signal detection
And after the cleaning in the previous step is finished, the signal measurement module performs signal acquisition and analysis on the signals to obtain corresponding concentration values, the total time is less than 20 minutes, and a standard curve chart is shown in figure 1.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A kit for detecting the content of soluble endothelial factor in human body fluid is characterized by comprising a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent and a luminescent substrate.
2. The kit for detecting the content of the soluble endoglin in the human body fluid according to claim 1, wherein the anti-reagent is an anti-soluble endoglin labeled antibody labeled with alkaline phosphatase.
3. The kit for detecting the content of the soluble endoglin in the human body fluid according to claim 2, wherein the magnetic particle reagent is a magnetic particle-anti-soluble endoglin coated antibody conjugate.
4. The kit for detecting the content of the soluble endoglin in the human body fluid according to claim 3, wherein the chemiluminescent substrate is AMPPD.
5. The kit of claim 4, wherein the sample source of the human body fluid is one or more of serum, plasma, cerebrospinal fluid, ascites, or amniotic fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011609713.XA CN112816702A (en) | 2020-12-29 | 2020-12-29 | Kit for detecting content of soluble endothelial factor in human body fluid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011609713.XA CN112816702A (en) | 2020-12-29 | 2020-12-29 | Kit for detecting content of soluble endothelial factor in human body fluid |
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| Publication Number | Publication Date |
|---|---|
| CN112816702A true CN112816702A (en) | 2021-05-18 |
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| CN202011609713.XA Pending CN112816702A (en) | 2020-12-29 | 2020-12-29 | Kit for detecting content of soluble endothelial factor in human body fluid |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103109192A (en) * | 2010-06-18 | 2013-05-15 | 塞尚公司 | Markers for the prognosis and risk assessment of pregnancy-induced hypertension and preeclampsia |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
| CN107543927A (en) * | 2017-11-03 | 2018-01-05 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of placenta growth factor |
| CN107807241A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of intravascular ErbB1 |
-
2020
- 2020-12-29 CN CN202011609713.XA patent/CN112816702A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103109192A (en) * | 2010-06-18 | 2013-05-15 | 塞尚公司 | Markers for the prognosis and risk assessment of pregnancy-induced hypertension and preeclampsia |
| WO2016127301A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Rt3 chemiluminescent immunological detection reagent kit, and detection method and application therefor |
| CN107543927A (en) * | 2017-11-03 | 2018-01-05 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of placenta growth factor |
| CN107807241A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of intravascular ErbB1 |
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Application publication date: 20210518 |