CN114252592A - 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
CN114252592A
CN114252592A CN202111388930.5A CN202111388930A CN114252592A CN 114252592 A CN114252592 A CN 114252592A CN 202111388930 A CN202111388930 A CN 202111388930A CN 114252592 A CN114252592 A CN 114252592A
Authority
CN
China
Prior art keywords
tyrosine kinase
soluble fms
antibody
detection
chemiluminescent
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.)
Granted
Application number
CN202111388930.5A
Other languages
Chinese (zh)
Other versions
CN114252592B (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

Images

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

Abstract

The invention relates to a soluble fms-like tyrosine kinase-1 detection kit and a preparation method and application thereof, the inventor adopts a double-antibody sandwich method combined with a magnetic particle chemiluminescence technology to detect the soluble fms-like tyrosine kinase-1, and finds that the immune micro-magnetic particle preparation method optimized by the inventor is adopted, the prepared placenta growth factor capture antibody magnetic particles and the placenta growth factor detection antibody marked by alkaline phosphatase further prepared by the inventor through cross-linking agents DTBP, DTT and a sealant MMTS with specific proportion form the soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit, a full-automatic chemiluminescence immunoassay analyzer can be used as a detection tool to automatically complete detection, the detection performance is also obviously improved, the detection sensitivity reaches 0.97pg/mL, the detection linear range is wide, can reach 10 pg/mL-85000 pg/mL. Indicating that the method has 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 biomedicine, 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 Vascular Endothelial Growth Factor Receptors (VEGFRs), and soluble fms-like tyrosine kinase-1 (sFlt-1) is a soluble form thereof, has no signal transduction function 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 type 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 it. Normally, placental vascular network formation is maintained in balance by VEGF and its antagonistic factors, including sFlt-1. The abnormal increase in sFlt-1 expression in the peripheral blood of preeclamptic patients disrupts this balance, resulting in decreased angiogenesis, leading to endothelial cell activation and injury. sFlt-1 has a role in arresting vascular growth, and studies have shown that sFlt-1 may be involved in the development of pre-eclampsia (PE). Thus, sFlt-1 can be used as a biological indicator for predicting the onset and progression of preeclampsia.
The placenta is the major source of sFlt-1 during pregnancy. The increase of sFlt-1 and the decrease of PLGF cause the dysequilibrium of the angiogenesis dynamic state of the placenta, so that the invasion of the trophoblast to the endometrium is insufficient, the recasting of the uterine spiral arteriole causes the ischemia and anoxia of the placenta, and the further increase of sFlt-1 causes the dysfunction of the endothelial cells of the whole body of the parent, thereby generating preeclampsia. The serum levels of sFlt-1 are elevated in pregnant women before eclampsia and the levels of PLGF are reduced, prior to the onset of the disease. The single measurement of the levels of sFlt-1 and PLGF to predict the occurrence of preeclampsia diseases has low coincidence rate and unsatisfactory effect, and the combined measurement of the ratio of sFlt-1 to PLGF in serum of pregnant women has more reference value in diagnosing the onset of preeclampsia than the single measurement of sFlt-1 and PLGF.
Preeclampsia (PE) is a serious complication of pregnancy whose major clinical manifestations are hypertension and proteinuria around 20 weeks after pregnancy. About 3-5% of pregnant women develop preeclampsia and can lead to maternal, fetal, or neonatal death. Preeclampsia, which is associated with thrombocytopenia and elevated liver enzyme activity, can manifest as thrombocytopenia syndrome (hemolysis, elevated liver enzymes, thrombocytopenia). Preeclampsia occurs due to endothelial dysfunction caused by the release of angiogenic factors from the placenta. Alterations in serum PLGF and sFlt-1 levels occur in women with pre-eclampsia. Furthermore, the levels of PLGF and sFlt-1 in the blood circulation may precede the appearance of clinical symptoms to identify normal pregnancy and pre-eclampsia. The level of pro-vascular factor PLGF increases in the first 6 months of normal pregnancy and gradually decreases as pregnancy progresses through termination. In contrast, the anti-vascular factor sFlt-1 levels remained stable during early and middle gestation, and did not rise smoothly until termination of pregnancy. Women with pre-eclampsia have higher than normal pregnancy levels of sFlt-1, while levels of PLGF are lower than normal pregnancy levels. Determination of the ratio of sFlt-1 to PLGF is more valuable than detection of sFlt-1 or PLGF alone. Placental endoglin is a member of the TGF- β family, which is upregulated in preeclampsia and released into the maternal circulation as soluble endoglin. Soluble endoglin has been shown to be significantly elevated in severe cases of preeclampsia.
Thus, detection of sFlt-1 levels in pregnant women's blood may be clinically used to identify the presence of ventilatory pressure on the trophoblast cells of the placental syncytium. Can be used for predicting and assisting diagnosis of gestational hypertension.
The traditional placenta growth factor measuring method includes enzyme linked immunosorbent assay and chemiluminescence assay, and most of the current domestic methods for detecting sFlt-1 are enzyme linked immunosorbent assay and fluorescence chromatography. The enzyme-linked immunosorbent assay is long in time consumption and complicated in operation. In addition, the two detection methods have poor detection sensitivity and narrow detection range, and are not fully-automatic tests. The foreign detection method for sFlt-1 is an electrochemiluminescence immunoassay method, but the detection cost of the method is high, so that the method cannot be applied to pregnancy of resource-limited areas.
Disclosure of Invention
Accordingly, an object of the present invention is to provide a method for producing a chemiluminescent-labeled protein.
The technical scheme is as follows:
(1) protein activation: carrying out activation treatment on the protein by using a cross-linking agent;
(2) activation of chemiluminescence substance: activating the chemiluminescent marker by using dithiothreitol;
(3) coupling: mixing and incubating the chemiluminescent marker activated in the step (2) and the protein activated in the step (1) to obtain a chemiluminescent marker-protein conjugate;
(4) and (3) sealing: adding a sealing agent into the chemiluminescent marker-protein conjugate obtained in the step (3) for sealing to obtain a chemiluminescent marker-protein conjugate final product;
wherein, the cross-linking agent in the step (1) is selected from any one of SPDP, DTBP, DTSSP and AMAS, and the blocking agent in the step (4) is MMTS.
It is also an object of the present invention to provide a chemiluminescent labeled protein obtained according to the above preparation method.
The invention also aims to provide the application of the chemiluminescence object labeling antibody in the preparation of a kit or a kit detection reagent.
It is another object of the present invention to provide an alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody prepared according to the above preparation method.
One of the purposes of the invention is to provide a soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit.
The technical scheme is as follows:
a soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit comprises the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody and a soluble fms-like tyrosine kinase-1 capture antibody magnetic particle.
It is also an object of the present invention to provide a method for the quantitative detection of soluble fms-like tyrosine kinase-1 for non-disease diagnostic purposes.
The technical scheme is as follows:
establishing a standard fitting curve according to the chemiluminescence immunoassay kit for the soluble fms-like tyrosine kinase-1;
and obtaining a sample to be detected, detecting by adopting the chemiluminescence immunoassay kit for the soluble fms-like tyrosine kinase-1, recording the luminous value of the sample to be detected, and substituting the luminous 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 detects sFlt-1 by adopting a double antibody sandwich method and a magnetic particle chemiluminescence technology, and in the preparation process of a chemiluminescence object marker sFlt-1 detection antibody, unexpectedly discovers a preparation method of the chemiluminescence object marker protein optimized by the inventor, particularly when the chemiluminescence object is alkaline phosphatase activated by DTT and the used protein cross-linking agent is selected from any one of SPDP, DTBP, DTSSP and AMAS, the detection antibody of the alkaline phosphatase marker sFlt-1 prepared by a specific proportion reaction and the inventor further use a specific proportion cross-linking agent BS (PEG)5The prepared magnetic particles of the sFlt-1 capture antibody can form an sFlt-1 chemiluminescence immunoassay kit, a full-automatic chemiluminescence immunoassay analyzer is used as a detection tool, the detection is automatically completed, meanwhile, the detection performance is also obviously improved, the detection sensitivity reaches 0.97pg/mL, the sensitivity is at least improved by 8 times compared with the traditional detection method of soluble fms-like tyrosine kinase-1, the detection linear range is wide, and the detection linear range can reach 10 pg/mL-85000 pg/mL. Indicating that the method has wide detection application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the soluble fms-like tyrosine kinase-1 chemiluminescence immunoassay kit of the invention.
FIG. 2 is a graph of the placental growth factor standards obtained from the test series of sFlt-1 standards in example 2.
FIG. 3 is a statistical chart showing the correlation analysis between the results of the chemiluminescence immunoassay kit for soluble fms-like tyrosine kinase-1 of the present invention and the results of the Roche kit in example 4.
Detailed Description
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals 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. As used herein, the term "and/or" 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 disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the 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.
In order that the invention may be more fully understood, reference will now be made to the following description. The present 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 present invention relates to abbreviations and terms 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 sulfur methanesulfonate ester
BMPS: N-beta-Maleimidopropylsuccinimidyl ester
BM(PEG)3:1, 11-bismaleimide-triethylene glycol
DTBP: dithio-alanine dimethyl ester dihydrochloride
DTSSP: 3, 3' -Dithiobis (sulfosuccinimidyl propionate)
AMAS: N-alpha-Maleimidooxysuccinimide ester
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: carrying out activation treatment on the protein by using a cross-linking agent;
(2) activation of chemiluminescence substance: activating the chemiluminescent marker by using dithiothreitol;
(3) coupling: mixing and incubating the chemiluminescent marker activated in the step (2) and the protein activated in the step (1) to obtain a chemiluminescent marker-protein conjugate;
(4) and (3) sealing: adding a sealing agent into the chemiluminescent marker-protein conjugate obtained in the step (3) for sealing to obtain a chemiluminescent marker-protein conjugate final product;
wherein, the cross-linking agent in the step (1) is selected from any one of SPDP, DTBP, DTSSP and AMAS, and the blocking agent in the step (4) is MMTS.
In some of the examples, the crosslinking agent in step (1) of the above preparation method is preferably DTBP.
In some embodiments, in the step (1), the mass ratio of the protein to the cross-linking agent is 100 (0.5-2.0). Further preferably, when the mass ratio of the protein to the cross-linking agent is 100:1.5, wherein the cross-linking agent is 5mg/mL DTBP solution, the DTBP is used as an antibody activator, the antibody can be activated with maximum efficiency, and the coupling efficiency of the subsequent antibody and the chemiluminescent marker can be greatly improved. Thereby improving the detection sensitivity.
In some of the examples, the protein and the cross-linking agent are used at equal concentrations in the activation treatment in step (1) by incubation for 1 hour at room temperature.
In some of these embodiments, the activation treatment of the chemiluminescent label in step (2) is DTT at a concentration of 5mg/mL and incubation at room temperature for 1 h.
In some embodiments, the blocking agent used in step (4) is MMTS of 5mg/mL, and further, desalting and purifying treatment is performed, preferably, the desalted and purified conjugate is recovered to 0.1mg/mL by using PBS buffer solution of 0.01M and pH 7.4, and then equal volume of glycerol is added and mixed uniformly to obtain the chemiluminescent label protein.
In some embodiments, in step (2), the chemiluminescent label is selected from one of acridinium ester, ruthenium terpyridyl, adamantane, luminol, a luminol derivative, isoluminol, an isoluminol derivative, horseradish peroxidase, and alkaline phosphatase. Of course, in other embodiments, the label in the antibody labeled with the label is not limited to the above, and may be other substances that can be used in the chemiluminescent immunoassay platform.
In some of these 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, and further, the protein is an antibody, and more preferably, a soluble fms-like tyrosine kinase-1 monoclonal antibody.
Some embodiments of the present invention also provide a chemiluminescent labeling protein, which is obtained according to the above preparation method.
Some embodiments of the invention also provide the use of the above-described chemiluminescent labeled antibody in the preparation of a kit or kit reagents.
Some embodiments of the present invention also provide an alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody, which is prepared according to the above preparation method.
Some embodiments of the present invention also provide a soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit comprising the above-described alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody and soluble fms-like tyrosine kinase-1 capture antibody magnetic microparticles.
In some embodiments, the above-mentioned soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit is characterized in that, in the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles, the soluble fms-like tyrosine kinase-1 capture antibody is activated by a cross-linking agent selected from the group consisting of BS (PEG)5BMPS and BM (PEG)3Any one of them.
In some embodiments, the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles are prepared according to the following steps:
(1) pretreatment: pretreating the magnetic particles to obtain a mixed solution 1;
(2) and (3) activation: 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) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (3) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
wherein the cross-linking agent in step (2) is selected from BS (PEG)5BMPS and BM (PEG)3Any one of the above; the mass ratio of the antibody to the cross-linking agent in the activation treatment is 1030.5 to 3.0. More preferably, the mass ratio of the antibody to the crosslinking agent is 103:1.5。
In some of the examples, the crosslinking agent in step (2) of the above preparation process is preferably BS (PEG)5Antibodies with crosslinking agent BS (PEG)5The mass ratio used was 1031.5, the steric hindrance is reduced by changing the conjugated structure of the antibody, so that the covalent bonding of the antibody and the magnetic particles is more efficient, the steric blockage of an active part for bonding the antibody and the antigen can be reduced, the antigen bonding activity of the antibody is improved, and the stability and the sensitivity of an immunoassay method are improved.
In some embodiments, in step (1) of the preparation method, the mixed solution 1 is obtained by washing and resuspending the magnetic particles with a buffer solution, and then uniformly dispersing the magnetic particles in an ammonium sulfate solution; further, the concentration of the ammonium sulfate solution is 2-5M, and the pH value is 8-10. More preferably, the ammonium sulfate solution used has a concentration of 3M and a pH of 9.5, and at this time, ammonium sulfate acts as a reaction accelerator to accelerate the binding of the subsequent magnetic beads to the protein.
In some embodiments, in step (1) of the preparation method, the buffer is preferably a PBS buffer and a BBS buffer; more preferably, the magnetic microparticles are washed with 0.01M PBS buffer at pH 7.4, the supernatant is magnetically separated, and then resuspended in 0.1M BBS buffer at pH 9.5.
In some embodiments, in step (1) of the preparation method, the magnetic particle is modified by tosyl, that is, when the active group of the magnetic particle is tosyl, the magnetic particle can be coupled with protein, so that the coupled protein can be combined with the substance to be detected, and then the high-efficiency separation and detection of the substance to be detected can be realized by the action of an external magnetic field.
In some embodiments, in step (1) of the above preparation method, the tosylated magnetic particles have a particle size of 0.9 μm to 1.8. mu.m.
In some embodiments, in step (4) of the above preparation method, the blocking solution is 0.05M Tris buffer containing 0.5% BSA and having a pH of 7.4.
In some embodiments, in the step (4) of the preparation method, in the final product of the antibody-magnetic bead coupled immunomagnetic particles, the mass ratio of the antibody to the magnetic bead is 1 (10-50). More preferably, the mass ratio of the antibody to the magnetic beads is 1: 25.
In some embodiments, the chemiluminescence immunoassay kit for the soluble fms-like tyrosine kinase-1 further comprises a soluble fms-like tyrosine kinase-1 calibration substance, and further, the soluble fms-like tyrosine kinase-1 calibration substance is prepared by using a calibration buffer solution, wherein the calibration buffer solution comprises 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, pH 7.5.
In some embodiments, the above-mentioned dish growth factor chemiluminescence immunoassay kit further comprises a chemiluminescence substrate solution, and further preferably an APS substrate solution.
Some embodiments of the 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 chemiluminescence immunoassay kit for the soluble fms-like tyrosine kinase-1;
and obtaining a sample to be detected, detecting by adopting the chemiluminescence immunoassay kit for the soluble fms-like tyrosine kinase-1, recording the luminous value of the sample to be detected, and substituting the luminous 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 comprises: and (2) taking 20-100 mu L of serum sample, adding 20-100 mu L of soluble fms-like tyrosine kinase-1 capture antibody magnetic particles 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, then adding 100-300 mu L of substrate solution, mixing uniformly, sending the reaction mixture into a dark room by an instrument, and finally recording the luminescence value. Preferably, 50 mu L of serum sample is taken, 50 mu L of soluble fms-like tyrosine kinase-1 capture antibody magnetic particles and 50 mu L of soluble fms-like tyrosine kinase-1 detection antibody marked by alkaline phosphatase are added, after reaction for 10min, magnetic separation is carried out, then 200 mu L of substrate solution is added, after uniform mixing, the reaction mixture is sent into a dark room by an instrument, and finally, the luminescence value is recorded.
EXAMPLE 1 preparation of sFlt-1 chemiluminescence immunoassay kit
(1) Preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles:
taking a suspension containing 90mg of magnetic particles (Magnosphere TM, cat # MS160) having a particle size of 0.9 to 1.8 μ M, magnetically separating the supernatant, washing the magnetic particles with 0.01M PBS buffer at pH 7.4, magnetically separating the supernatant, then resuspending the supernatant with 0.1M BBS buffer at pH 9.5, adding 4mL of 3M ammonium sulfate solution at pH 9.5, dispersing the mixture uniformly to obtain a mixed solution 1, and adding 5.4. mu.g of BS (PEG) to 3.6mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (Kyoto Biotechnology, Guangzhou, cat # sFlt-1-10)5(Pegylated bis (sulfosuccinimidyl) suberic acid, ThermoFisher SCIENTIFIC) activates sFlt-1 monoclonal antibody, and the activated antibody is added to the mixed solution 1 to obtain a mixed solution 2. Then placing the mixed solution 2 on a rotary shaking bed for carrying out a first incubation treatment, washing with 0.05M Tris buffer solution with pH 7.4 after magnetically separating a supernatant, removing the supernatant, then adding 0.5% BSA solution for carrying out a second incubation treatment, washing with 0.05M Tris buffer solution with pH 7.4 after magnetically separating the supernatant, finally suspending to 10mg/mL with 0.05M Tris buffer solution with 0.5% BSA and with pH 7.4 to obtain the magnetic Flt particles of the safroxylation coated by the s1 monoclonal antibody, wherein the corresponding magnetic bead mother solution is named as:sFlt-1-R1-R; stored at 4 ℃ for later 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 (Kyofu Biotechnology Co., Ltd., Guangzhou, cat # sFlt-1-12), centrifuging and removing the supernatant, then replacing the liquid in the original antibody with 0.01M PBS buffer solution with pH of 7.4 to 5mg/mL, then adding 3.6. mu.L of DTBP (dimethyl dithioalaninate dihydrochloride, ThermoFisher SCIENTIFIC) with the concentration of 5mg/mL, incubating for 1h at room temperature, respectively, and activating the antibody; then adding 2.88. mu.L of DTT (dithiothreitol) with the concentration of 5mg/mL to 72. mu.L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. Finally, 6.48. mu.L of MMTS (methyl sulfur methanesulfonate, ThermoFisher SCIENTIFIC) with a concentration of 5mg/mL was added to the crosslinked mixture to block, followed by desalting purification, and the desalted and purified conjugate was recovered to 0.1mg/mL with 0.01M PBS buffer solution with a pH of 7.4, followed by adding an equal volume of glycerol, and mixing to obtain a soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate labeled with alkaline phosphatase, and the corresponding enzyme-labeled stock solution was named: sFlt-1-R2-R, stored at-20 ℃ for use.
(3) Preparation of sFlt-1 standards:
soluble fms-like tyrosine kinase-1 (R & D Systems, Inc.) was prepared to a concentration of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, 5000pg/mL, 8000pg/mL, 11000pg/mL using a calibrator buffer (50mM Tris, 0.1% BSA, 0.9% NaCl, pH7.5), and dispensed at 0.5mL per tube and stored at-20 ℃ for future use.
EXAMPLE 2 detection method of sFlt-1 chemiluminescence immunoassay kit
The detection principle of the kit is as follows: magnetic microparticles coated with a soluble fms-like tyrosine kinase-1 (sFlt-1) capture antibody are combined with an alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 (sFlt-1) detection antibody and placental growth factor in a 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. Meanwhile, the luminous marker alkaline phosphatase is not consumed basically, the luminous agent in the reaction system is excessive enough, 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 the 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, Vanfu Biotechnology Ltd., Guangzhou) is used as a detection tool, the methodology 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 soluble fms-like tyrosine kinase-1 monoclonal antibody marked by alkaline phosphatase are added, after reaction for 10min, magnetic separation is carried out, then 200 mu L of substrate liquid is added into the mixture, after uniform mixing, the analyzer sends the reaction mixture into a dark room, and finally, the luminescence value is recorded.
The sFlt-1 calibration sample prepared in example 1 was tested by the above method, and the standard curve was plotted as shown in FIG. 2.
EXAMPLE 3 optimization of sFlt-1 chemiluminescence immunoassay kit
(1) screening of antibody activators in the preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles:
mixed solution 1 was prepared as described in reference example 1, followed by adding 5.4. mu.g of BS (PEG) to 3.6mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (product No.: sFlt-1-10#) respectively55.4. mu.g of BMPS (N-. beta. -maleimidopropylsuccinimidyl ester, ThermoFisher SCIENTIFIC) and 5.4. mu.g of BM (PEG)3(1, 11-bismaleimide groupTriethylene glycol, ThermoFisher SCIENTIFIC) activates soluble fms-like tyrosine kinase-1 monoclonal antibodies, and then the activated antibodies are added into the mixed solution 1 respectively to obtain 3 mixed solutions 2. Then, the 3 mixed solutions 2 were placed on a rotary shaking table to carry out a first incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, after removal of the supernatant, a solution containing 0.5% BSA was then added to carry out a second incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, and finally resuspended in 10mg/mL Tris buffer at pH 7.4 containing 0.5% BSA to obtain 3 tosylated magnetic particles coated with the soluble fms-like tyrosine kinase-1 monoclonal antibody (wherein, BS (PEG))5The mother liquor of magnetic beads corresponding to the coupling agent is named as: sFlt-1-R1-R; the mother liquor of magnetic beads corresponding to the BMPS coupling agent is named as: sFlt-1-R1- ②; BM (PEG)3The mother liquor of magnetic beads corresponding to the coupling agent is named as: sFlt-1-R1- ③) and stored at 4 ℃ for later use, the three mother liquid of the magnetic beads are used subsequently and matched with corresponding soluble fms-like tyrosine kinase-1 monoclonal antibody marked by alkaline phosphatase to test and verify the sensitivity, repeatability and linearity indexes of the reagent, and the specific results are shown in a table 3-1.
TABLE 3-1 results of performance verification of sFlt-1 antibody coated with magnetic particles of different coupling agents
Figure BDA0003368104920000141
Figure BDA0003368104920000151
As can be seen from the above table 3-1, when comparing the sFlt-1-R1-R with the sFlt-1-R1-R and the sFlt-1-R1-magnetic bead coating, the signal to noise ratio (S/N) of the sFlt-1-R1-R magnetic bead coating is the largest, the repeatability is within 5%, and the linearity R is the best.
Thus, in the preparation of sFlt-1 monoclonal antibody coated tosylated magnetic microparticles, 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:
mixed solution 1 was prepared as described in reference example 1, followed by adding 0.8. mu.g of BS (PEG) to 1mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (product number: sFlt-1-10#, Van. guangzhou, Mobil Biotechnology Co., Ltd.)51.5. mu.g of BS (PEG)5And 3.0. mu.g of BS (PEG)5Activating soluble fms-like tyrosine kinase-1 monoclonal antibodies, and adding the activated antibodies into the mixed solution 1 to obtain 3 mixed solutions 2. Then, the 3 mixed solutions 2 were placed on a rotary shaking table to carry out a first incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, after removal of the supernatant, a solution containing 0.5% BSA was then added to carry out a second incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, and finally resuspended in 10mg/mL Tris buffer at pH 7.4 containing 0.5% BSA to obtain 3 tosylated magnetic particles coated with the soluble fms-like tyrosine kinase-1 monoclonal antibody (wherein 0.8. mu.g of BS (PEG))5The mother liquor of magnetic beads corresponding to the coupling agent is named as: sFlt-1-R1-tetra; 1.5 μ g of BS (PEG)5The mother liquor of magnetic beads corresponding to the coupling agent is named as: sFlt-1-R1-fifthly; 3.0 μ g of BS (PEG)5The mother liquor of magnetic beads corresponding to the coupling agent is named as: sFlt-1-R1-sixth), stored at 4 ℃ for standby, and then the three magnetic bead mother solutions are used respectively, matched with corresponding alkaline phosphatase labeled soluble fms-like tyrosine kinase-1 monoclonal antibodies, and test verification is carried out on the sensitivity, repeatability and linearity indexes of the reagent, and the specific results are shown in tables 3-2.
TABLE 3-2 different concentrations of BS (PEG)5Performance verification result of coupling agent magnetic particle coated sFlt-1 antibody
Figure BDA0003368104920000161
Figure BDA0003368104920000171
As can be seen from Table 3-2, the signal to noise ratio (S/N), reproducibility and linearity of the sFlt-1-R1-o and sFlt-1-R1-o beads coated with the sFlt-1-R1-o and sFlt-1-R1-o beads coated with the sFlt-1-R1-o beads coated with the sFlt-1-R1-o beads are almost the same as those of the sFlt-1-R1-o beads coated with the sFlt-1-R1-o beads coated with the sFlt-1-R3678-o beads coated with the sFlt-1-R1-o beads coated with the sFlt-1-R1-o beads coated with the sFlt-1-R1-o beads coated with the highest priority.
Thus, in the placental growth factor monoclonal antibody-coated tosylated superparamagnetic microparticles, the antibody and antibody activator BS (PEG) used5Is preferably 103:1.5。
(3) Screening of the crosslinking agent in the preparation of the alkaline phosphatase-labeled sFlt-1 monoclonal antibody:
1.2mg of a soluble fms-like tyrosine kinase-1 monoclonal antibody (Kyofu Biotechnology Co., Ltd., Guangzhou, cat # sFlt-1-12) was substituted with 0.01M of PBS buffer at pH 7.4 to 5mg/mL, followed by addition of 3.6. mu.L of DTBP having a concentration of 5mg/mL, 3.6. mu.L of SPDP (succinimide 3- (2-pyridyldithio) -propionate, ThermoFisher SCIENTIFIC) having a concentration of 5mg/mL, 3.6. mu.L of DTSSP (3, 3' -dithiobis (sulfosuccinimidyl propionate), ThermoFisher SCIENTIFIC) having a concentration of 5mg/mL, and 3.6. mu.L of AMAS (N-. alpha. -maleimidoxysuccinimide ester, ThermoFisher SCIFIC) having a concentration of 5mg/mL, respectively, and the antibody was incubated at room temperature for 1h to activate the antibody; then adding 2.88 μ L of DTT with the concentration of 5mg/mL into 72 μ L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. 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 combination to 0.1mg/mL by using PBS buffer solution with the pH value of 7.4 and 0.01M, then adding isovolumetric glycerol, uniformly mixing to obtain soluble fms-like tyrosine kinase-1 monoclonal antibody combination labeled by alkaline phosphatase (wherein, the enzyme-labeled mother liquor corresponding to the DTBP crosslinking agent is named as sFlt-1-R2-R, the magnetic bead mother liquor corresponding to the SPDP crosslinking agent is named as sFlt-1-R2-R, the magnetic bead mother liquor corresponding to the DTBP crosslinking agent is named as sFlt-1-R2-R, the magnetic bead mother liquor corresponding to the AMAS crosslinking agent is named as sFlt-1-R2-Flt), preserving at-20 ℃ for later use, respectively using the three enzyme-labeled mother liquors, the kit is matched with tosylated magnetic particles coated by corresponding soluble fms-like tyrosine kinase-1 monoclonal antibodies to test and verify the sensitivity, repeatability and linearity indexes of the reagent, and the specific results are shown in tables 3-3.
TABLE 3-3 results of performance verification of different cross-linker alkaline phosphatase-labeled sFlt-1 antibodies
Figure BDA0003368104920000181
Figure BDA0003368104920000191
As can be seen from the above table 3-3, when comparing the sFlt-1-R2-R with the sFlt-1-R2-c, sFlt-1-R2-c and the sFlt-1-R2-c, the signal-to-noise ratio (S/N) of the sFlt-1-R2-c is the largest, the repeatability is within 5%, and the linearity R is the best.
Therefore, in the preparation of the soluble fms-like tyrosine kinase-1 monoclonal antibody labeled with alkaline phosphatase, the used cross-linking agent is 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 a soluble fms-like tyrosine kinase-1 monoclonal antibody (Kyofu Biotechnology Co., Ltd., Guangzhou, cat # sFlt-1-12) was subjected to a displacement of the liquid in the original antibody to 5mg/mL with 0.01M of a PBS buffer solution having a pH of 7.4, followed by addition of 1.2. mu.L, 2.4. mu.L, 3.6. mu.L and 4.8. mu.L of DTBP having a concentration of 5mg/mL, respectively, and incubation at room temperature for 1 hour, respectively, to activate the antibody; then adding 2.88 μ L of DTT with the concentration of 5mg/mL into 72 μ L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. 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 0.01M PBS buffer solution with the pH value of 7.4, adding isovolumetric glycerol, uniformly mixing to obtain soluble fms-like tyrosine kinase-1 monoclonal antibody conjugate labeled by alkaline phosphatase (wherein, enzyme labeled mother liquor corresponding to 2.4 mu L of DTBP crosslinking agent with the concentration of 5mg/mL is named as sFlt-1-R2-; enzyme labeled mother liquor corresponding to 3.6 mu L of DTBP crosslinking agent with the concentration of 5mg/mL is named as sFlt-1-R2-; enzyme labeled mother liquor corresponding to 4.8 mu L of DTBP crosslinking agent with the concentration of 5mg/mL is named as sFlt-1-R2-), and preserving at-20 ℃ for later use, and then, the three enzyme-labeled mother solutions are matched with corresponding tosylated magnetic particles coated by soluble fms-like tyrosine kinase-1 monoclonal antibodies, and the sensitivity, repeatability and linearity indexes of the reagent are tested and verified, wherein the specific results are shown in tables 3-4.
TABLE 3-4 Performance test results of DTBP crosslinker alkaline phosphatase-labeled sFlt-1 antibody at different concentrations
Figure BDA0003368104920000201
Figure BDA0003368104920000211
As can be seen from tables 3-4, the comparison of the sFlt-1-R2-c, sFlt-1-R2-c and sFlt-1-R2-c enzyme markers shows that the signal to noise ratio (S/N), repeatability and linearity of the sFlt-1-R2-c and sFlt-1-R2-c magnetic bead coating are almost the same as those of the sFlt-1-R2-c, but the sFlt-1-R2-c is preferable.
Therefore, in the preparation of the placenta growth factor monoclonal antibody labeled with alkaline phosphatase, the mass ratio of the antibody to the crosslinking agent SPDP used is preferably 100: 1.5.
EXAMPLE 4 evaluation of sFlt-1 chemiluminescence immunoassay kit Performance
The soluble fms-like tyrosine kinase-1 calibration samples were assayed using the method of example 2 and a standard curve was plotted as shown in FIG. 2.
Then, the actual sample is tested, and the concentration of the sample is calculated according to the luminous value of the sample.
(1) Detection of sensitivity:
the sensitivity of the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit was calculated according to the recommended protocol of CLSI EP17-A and was found to be 0.97 pg/mL.
(2) And (3) linear detection:
the linear analysis is carried out on the calibration samples with the concentrations of 0pg/mL, 15pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 5000pg/mL, 10000pg/mL, 20000pg/mL, 40000pg/mL and 86000pg/mL, the linear correlation coefficient is calculated, r is 0.9993, and in addition, the linear range of the detection of the kit on the soluble fms-like tyrosine kinase-1 sample is 10 pg/mL-85000 pg/mL.
(3) And (3) detection of precision:
two soluble fms-like tyrosine kinase-1 samples with the concentrations of 200pg/mL and 40000pg/mL are taken, each sample is tested for 10 times, three batches of kits are used for detection, the intra-batch difference and the inter-batch difference of the kits are calculated, the result shows that the intra-batch difference of the kits is less than 5%, the inter-batch difference is less than 10%, and the specific result is shown in the following table 4.
(4) Interference experiments:
taking mixed serum and adding interferents respectively comprises the following steps: bilirubin (10mg/dL), hemoglobin (500mg/dL), and triglyceride (1000mg/dL) were added at a mass ratio of 1:20, and the measurement values of the mixed serum and the mixed serum to which various interfering substances were added were measured. The deviation between the two was calculated to be within ± 10% of the acceptable range, and the specific results are shown in table 4 below. The result shows that the interference performance reaches the file standard of NCCLS, and the method can be used for accurately evaluating the condition of soluble fms-like tyrosine kinase-1 in a clinical laboratory.
TABLE 4 detection results of sFlt-1 chemiluminescence immunoassay kit
Figure BDA0003368104920000221
(5) And (3) detection of clinical performance:
128 clinical samples were taken and tested using the kit of the present invention, and the concentration was calculated from the standard curve (shown in FIG. 2).
Meanwhile, the concentration of the clinical sample is measured by using a soluble fms-like tyrosine kinase-1 detection kit (electrochemiluminescence method) of Roche.
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, and the clinical correlation result is shown in figure 3, wherein the clinical correlation of the soluble fms-like tyrosine kinase-1 is R20.9757, indicating that the kit of the invention has good correlation with the Roche kit.
EXAMPLE 5 comparative experiment with sFlt-1 chemiluminescence immunoassay kit
Soluble fms-like tyrosine kinase-1 samples at concentrations of 0pg/mL and 15pg/mL were tested using chemiluminescence detection methods and conventional enzyme-linked immunosorbent assay (R & D Systems, Inc., cat # DVR100C), respectively, and the sensitivity of detection was compared in the two methods, as shown in Table 5-1 below:
TABLE 5-1
Figure BDA0003368104920000231
Figure BDA0003368104920000241
As can be seen from the above table, the sensitivity of the chemiluminescence detection method is improved by about 8 times compared with that of the enzyme-linked immunosorbent assay.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (15)

1. A method for preparing a chemiluminescent labeled protein, comprising the steps of:
(1) protein activation: carrying out activation treatment on the protein by using a cross-linking agent;
(2) activation of chemiluminescence substance: activating the chemiluminescent marker by using dithiothreitol;
(3) coupling: mixing and incubating the chemiluminescent marker activated in the step (2) and the protein activated in the step (1) to obtain a chemiluminescent marker-protein conjugate;
(4) and (3) sealing: adding a sealing agent into the chemiluminescent marker-protein conjugate obtained in the step (3) for sealing to obtain a chemiluminescent marker-protein conjugate final product;
wherein, the cross-linking agent in the step (1) is selected from any one of SPDP, DTBP, DTSSP and AMAS, and the blocking agent in the step (4) is MMTS.
2. The preparation method according to claim 1, wherein in the step (1), the mass ratio of the protein to the crosslinking agent is 100 (0.5-2.0).
3. The method according to any one of claims 1 to 2, wherein in the step (2), the chemiluminescent label is selected from any one of luminol, isoluminol, alkaline phosphatase, ruthenium terpyridyl, and acridinium ester.
4. The method according to claim 3, wherein the chemiluminescent label is alkaline phosphatase.
5. The method according to any one of claims 1 to 4, wherein the protein is an antibody, and further, a soluble fms-like tyrosine kinase-1 monoclonal antibody.
6. A chemiluminescent labeled protein obtained by the production method according to any one of claims 1 to 5.
7. Use of the chemiluminescent-tagged antibody of claim 6 in the preparation of a kit or a kit detection reagent.
8. An alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody, wherein the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody is prepared according to the preparation method of claim 5.
9. A soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit comprising the alkaline phosphatase-labeled soluble fms-like tyrosine kinase-1 detection antibody of claim 8 and magnetic microparticles of a soluble fms-like tyrosine kinase-1 capture antibody.
10. The kit according to claim 9, wherein the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles are characterized in that the soluble fms-like tyrosine kinase-1 capture antibody is activated by a cross-linking agent,
the cross-linking agent is selected from BS (PEG)5BMPS and BM (PEG)3Any one of them.
11. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of claim 10, wherein the mass ratio of the soluble fms-like tyrosine kinase-1 capture antibody to the cross-linking agent used in the activation process is 103:(0.5～3.0)。
12. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of any of claims 9-11, wherein said magnetic particle is modified with tosyl.
13. The soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of claim 12, wherein said tosylated magnetic particles have a particle size of 0.9 μm to 1.8 μm.
14. The kit for chemiluminescence immunoassay of soluble fms-like tyrosine kinase-1 according to any one of claims 9 to 13, wherein the mass ratio of the soluble fms-like tyrosine kinase-1 capture antibody to the magnetic particles in the soluble fms-like tyrosine kinase-1 capture antibody magnetic particles is 1 (10-50).
15. A method for the quantitative detection of soluble fms-like tyrosine kinase-1 for non-disease diagnostic purposes comprising the steps of:
the soluble fms-like tyrosine kinase-1 chemiluminescent immunoassay kit of any of claims 9-14, establishing a standard fit curve;
obtaining a sample to be detected, detecting by using the chemiluminescence immunoassay kit for soluble fms-like tyrosine kinase-1 according to any one of claims 8 to 12, recording the luminous value of the sample to be detected, and substituting the luminous value into the standard fitting curve to obtain the concentration of the soluble fms-like tyrosine kinase-1 in the sample.
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 true CN114252592A (en) 2022-03-29
CN114252592B 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)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115561230A (en) * 2022-11-02 2023-01-03 江苏三联生物工程股份有限公司 Application of dithiothreitol in preparation of product based on electrochemiluminescence immunoassay
CN115791340A (en) * 2023-01-17 2023-03-14 北京水木济衡生物技术有限公司 Composite quality control product for epilepsy as well as preparation method and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100266575A1 (en) * 2008-12-23 2010-10-21 Abbott Laboratories SOLUBLE FMS-LIKE TYROSINE KINASE-1 (sFLT-1) ANTIBODY AND RELATED COMPOSITION, KIT, METHODS OF USING, AND MATERIALS AND METHOD FOR MAKING
US20160347843A1 (en) * 2015-03-31 2016-12-01 University Of Massachusetts Isoform specific soluble fms-like tyrosine kinase (sflt) binding agents and uses thereof
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
US20200040086A1 (en) * 2017-03-10 2020-02-06 Berlin-Chemie Ag Pharmaceutical combinations comprising an anti-ly75 antibody
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
US20210172938A1 (en) * 2019-12-04 2021-06-10 Progenity, Inc. Assessment of preeclampsia using assays for free and dissociated placental growth factor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100266575A1 (en) * 2008-12-23 2010-10-21 Abbott Laboratories SOLUBLE FMS-LIKE TYROSINE KINASE-1 (sFLT-1) ANTIBODY AND RELATED COMPOSITION, KIT, METHODS OF USING, AND MATERIALS AND METHOD FOR MAKING
US20160347843A1 (en) * 2015-03-31 2016-12-01 University Of Massachusetts Isoform specific soluble fms-like tyrosine kinase (sflt) binding agents and uses thereof
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
US20200040086A1 (en) * 2017-03-10 2020-02-06 Berlin-Chemie Ag Pharmaceutical combinations comprising an anti-ly75 antibody
US20210172938A1 (en) * 2019-12-04 2021-06-10 Progenity, Inc. Assessment of preeclampsia using assays for free and dissociated placental growth factor
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
VARAPRASAD KOLLA等: "Quantitative Proteomic (iTRAQ) Analysis of 1st Trimester Maternal Plasma Samples in Pregnancies at Risk for Preeclampsia", BIOMED RESEARCH INTERNATIONAL, vol. 2012, pages 305964 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115561230A (en) * 2022-11-02 2023-01-03 江苏三联生物工程股份有限公司 Application of dithiothreitol in preparation of product based on electrochemiluminescence immunoassay
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
CN115791340A (en) * 2023-01-17 2023-03-14 北京水木济衡生物技术有限公司 Composite quality control product for epilepsy as well as preparation method and application thereof

Also Published As

Publication number Publication date
CN114252592B (en) 2024-05-31

Similar Documents

Publication Publication Date Title
US11959912B2 (en) Fluorescence immunochromatographic detection card and a preparation method therefor and use thereof
CN114252594B (en) Placenta growth factor detection kit and preparation method and application thereof
WO2016127318A1 (en) Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor
EP0565541B1 (en) Diagnostic method for detecting the rupture of fetal membranes and test kit employing the method
US9128099B2 (en) Determination of sFlt-1:angiogenic factor complex
CN107907691B (en) Myoglobin detection kit and use method thereof
CN107942051B (en) D-dimer detection kit and use method thereof
CN114252592B (en) Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof
CN107918022B (en) cTnI detection kit and use method thereof
JP4950879B2 (en) Biomarker for diagnosis of heart disease and use thereof
JP2023017986A (en) Direct immunoassay measurement of autoantibodies
CN115097129B (en) Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1
CN116973574B (en) Determination kit for soluble growth stimulation expression gene 2 protein and detection method thereof
JPH11223631A (en) Improved measuring method for prostate specific antigenalpha1-antichymotrypsin complex
CN112710841B (en) ST2 protein detection kit and detection method
CN112129933A (en) Reagent, kit and method for resisting biotin interference in immunoassay system
CN114371292A (en) Kit for detecting soluble ST2 protein
JP3709078B2 (en) Method and kit for measuring diacetylpolyamine
CN114324877B (en) Latex immunonephelometry reagent with low value precision and HOOK resistance
JPH06148193A (en) Anti-phospholipid antibody bonding carrier and immunological measuring method using this carrier
JP3896303B2 (en) Test reagent for multiple organ failure in SIRS by cytochrome C measurement
CN117269503A (en) Soluble growth stimulation expressed gene 2 protein (ST 2) detection kit
CN116466091A (en) Kit for detecting IgA immune complex based on chemiluminescence method
CN118191342A (en) Quantitative detection kit and detection method for soluble fms-like tyrosine kinase-1
CN114705870A (en) Thrombin-antithrombin III compound determination kit and preparation method and application thereof

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