CN114252592B - Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof - Google Patents

Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof Download PDF

Info

Publication number
CN114252592B
CN114252592B CN202111388930.5A CN202111388930A CN114252592B CN 114252592 B CN114252592 B CN 114252592B CN 202111388930 A CN202111388930 A CN 202111388930A CN 114252592 B CN114252592 B CN 114252592B
Authority
CN
China
Prior art keywords
tyrosine kinase
soluble fms
sflt
chemiluminescent
kit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111388930.5A
Other languages
Chinese (zh)
Other versions
CN114252592A (en
Inventor
秦静
吴晗琪
詹雯雯
王保升
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Wondfo Biotech Co Ltd
Original Assignee
Guangzhou Wondfo Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Wondfo Biotech Co Ltd filed Critical Guangzhou Wondfo Biotech Co Ltd
Priority to CN202111388930.5A priority Critical patent/CN114252592B/en
Publication of CN114252592A publication Critical patent/CN114252592A/en
Application granted granted Critical
Publication of CN114252592B publication Critical patent/CN114252592B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Endocrinology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a soluble fms-like tyrosine kinase-1 detection kit, a preparation method and application thereof, wherein the inventor adopts a double-antibody sandwich method and magnetic particle chemiluminescence technology to detect the soluble fms-like tyrosine kinase-1, and discovers that the detection performance is obviously improved, the detection sensitivity reaches 0.97pg/mL, the detection linear range is wide, and the detection linear range can reach 10 pg/mL-85000 pg/mL. It is predicted to have wide detection application prospect.

Description

Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a soluble fms-like tyrosine kinase-1 detection kit and a preparation method and application thereof.
Background
VEGFR-1 (Flt-1) is one of the vascular endothelial growth factor receptors (vascular endothelial growth factor receptor, VEGFR), and soluble fms-like tyrosine kinase-1 (sFlt-1) is a soluble form thereof, which is devoid of signal transduction functions due to lack of transmembrane region and tyrosine kinase activity, and can be used as a VEGF antagonist. Soluble fms-like tyrosine kinase-1 (sFlt-1) is a glycoprotein with tyrosine kinase activity, belongs to the class III receptor tyrosine kinase family, is a potent inhibitor of Vascular Endothelial Growth Factor (VEGF) and placental growth factor (PLGF), and can bind to VEGF and PLGF to inactivate the same. Normally, placental vascular network formation is maintained in equilibrium by VEGF and its antagonistic factors (including sFlt-1). Peripheral blood sFlt-1 expression in preeclampsia patients is abnormally elevated, disrupting the balance, causing reduced angiogenesis, leading to endothelial cell activation and injury. sFlt-1 has the effect of preventing the growth of blood vessels, and research shows that sFlt-1 probably participates in the development of Preeclampsia (PE). Thus, sFlt-1 may be used as a biological indicator for predicting the onset and progression of preeclampsia.
Placenta is the major source of sFlt-1 during pregnancy. Increased sFlt-1 is accompanied by imbalance of the dynamic balance of placenta angiogenesis caused by PLGF decrease, so that the invasion of trophoblasts to endometrium is insufficient, thus the uterine spiral arteriole recasting disorder causes ischemia hypoxia of placenta, and further increased sFlt-1 also causes dysfunction of maternal whole-body vascular endothelial cells, thereby preeclampsia occurs. The serum level of sFlt-1 in preeclampsia pregnant women was elevated and the PLGF level was decreased prior to the onset of the disease. The combined measurement of the ratio of serum sFlt-1 to PLGF in pregnant women is more valuable than the combined measurement of sFlt-1 and PLGF in diagnosing the onset of preeclampsia.
Preeclampsia (PE) is a serious complication of pregnancy with the main clinical manifestations being hypertension and proteinuria around 20 weeks after pregnancy. About 3-5% of pregnant women develop preeclampsia and can lead to death of the parturient, fetus, or neonate. Preeclampsia is associated with thrombocytopenia and elevated liver enzyme activity and may manifest as thrombocytopenia syndrome (hemolysis, elevated liver enzyme, thrombocytopenia) with varying degrees of clinical symptoms. Preeclampsia occurs due to endothelial dysfunction caused by the release of angiogenic factors from the placenta. Serum PLGF and sFlt-1 concentration levels in women suffering from preeclampsia are altered. In addition, levels of PLGF and sFlt-1 in the blood circulation may be used to identify normal pregnancy and preeclampsia prior to the onset of clinical symptoms. The level of the pro-vascular factor PLGF increases in the first 6 months of normal pregnancy and gradually decreases as pregnancy progresses until termination. In contrast, the level of the anti-vascular factor sFlt-1 remained stable in early and mid gestation and did not rise smoothly until gestation was terminated. Women suffering from preeclampsia have sFlt-1 levels above normal pregnancy levels while PLGF levels are below normal pregnancy levels. Determining the ratio of sFlt-1 to PLGF is more valuable than detecting sFlt-1 or PLGF alone. Placental endothelial factor is a member of the TGF- β family that is upregulated in preeclampsia and released into the maternal blood circulation in the form of soluble endothelial factor. Soluble endoglin has been shown to be significantly elevated in severe cases of preeclampsia.
Therefore, the detection of blood sFlt-1 level in pregnant women can be clinically used for identifying the existence of oxygen supply pressure in placenta-syngeneic trophoblast cells. Can be used for pregnancy-induced hypertension prediction and auxiliary diagnosis.
The traditional placenta growth factor determination method comprises an enzyme-linked immunosorbent assay and a chemiluminescence method, and most of the current domestic sFlt-1 detection methods are the enzyme-linked immunosorbent assay and the fluorescence chromatography. The ELISA method is long in time consumption and complex in operation. In addition, the two detection methods have poor detection sensitivity and narrow detection range, and are not fully automatic. The foreign sFlt-1 detection method has an electrochemiluminescence immunoassay method, but the detection cost of the method is high, so that the method cannot be suitable for pregnancy in resource-limited areas.
Disclosure of Invention
Based on the above, an object of the present invention is to provide a method for preparing a chemiluminescent labeling protein.
The method comprises the following technical scheme:
(1) Protein activation: activating the protein by using a cross-linking agent;
(2) Chemiluminescent activation: activating the chemiluminescent label with dithiothreitol;
(3) Coupling: mixing the chemiluminescent label activated in the step (2) with the activated protein in the step (1), and incubating to obtain a chemiluminescent label-protein conjugate;
(4) Closing: adding a blocking agent into the chemiluminescent label-protein conjugate obtained in the step (3) for blocking to obtain a chemiluminescent label-protein conjugate final product;
Wherein the cross-linking agent in the step (1) is selected from any one of SPDP, DTBP, DTSSP, AMAS, and the blocking agent in the step (4) is MMTS.
It is also an object of the present invention to provide a chemiluminescent label protein obtainable according to the above-described preparation method.
The invention also aims to provide the application of the chemiluminescent labeling antibody in preparing a kit or a kit detection reagent.
The invention also aims at providing the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody prepared by the preparation method.
The invention also aims at providing a soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit.
The method comprises the following technical scheme:
a chemiluminescent immunoassay kit for soluble fms-like tyrosine kinase-1 comprises the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody and soluble fms-like tyrosine kinase-1 capture antibody magnetic particles.
It is also an object of the present invention to provide a method for quantitative detection of soluble fms-like tyrosine kinase-1 for non-disease diagnostic purposes.
The method comprises the following technical scheme:
establishing a standard fitting curve according to the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit;
Obtaining a sample to be detected, detecting by adopting the soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit, recording the luminescence value of the sample to be detected, and substituting the luminescence value into the standard fitting curve to obtain the concentration of the soluble fms-like tyrosine kinase-1 in the sample.
The inventor of the invention aims to solve the problems of poor sensitivity, narrow detection range and the like of the detection of soluble fms-like tyrosine kinase-1 (sFlt-1) in the prior art, adopts a double-antibody sandwich method and combines a magnetic particle chemiluminescence technology to detect the sFlt-1, and in the preparation process of a chemiluminescent substance marked sFlt-1 detection antibody, the inventor surprisingly finds that the preparation method of a chemiluminescent substance marked protein after optimization is adopted, particularly when the chemiluminescent substance is alkaline phosphatase after DTT activation, and a protein cross-linking agent is selected from SPDP, DTBP, DTSSP, AMAS, the alkaline phosphatase marked sFlt-1 detection antibody prepared by a specific proportion reaction and the sFlt-1 capture antibody magnetic particle prepared by the inventor through a specific proportion cross-linking agent BS (PEG) 5 can automatically finish the detection by taking a full-automatic chemiluminescent immunoassay analyzer, and the detection performance is also obviously improved, and the detection sensitivity reaches 0.97/mL, compared with the traditional method of detecting the alkaline phosphatase marked sFlt-1 with the sensitivity of at least 10 pg/85 mL, and the sensitivity of the sample is improved by at least 10 pg/85 mL. It is predicted to have wide detection application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation flow of a soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of the present invention.
FIG. 2 is a standard graph of placenta growth factor obtained from the test series sFlt-1 standard in example 2.
FIG. 3 is a statistical chart of correlation analysis of the detection results of the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit and the Roche kit of the present invention in example 4.
Detailed Description
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present invention does not necessarily refer to the same embodiment, although it may. Furthermore, the phrase "in another embodiment" as used in the present invention does not necessarily refer to a different embodiment, although it may. Accordingly, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention.
Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification, the meaning of "a", "an", and "the" include plural referents. The meaning of "in" is included "in" and "on".
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The abbreviations and terms involved in the present invention are defined as follows:
BS (PEG) 5: pegylated bis (sulfosuccinimidyl) suberic acid
SFlt-1: soluble fms-like tyrosine kinase-1
SPDP: succinimide 3- (2-pyridyldithio) -propionic acid ester
MMTS: methyl thiomethane sulfonate
BMPS: n-beta-maleimidopropyl succinimidyl oxide ester
BM (PEG) 3: 1, 11-bismaleimide-triethylene glycol
DTBP: dithiopropyliminoacid dimethyl ester dihydrochloride
DTSSP:3,3' -dithiobis (sulfosuccinimidyl) propionate
AMAS: n-alpha-maleimidosuccinimide esters
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the present invention provide a method for preparing a chemiluminescent labeling protein, comprising the steps of:
(1) Protein activation: activating the protein by using a cross-linking agent;
(2) Chemiluminescent activation: activating the chemiluminescent label with dithiothreitol;
(3) Coupling: mixing the chemiluminescent label activated in the step (2) with the activated protein in the step (1), and incubating to obtain a chemiluminescent label-protein conjugate;
(4) Closing: adding a blocking agent into the chemiluminescent label-protein conjugate obtained in the step (3) for blocking to obtain a chemiluminescent label-protein conjugate final product;
Wherein the cross-linking agent in the step (1) is selected from any one of SPDP, DTBP, DTSSP, AMAS, and the blocking agent in the step (4) is MMTS.
In some embodiments, the crosslinking agent in step (1) of the above preparation method is preferably DTBP.
In some embodiments, in the preparation method step (1), the mass ratio of the protein to the cross-linking agent is 100 (0.5-2.0). It is further preferred that when the mass ratio of the protein to the crosslinking agent is 100:1.5, and when the crosslinking agent is a 5mg/mL DTBP solution, DTBP is used as an antibody activator, so that the antibody can be activated with maximum efficiency, and the coupling efficiency of the subsequent antibody and the chemiluminescent label can be greatly improved. Thereby enabling to improve the detection sensitivity.
In some embodiments, the protein and the cross-linking agent are used in equal concentrations during the activation treatment in step (1), in a manner that the activation is performed at room temperature for 1 hour.
In some of these embodiments, the activation of the chemiluminescent label in step (2) is performed using DTT at a concentration of 5mg/mL and incubated for 1h at room temperature.
In some embodiments, the blocking agent used in step (4) is MMTS at 5mg/mL, and further, a desalting purification treatment is performed, preferably, the above desalted and purified conjugate is recovered to 0.1mg/mL with 0.01M PBS buffer at pH 7.4, and then an equal volume of glycerol is added and mixed to obtain the chemiluminescent label-tagged protein.
In some embodiments, in the above preparation method step (2), the chemiluminescent label is selected from one of acridinium ester, ruthenium terpyridyl, adamantane, luminol, a derivative of luminol, isoluminol, a derivative of isoluminol, horseradish peroxidase and alkaline phosphatase. Of course, in other embodiments, the labels in the label-labeled antibodies are not limited to the above, but may be other substances useful in chemiluminescent immunoassay platforms.
In some embodiments, the chemiluminescent label is alkaline phosphatase.
In some embodiments, in step (1) of the above preparation method, the protein comprises an antigen and an antibody, further the protein is an antibody, more preferably a soluble fms-like tyrosine kinase-1 monoclonal antibody.
Some embodiments of the invention also provide a chemiluminescent label protein obtained according to the preparation method described above.
Some embodiments of the invention also provide the use of the chemiluminescent labeled antibody described above in the preparation of a kit or kit reagent.
Some embodiments of the invention also provide an alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody, which is prepared according to the preparation method described above.
Some embodiments of the invention also provide a soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit comprising the above alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody and soluble fms-like tyrosine kinase-1 capture antibody magnetic particles.
In some embodiments, the above-mentioned soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit is characterized in that the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles are activated by a cross-linking agent selected from any one of BS (PEG) 5, BMPS and BM (PEG) 3.
In some embodiments, the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles are prepared according to the following steps:
(1) Pretreatment: pretreating magnetic particles to obtain a mixed solution 1;
(2) Activating: activating the antibody by using a cross-linking agent;
(3) Coupling: adding the activated antibody in the step (2) into the mixed solution 1 obtained in the step (1) to obtain a mixed solution 2, and incubating to obtain an antibody-magnetic bead conjugate;
(4) Closing: adding a blocking solution into the antibody-magnetic bead conjugate obtained in the step (3) to block to obtain an immune magnetic particle end product of the antibody-magnetic bead conjugate;
Wherein the cross-linking agent in step (2) is selected from any one of BS (PEG) 5, BMPS and BM (PEG) 3; the mass ratio of the antibody to the cross-linking agent used in the activation treatment is 10 3 (0.5-3.0). It is further preferable that the mass ratio of the antibody to the crosslinking agent is 10 3:1.5.
In some embodiments, the cross-linking agent in the step (2) of the preparation method is preferably BS (PEG) 5, and when the mass ratio of the antibody to the cross-linking agent BS (PEG) 5 is 10 3:1.5, the steric hindrance is reduced by changing the conjugated structure of the antibody, so that the covalent binding of the antibody to the magnetic particles is more efficient, and the prepared immunomagnetic particles can reduce the blocking of the active site of the antibody bound to the antigen, thereby improving the binding antigen activity of the antibody, and further improving the stability and sensitivity of the immunoassay method.
In some embodiments, in the step (1) of the preparation method, the mixed solution 1 is obtained by uniformly dispersing magnetic particles in an ammonium sulfate solution after washing and resuspension with a buffer solution; further, the concentration of the ammonium sulfate solution is 2-5M, and the pH value is 8-10. More preferably, the concentration of the ammonium sulfate solution used is 3M and the pH is 9.5, and at this time, ammonium sulfate is used as a reaction accelerator, so that the subsequent binding speed of the magnetic beads and the protein can be increased.
In some embodiments, in the above preparation method step (1), the buffer is preferably a PBS buffer or a BBS buffer; more preferably, the magnetic particles are washed with 0.01M PBS buffer at pH 7.4, and the supernatant is magnetically separated and resuspended in 0.1M BBS buffer at pH 9.5.
In some embodiments, in the step (1) of the preparation method, the magnetic particles are modified by tosyl, that is, when the active group of the magnetic particles is tosyl, the magnetic particles can be coupled with protein, so that the coupled protein can be combined with the to-be-detected object to realize efficient separation and detection of the to-be-detected object by using the action of an external magnetic field.
In some embodiments, in the above preparation method step (1), the tosylated magnetic particles have a particle diameter of 0.9 μm to 1.8 μm.
In some of these embodiments, in step (4) of the above preparation method, the blocking solution is 0.05M Tris buffer with 0.5% BSA, pH 7.4.
In some embodiments, in the preparation method step (4), the mass ratio of the antibody to the magnetic beads in the antibody-magnetic bead coupled immunomagnetic particle end product is 1 (10-50). More preferably, the mass ratio of the antibody to the magnetic beads is 1:25.
In some embodiments, the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit further comprises a soluble fms-like tyrosine kinase-1 standard, further wherein the soluble fms-like tyrosine kinase-1 standard is formulated with a calibration buffer comprising 45 mM-55 mM Tris,0.05% -0.15% BSA and 0.9% NaCl. Preferably, the calibration buffer comprises 50mM Tris,0.1%BSA,0.9%NaCl,pH7.5.
In some of these embodiments, the disc growth factor chemiluminescent immunoassay kit further comprises a chemiluminescent substrate solution, further preferably an APS substrate solution.
Some embodiments of the present invention also provide a method for quantitatively detecting soluble fms-like tyrosine kinase-1 for non-disease diagnostic purposes, comprising the steps of:
establishing a standard fitting curve according to the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit;
Obtaining a sample to be detected, detecting by adopting the soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit, recording the luminescence value of the sample to be detected, and substituting the luminescence value into the standard fitting curve to obtain the concentration of the soluble fms-like tyrosine kinase-1 in the sample.
In some embodiments, the sample to be tested is serum.
In some embodiments, the quantitative detection method specifically includes: taking 20-100 mu L of serum sample, adding 20-100 mu L of magnetic particles of soluble fms-like tyrosine kinase-1 capture antibody and 20-100 mu L of alkaline phosphatase labeled soluble fms-like tyrosine kinase-1 detection antibody, reacting for 5-15 min, performing magnetic separation, adding 100-300 mu L of substrate solution, mixing, sending the reaction mixture into a darkroom by an instrument, and finally recording the luminescence value. Preferably, 50. Mu.L of serum sample is taken, 50. Mu.L of magnetic particles of the soluble fms-like tyrosine kinase-1 capture antibody and 50. Mu.L of alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody are added, the reaction is carried out for 10min, then magnetic separation is carried out, 200. Mu.L of substrate solution is added, after uniform mixing, the reaction mixture is sent into a darkroom by an instrument, and finally the luminescence value is recorded.
EXAMPLE 1 preparation of sFlt-1 chemiluminescent immunoassay kit
(1) Preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles:
A suspension containing 90mg of tosylated magnetic particles (MagnosphereTM, cat# MS 160) with a particle size of 0.9 μm-1.8 μm was taken, the supernatant was magnetically separated, the magnetic particles were washed with 0.01M PBS buffer with a pH of 7.4, the supernatant was magnetically separated, resuspended with 0.1M BBS buffer with a pH of 9.5, 4mL of 3M ammonium sulfate solution with a pH of 9.5 was added, the mixture was uniformly dispersed to give a mixed solution 1, and then 5.4. Mu.g of BS (PEG) 5 (PEGylated bis (sulfosuccinimidyl) suberic acid, thermoFisher SCIENTIFIC) was added to 3.6mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (Kyowa Biotechnology Co., ltd., cat# sFlt.; cat# sFlt-1-10#) to activate sFlt-1 monoclonal antibody, and the activated antibody was added to the mixed solution 1 to give a mixed solution 2. Then placing the mixed solution 2 on a rotary shaking table for the first incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH value of 7.4 after magnetically separating, adding a BSA solution with the concentration of 0.5 percent after removing the supernatant, carrying out the second incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH value of 7.4 after magnetically separating, and finally, resuspending the supernatant to 10mg/mL by using a Tris buffer solution with the concentration of 0.05M and the pH value of 7.4, thereby obtaining toluene yellow acylated magnetic particles coated by the sFlt-1 monoclonal antibody, wherein the corresponding magnetic bead mother liquor is named as follows: sFlt-1-R1- ①; stored at 4℃for further use.
(2) Preparation of alkaline phosphatase-labeled sFlt-1 monoclonal antibody:
Taking a solution containing 1.2mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (Wanfu Biotechnology Co., ltd., guangzhou, cat# sFlt-1-12), centrifuging, removing the supernatant, replacing the liquid in the original antibody to 5mg/mL with 0.01M PBS buffer with pH of 7.4, adding 3.6 mu L of DTBP (dimethyl dithiopropyliminoate dihydrochloride, thermoFisher SCIENTIFIC), and incubating for 1h at room temperature to activate the antibody; then, 2.88. Mu.L of DTT (dithiothreitol) at a concentration of 5mg/mL was added to 72. Mu.L of alkaline phosphatase at a concentration of 20mg/mL, and incubated at room temperature for 1 hour to activate alkaline phosphatase; the activated antibody and alkaline phosphatase were then mixed together and incubated at room temperature for 1h to crosslink. Finally, adding 6.48 mu L of MMTS (methyl thiomethanesulfonate, thermoFisher SCIENTIFIC) with the concentration of 5mg/mL into the crosslinked mixture for sealing, desalting and purifying, recovering the desalted and purified conjugate to 0.1mg/mL by using a PBS buffer with the pH of 7.4 and 0.01M, adding equal volume of glycerol, and uniformly mixing to obtain an alkaline phosphatase marked soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate, wherein the corresponding enzyme-marked mother solution is named as: sFlt-1-R2- ①, stored at-20deg.C for use.
(3) Preparation of sFlt-1 standard:
The soluble fms-like tyrosine kinase-1 (R & D Systems, inc.) was formulated with a standard buffer (50mM Tris,0.1%BSA,0.9%NaCl,pH7.5) at a concentration of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, 5000pg/mL, 8000pg/mL, 11000pg/mL, and dispensed at 0.5mL per tube for storage at-20 ℃.
Example 2 detection method of sFlt-1 chemiluminescent immunoassay kit
The detection principle of the kit of the invention is as follows: the magnetic particles coated with the soluble fms-like tyrosine kinase-1 (sFlt-1) capture antibody are combined with alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 (sFlt-1) detection antibody to form a "sandwich" complex with placenta growth factor in a sample, calibrator or quality control. Under the action of an externally applied magnetic field, separating the compound formed by the immune reaction from other unbound substances, cleaning the compound, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, and photons are emitted when the excited state intermediate returns to the ground state to form a luminescence reaction, so that the luminescence intensity of the reaction can be detected by using a chemiluminescent instrument. Meanwhile, the luminous marker alkaline phosphatase is not basically consumed, and the luminous agent in the reaction system is sufficiently excessive, so that the luminous signal is strong and stable, the luminous time is long, the luminous intensity is in direct proportion to the content of placenta growth factor in the sample in the detection range, and the concentration of sFlt-1 in the sample can be calculated by referring to a standard curve.
A full-automatic chemiluminescence immunoassay analyzer (model FC-302, kwando Biotechnology Co., ltd.) is used as a detection tool, the method mode is double antibody sandwich, namely 50 mu L of serum sample is sequentially added into the analyzer, 50 mu L of toluene sulfonylation magnetic particles coated by soluble fms-like tyrosine kinase-1 monoclonal antibody and 50 mu L of alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody are added into the analyzer, after 10min of reaction, magnetic separation is carried out, then 200 mu L of substrate solution is added into the analyzer, after the mixture is uniformly mixed, the analyzer sends the reaction mixture into a darkroom, and finally the luminescence value is recorded.
The sFlt-1 standard prepared in example 1 was tested by the method described above to give a standard curve as shown in FIG. 2.
Example 3 optimization of sFlt-1 chemiluminescent immunoassay kit
(1) Screening of antibody activators in the preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles:
A mixed solution 1 was prepared as described in reference example 1, followed by adding 5.4. Mu.g of BS (PEG) 5, 5.4. Mu.g of BMPS (N-. Beta. -maleimidopropyl-succinimidyl oxide, thermoFisher SCIENTIFIC) and 5.4. Mu.g of BM (PEG) 3 (1, 11-bismaleimidyl-triethylene glycol, thermoFisher SCIENTIFIC) to 3.6mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (Wanfu Biotechnology Co., ltd., guangzhou) respectively, and adding the activated antibodies to the mixed solution 1 to obtain 3 kinds of mixed solutions 2. Then placing the 3 mixed liquids 2 on a rotary shaking table for first incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH of 7.4, removing the supernatant, then adding a BSA solution with the concentration of 0.5%, performing second incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH of 7.4 after magnetic separation, finally resuspending the supernatant to 10mg/mL by using a Tris buffer solution with the concentration of 0.5% BSA and the pH of 7.4, respectively obtaining toluene-yellow acylated magnetic particles coated by 3 soluble fms-like tyrosine kinase-1 monoclonal antibodies (wherein, the name of the magnetic bead mother liquor corresponding to a BS (PEG) 5 coupling agent is sFlt-1-R1- ①, the name of the magnetic bead mother liquor corresponding to a BMPS coupling agent is sFlt-1-R1- ③), storing the magnetic bead mother liquor corresponding to the sFlt-1-R1- ③ at the temperature of 4 ℃, and carrying out linear assay on the three monoclonal antibodies by using the magnetic beads, respectively carrying out the linear assay of the magnetic beads, and carrying out the linear assay on the alkaline assay of the monoclonal antibodies.
TABLE 3-1 results of Performance verification of magnetic particles coated with sFlt-1 antibodies by different coupling agents
As can be seen from Table 3-1 above, the signal to noise ratio (S/N) of the sFlt-1-R1- ① was maximized, the repeatability was within 5% and the linearity R was optimal for the sFlt-1-R1- ① bead coatings compared to the sFlt-1-R1- ② and the sFlt-1-R1- ③ bead coatings.
Thus, in the preparation of the tosylated magnetic particles coated with sFlt-1 monoclonal antibody, the antibody activator used is preferably BS (PEG) 5.
(2) Screening of antibody activator concentration in preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles:
A mixed solution 1 was prepared as described in reference example 1, then 0.8. Mu.g of BS (PEG) 5, 1.5. Mu.g of BS (PEG) 5 and 3.0. Mu.g of BS (PEG) 5 were added to 1mg of the soluble fms-like tyrosine kinase-1 monoclonal antibody (sFlt-1-10 # of Kyowa Biotechnology Co., ltd.) to activate the soluble fms-like tyrosine kinase-1 monoclonal antibody, and 3 kinds of mixed solutions 2 were obtained by adding the activated antibodies to the mixed solution 1, respectively. Then placing the 3 mixed liquids 2 on a rotary shaking table for first incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH of 7.4, adding a BSA solution with the concentration of 0.5 percent for second incubation treatment, washing the supernatant by using a Tris buffer solution with the concentration of 0.05M and the pH of 7.4 after magnetic separation, finally re-suspending the supernatant to 10mg/mL by using a Tris buffer solution with the concentration of 0.5 percent BSA and the pH of 7.4 to obtain toluene-yellow acylated magnetic particles coated by 3 soluble fms-like tyrosine kinase-1 monoclonal antibodies (wherein the name of a magnetic bead mother liquor corresponding to 0.8 mu g of BS (PEG) 5 coupling agent is sFlt-1-R1- ④, the name of a magnetic bead mother liquor corresponding to 1.5 mu g of BS (PEG) 5 coupling agent is sFlt-1-R1- ⑤, the name of a BS (PEG) 5 ℃ C. Corresponds to sFlt-1-R1-35), and carrying out linear assay on the magnetic beads by using the magnetic bead buffer solution, and carrying out repeated test on the magnetic beads coated by the magnetic beads, respectively carrying out alkaline assay on the magnetic beads, wherein the magnetic beads are coated by the magnetic beads coated by 3 soluble fms-like tyrosine kinase-1 monoclonal antibodies, and the magnetic beads are subjected to the alkaline assay.
TABLE 3-2 results of Performance verification of BS (PEG) 5 coupling agent magnetic particle coated sFlt-1 antibodies at different concentrations
As can be seen from Table 3-2, the signal to noise ratio (S/N), repeatability and linearity performance of the sFlt-1-R1- ④ compared to the sFlt-1-R1- ⑤ and the sFlt-1-R1- ⑥ bead coatings, the sFlt-1-R1- ⑤ and the sFlt-1-R1- ⑥ bead coatings were comparable, but were all superior to the performance of sFlt-1-R1- ④, and finally the sFlt-1-R1- ⑤ was preferred.
Thus, in the placenta growth factor monoclonal antibody-coated tosylated superparamagnetic particles, the mass ratio of the antibody used to the antibody activator BS (PEG) 5 is preferably 10: 10 3:1.5.
(3) Screening of crosslinking agent in preparation of alkaline phosphatase-labeled sFlt-1 monoclonal antibody:
1.2mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (product number: sFlt-1-12# by Wanfu Biotechnology Co., ltd., guangzhou) was used to replace the original antibody with 0.01M PBS buffer at pH 7.4 to 5mg/mL, followed by addition of 3.6. Mu.L of DTBP at 5mg/mL, 3.6. Mu.L of SPDP at 5mg/mL (succinimid 3- (2-pyridyldithio) -propionate, thermoFisher SCIENTIFIC), 3.6. Mu.L of DTSSP (3, 3' -dithiobis (sulfosuccinimidpropionate), thermoFisher SCIENTIFIC) and 3.6. Mu.L of AMAS (N-. Alpha. -maleimidosuccinimidyl ester, thermoFisher SCIENTIFIC) at 5mg/mL, respectively, and incubation of the activated antibody at room temperature for 1 h; then adding 2.88 mu L of DTT with the concentration of 5mg/mL into 72 mu L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate alkaline phosphatase; the activated antibody and alkaline phosphatase were then mixed together and incubated at room temperature for 1h to crosslink. Finally, adding 6.48 mu L of MMTS with the concentration of 5mg/mL into the crosslinked mixture for sealing, desalting and purifying, recovering the desalted and purified conjugate to 0.1mg/mL by using a PBS buffer with the pH of 7.4 and 0.01M, adding glycerol with the same volume, uniformly mixing to obtain an alkaline phosphatase marked soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate (wherein an enzyme-labeled mother solution corresponding to a DTBP crosslinking agent is named as sFlt-1-R2- ①, a magnetic bead mother solution corresponding to a SPDP crosslinking agent is named as sFlt-1-R2- ②, a magnetic bead mother solution corresponding to a DTSSP crosslinking agent is named as sFlt-1-R2- ③, a magnetic bead mother solution corresponding to an AMAS crosslinking agent is named as sFlt-1-R2- ④), storing the alkaline phosphatase marked soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate at the temperature of-20 ℃, and carrying out repeated test on toluene sulfonyl sensitivity test reagents of the toluene-like tyrosine-like monoclonal antibody corresponding to the three enzyme-labeled toluene-like tyrosine kinase-1 monoclonal antibody respectively at the temperature of-20 ℃ for later use, and carrying out repeated test on the test to obtain the test results of the toluene-coated monoclonal antibody.
TABLE 3-3 results of Performance validation of alkaline phosphatase-labeled sFlt-1 antibodies with different crosslinking agents
/>
As can be seen from the above tables 3-3, the signal to noise ratio (S/N) of the sFlt-1-R2- ① enzyme markers is maximum, the repeatability is within 5% and the linearity R is optimal when compared with the sFlt-1-R2- ②、sFlt-1-R2-③ and sFlt-1-R2- ④ enzyme markers.
Thus, in the preparation of alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 monoclonal antibodies, the cross-linking agents used are preferably DTBP and DTT, and the blocking agent is MMTS.
(4) Screening of crosslinker concentration in preparation of alkaline phosphatase-labeled sFlt-1 monoclonal antibody:
1.2mg of soluble fms-like tyrosine kinase-1 monoclonal antibody (product number: sFlt-1-12# of Kwando Biotechnology Co., ltd., guangzhou) was used to replace the original antibody with 0.01M PBS buffer at pH 7.4 to 5mg/mL, then 1.2. Mu.L, 2.4. Mu.L, 3.6. Mu.L and 4.8. Mu.L of DTBP at 5mg/mL were added, and incubated for 1h at room temperature, respectively, to activate the antibody; then adding 2.88 mu L of DTT with the concentration of 5mg/mL into 72 mu L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate alkaline phosphatase; the activated antibody and alkaline phosphatase were then mixed together and incubated at room temperature for 1h to crosslink. Finally, adding 6.48 mu L of MMTS with the concentration of 5mg/mL into the crosslinked mixture for sealing, desalting and purifying, recovering the desalted and purified conjugate to 0.1mg/mL by using a PBS buffer with the pH of 7.4 and 0.01M, adding glycerol with the same volume, uniformly mixing to obtain an alkaline phosphatase marked soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate (wherein 2.4 mu L of enzyme marked mother liquor corresponding to the DTBP crosslinking agent with the concentration of 5mg/mL is named sFlt-1-R2- ⑤, 3.6 mu L of enzyme marked mother liquor corresponding to the DTBP crosslinking agent with the concentration of 5mg/mL is named sFlt-1-R2- ⑥, and 4.8 mu L of enzyme marked mother liquor corresponding to the DTBP crosslinking agent with the concentration of 5mg/mL is named sFlt-1-R2- ⑦), storing the alkaline phosphatase marked soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate at the temperature of minus 20 ℃, and subsequently carrying out repeated test on the three enzyme marked soluble fms-like tyrosine kinase-like monoclonal antibody with the concentration of 5mg/mL respectively, and carrying out repeated test on the magnetic sensitive assay to the ligand-like toluene-like ligand with the ligand, namely, the magnetic assay table-like ligand, and the magnetic assay reagent.
Tables 3-4 results of performance verification of alkaline phosphatase-labeled sFlt-1 antibodies with DTBP crosslinking agent at various concentrations
As can be seen from tables 3-4, the signal to noise ratio (S/N), repeatability and linearity of the magnetic bead coatings of sFlt-1-R2- ⑤、sFlt-1-R2-⑥ and sFlt-1-R2- ⑦ enzyme markers are comparable, but are better than those of sFlt-1-R2- ⑤, and finally sFlt-1-R2- ⑥ is preferred.
Therefore, in the preparation of the alkaline phosphatase-labeled placenta growth factor monoclonal antibody, the mass ratio of the antibody to the cross-linking agent SPDP is preferably 100:1.5.
EXAMPLE 4 evaluation of the Performance of sFlt-1 chemiluminescent immunoassay kit
The soluble fms-like tyrosine kinase-1 standard was tested using the procedure of example 2 to give a standard curve as shown in FIG. 2.
And then testing the actual sample, and calculating the sample concentration according to the sample luminescence value.
(1) Detection of sensitivity:
Referring to the CLSI EP17-A document recommended experimental protocol, the sensitivity of the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit was calculated to be 0.97pg/mL.
(2) Detection of linearity:
Linear analyses were performed on the standards of 0pg/mL, 15pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 5000pg/mL, 10000pg/mL, 20000pg/mL, 40000pg/mL, 86000pg/mL, and the linear correlation coefficient was calculated, r=0.9993, and the linear range of the kit for detection of the soluble fms-like tyrosine kinase-1 sample was 10pg/mL to 85000pg/mL.
(3) And (3) detecting precision:
Two soluble fms-like tyrosine kinase-1 samples were taken at concentrations of 200pg/mL and 40000pg/mL, each sample was tested 10 times, each sample was tested with three kits, and the intra-and inter-batch differences of the kit were calculated, indicating that the intra-batch differences were less than 5% and the inter-batch differences were less than 10%, as shown in Table 4 below.
(4) Interference experiments:
Taking mixed serum and adding interferents respectively comprises the following steps: bilirubin (10 mg/dL), hemoglobin (500 mg/dL) and triglyceride (1000 mg/dL) were added in a mass ratio of 1:20, and the measurement values of the mixed serum and the mixed serum after adding various interferents were measured, respectively. The deviation between the two was calculated to be within an acceptable range of + -10%, and the specific results are shown in Table 4 below. The results show that the interference reaches the file standard of NCCLS, and can be used for accurately evaluating the conditions of the soluble fms-like tyrosine kinase-1 in clinical laboratories.
TABLE 4sFlt-1 chemiluminescent immunoassay kit detection results
(5) Detection of clinical performance:
128 clinical samples were taken and tested with the kit of the present invention, luminescence values were determined, and concentrations were calculated according to a standard curve (as shown in fig. 2).
The concentration of the clinical samples was also determined using the soluble fms-like tyrosine kinase-1 detection kit (electrochemiluminescence method) from Roche Inc.
The detection concentration of the kit prepared in the embodiment of the invention is analyzed and compared with the concentration measurement result of a soluble fms-like tyrosine kinase-1 detection kit (electrochemiluminescence method) of Roche company, the clinical correlation result is shown in figure 3, wherein the clinical correlation of the soluble fms-like tyrosine kinase-1 is R 2 = 0.9757, and the kit has good correlation with the Roche kit.
Example 5 comparative experiments with sFlt-1 chemiluminescent immunoassay kit
Soluble fms-like tyrosine kinase-1 samples at concentrations of 0pg/mL and 15pg/mL were tested by chemiluminescence detection and conventional enzyme-linked immunosorbent assay (R & D Systems, inc.; product number: DVR 100C), respectively, and the data for the comparison of the detection sensitivities of the two methods are shown in Table 5-1 below:
TABLE 5-1
/>
As can be seen from the above table, the sensitivity of the chemiluminescent detection method is improved by about 8 times compared with that of the ELISA method.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (12)

1. A chemiluminescent immunoassay kit for soluble fms-like tyrosine kinase-1 is characterized by comprising an alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody and soluble fms-like tyrosine kinase-1 capture antibody magnetic particles,
The preparation method of the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody comprises the following steps:
(1) Protein activation: activating a protein by using a cross-linking agent, wherein the protein is a soluble fms-like tyrosine kinase-1 monoclonal antibody, and the cross-linking agent is selected from any one of SPDP, DTBP, DTSSP, AMAS;
(2) Chemiluminescent activation: activating a chemiluminescent label by dithiothreitol, wherein the chemiluminescent label is alkaline phosphatase;
(3) Coupling: mixing and incubating the alkaline phosphatase activated in the step (2) with the soluble fms-like tyrosine kinase-1 monoclonal antibody activated in the step (1) to obtain a chemiluminescent label-protein conjugate;
(4) Closing: adding a blocking agent into the chemiluminescent label-protein conjugate obtained in the step (3) for blocking to obtain a chemiluminescent label-protein conjugate final product, wherein the blocking agent is MMTS.
2. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of claim 1 wherein the cross-linking agent is DTBP.
3. The kit for chemiluminescent immunoassay of claim 1 wherein in step (1) the mass ratio of protein to cross-linking agent is 100 (0.5-2.0).
4. The kit for chemiluminescent immunoassay of claim 3 wherein in step (1) the mass ratio of protein to cross-linking agent is 100:1.5.
5. The kit for chemiluminescent immunoassay of claim 1 wherein the soluble fms-like tyrosine kinase-1 capture antibody is activated by a cross-linking agent in the magnetic particles of the soluble fms-like tyrosine kinase-1 capture antibody,
The cross-linking agent is selected from any one of BS (PEG) 5, BMPS and BM (PEG) 3.
6. The kit for chemiluminescent immunoassay of claim 5 wherein the mass ratio of the soluble fms-like tyrosine kinase-1 capture antibody to the cross-linking agent in the activation is 10 3 (0.5-3.0).
7. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of any one of claims 1-6 wherein the magnetic particles are modified with tosyl.
8. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of claim 7 wherein the tosylated magnetic particles have a particle size of 0.9 μm to 1.8 μm.
9. The chemiluminescent immunoassay kit of any one of claims 1-6 and 8 wherein the mass ratio of the soluble fms-like tyrosine kinase-1 capture antibody to the magnetic particles in the magnetic particles of the soluble fms-like tyrosine kinase-1 capture antibody is 1 (10-50).
10. The kit for chemiluminescent immunoassay of claim 9 wherein the mass ratio of soluble fms-like tyrosine kinase-1 capture antibody to magnetic particles in the magnetic particles of the soluble fms-like tyrosine kinase-1 capture antibody is 1:25.
11. The chemiluminescent immunoassay kit for the soluble fms-like tyrosine kinase-1 of claim 7 wherein the mass ratio of the soluble fms-like tyrosine kinase-1 capture antibody to the magnetic particles is 1 (10-50).
12. The kit for chemiluminescent immunoassay of claim 11 wherein the mass ratio of soluble fms-like tyrosine kinase-1 capture antibody to magnetic particles is 1:25.
CN202111388930.5A 2021-11-22 2021-11-22 Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof Active CN114252592B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111388930.5A CN114252592B (en) 2021-11-22 2021-11-22 Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111388930.5A CN114252592B (en) 2021-11-22 2021-11-22 Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN114252592A CN114252592A (en) 2022-03-29
CN114252592B true CN114252592B (en) 2024-05-31

Family

ID=80791052

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111388930.5A Active CN114252592B (en) 2021-11-22 2021-11-22 Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114252592B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115561230B (en) * 2022-11-02 2023-12-05 江苏三联生物工程股份有限公司 Application of dithiothreitol in preparation of electrochemiluminescence immunoassay-based product
CN115791340B (en) * 2023-01-17 2023-05-02 北京水木济衡生物技术有限公司 Epilepsy composite quality control product and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107044977A (en) * 2016-06-30 2017-08-15 深圳市亚辉龙生物科技股份有限公司 A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof
CN112098640A (en) * 2020-09-16 2020-12-18 浙江正熙生物医药有限公司 Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8349325B2 (en) * 2008-12-23 2013-01-08 Abbott Laboratories Soluble FMS-like tyrosine kinase-1 (sFLT-1) antibody and related composition, kit, methods of using, and materials and method for making
US10421812B2 (en) * 2015-03-31 2019-09-24 University Of Massachusetts Isoform specific soluble FMS-like tyrosine kinase (sFlt) binding agents and uses thereof
GB201703876D0 (en) * 2017-03-10 2017-04-26 Berlin-Chemie Ag Pharmaceutical combinations
EP4070113A4 (en) * 2019-12-04 2023-12-20 Biora Therapeutics, Inc. Assessment of preeclampsia using assays for free and dissociated placental growth factor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107044977A (en) * 2016-06-30 2017-08-15 深圳市亚辉龙生物科技股份有限公司 A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof
CN112098640A (en) * 2020-09-16 2020-12-18 浙江正熙生物医药有限公司 Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Quantitative Proteomic (iTRAQ) Analysis of 1st Trimester Maternal Plasma Samples in Pregnancies at Risk for Preeclampsia;Varaprasad Kolla等;BioMed Research International;第2012卷;Article ID 305964 *

Also Published As

Publication number Publication date
CN114252592A (en) 2022-03-29

Similar Documents

Publication Publication Date Title
US11959912B2 (en) Fluorescence immunochromatographic detection card and a preparation method therefor and use thereof
CN114252592B (en) Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof
AU2018374469B2 (en) Target interference suppressed anti-drug antibody assay
US20020106708A1 (en) Assays reagents and kits for detecting or determining the concentration of analytes
CN114252594B (en) Placenta growth factor detection kit and preparation method and application thereof
Kaul et al. Detection of immunoglobulin M antibodies specific for Toxoplasma gondii with increased selectivity for recently acquired infections
JP2000508075A (en) Luminescence-specific binding assay
CN108398554B (en) anti-TORCH-IgM antibody spectrum chip, preparation method thereof and TORCH detection kit
CN108398564B (en) anti-TORCH-IgG type antibody spectrum chip, preparation method thereof and TORCH detection kit
JP4950879B2 (en) Biomarker for diagnosis of heart disease and use thereof
JP2023017986A (en) Direct immunoassay measurement of autoantibodies
CN114487442A (en) Mouse monoclonal antibody coated magnetic bead, preparation method and kit for determining high-sensitivity cardiac troponin I
CN101504410A (en) Blood serum sample treatment preparation used for protein suspending chip detection
US6825000B1 (en) Immunoassay reagent and immunoassay method
JP5714791B2 (en) Non-specific reaction inhibitor
CN111208292B (en) Mycoplasma pneumoniae antibody IgM immunoassay kit, preparation method and use method thereof
CN114371292A (en) Kit for detecting soluble ST2 protein
JP3228791B2 (en) Measuring method of antigen or antibody in sample
CN112129933A (en) Reagent, kit and method for resisting biotin interference in immunoassay system
JP3709078B2 (en) Method and kit for measuring diacetylpolyamine
US20100304411A1 (en) Endogenous Morphine or a Naturally Occurring Metabolite Thereof as a Marker for Infection
CN114324877B (en) Latex immunonephelometry reagent with low value precision and HOOK resistance
IMAGAWA et al. Comparison of β-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab′ by sandwich enzyme immunoassay technique
CN116466091A (en) Kit for detecting IgA immune complex based on chemiluminescence method
CN117269503A (en) Soluble growth stimulation expressed gene 2 protein (ST 2) detection kit

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant