CN109324189B - Application of polyprotein composition and congenital heart disease screening kit - Google Patents
Application of polyprotein composition and congenital heart disease screening kit Download PDFInfo
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- CN109324189B CN109324189B CN201810927548.9A CN201810927548A CN109324189B CN 109324189 B CN109324189 B CN 109324189B CN 201810927548 A CN201810927548 A CN 201810927548A CN 109324189 B CN109324189 B CN 109324189B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
Application of a polyprotein composition and a congenital heart disease screening kit. The invention utilizes modern biological technology and bioinformatics analysis to research the detection and functions of platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin, finds that the combined application has the application of predicting the patent ductus arteriosus of congenital heart disease, can be applied to preparing targeted drugs for early intervention of related diseases, and can also be applied to preparing a screening kit for congenital heart disease, and the kit has potential huge application value for preventing the patent ductus arteriosus.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to application of a polyprotein composition and a congenital heart disease screening kit.
Background
Birth defects are the first cause of death in infants and children, and congenital heart disease has become the first disease category of birth defects in China. How to reduce the incidence rate of the congenital heart disease and improve the curative effect of the congenital heart disease become an important challenge for the health of the population in China in the new century. Internationally, birth defects are also the first cause of life and quality of life threatening for children. The worldwide birth defect report released in the united states in 2006 estimates that more than 800 million birth defects are newly added worldwide each year, with 90% occurring in mid-to-low income countries. Approximately 330 and over ten thousand children under 5 die of birth defects each year, with 320 thousands of children having disabilities for their lifetime. Wherein the congenital heart disease is at the first of birth defects.
Patent Ductus Arteriosus (PDA) is a common type of congenital heart disease. The main research of the project focuses on patent ductus arteriosus, which is a congenital abnormal channel between main pulmonary arteries, is a complex disease involving multiple factors, the pathogenesis of which is not completely elucidated, and is mainly related to factors such as vasoactive substances, hypoxia, ion channels, heredity and the like. Recent studies on the pathogenesis of PDA mainly include: (1) blood vasodilation and contractile factor levels, prostaglandin e (PGE) has vasodilating action, fetal arterial Duct (DA) patency is maintained by PGE2, not PGI2, hypoxia-associated endothelin (ET-1); thromboxane a2(TXA2) that indirectly affects DA occlusion; (2) increased blood oxygen levels are associated with DA shutdown after the fetus; (3) oxygen-sensitive Kv regulates cardiovascular tissue responsiveness, and gene conversion at birth is not only involved in ion channel expression during DA-off in premature infants, but also can improve oxygen sensitivity. In addition, PDA was found to be associated with the polymorphism of5, 10-methylenetetrahydrofolate reductase (MTHFR) gene.
Although much research has been conducted on the etiology and the genetics of PDA, the relationship between the protein substances and diseases related to the diseases has not been studied much and deeply and comprehensively, and the expected effect of people is far from being achieved. The key protein related to the disease is searched, the disease occurrence mechanism is further deeply, comprehensively and directly clarified, and scientific evidence can be provided for clinical medication guidance, treatment effect evaluation and prognosis to a certain extent.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an application of a multi-protein composition.
The invention aims to provide a congenital heart disease screening kit.
In order to solve the technical problems, the technical scheme of the invention is as follows:
use of a polyprotein composition consisting of platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-associated serine protease 2, and fibronectin for the manufacture of a targeted drug or device for congenital heart disease.
Preferably, the polyprotein composition is used, and the congenital heart disease is patent ductus arteriosus.
Preferably, the polyprotein composition is used, and the instrument is a congenital heart disease screening kit.
A congenital heart disease screening kit comprising 6 protein detection groups:
a first detection group comprising: a perforated plate coated with platelet factor 4 antibody, a standard product (platelet factor 4 pure product), a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the platelet factor 4 standard substance is 0,6.25,12.5,25,50,100,200 and 400 ng/ml;
the biotin labeled antibody is a biotinylated anti-platelet factor 4 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 20 (0.5%) 5ml, adding distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) taking an isolated plasma sample, melting overnight at 4 ℃ before detection, centrifuging at 2500rpm for 10min, diluting with a sample diluent by 1:100 times, and detecting;
2) preparing a standard substance: centrifuging 2 parts of standard substance for 30S, fully dissolving the standard substance with 1ml of sample diluent (S8), repeatedly blowing and beating for at least 5 times by using a pipette, taking 7 centrifuge tubes (S1-S7) of 1.5ml, adding 250ul of sample diluent into each centrifuge tube, sucking 250 mu l of the standard substance S8 into the first centrifuge tube (S7), lightly blowing and beating, and uniformly mixing; sucking 250. mu.l from S7 into a second EP tube (S6), and gently pipetting and mixing; performing multiple dilution on the standard product by analogy; s1 is a sample diluent;
3) the method comprises the following operation steps:
a) aiming at a standard substance hole and a sample hole to be detected which are arranged in the kit, adding 100 mu l of the standard substance or the sample to be detected into each hole respectively, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 3 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 1 hour at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction;
4) data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
A second detection group comprising: a perforated plate coated with von willebrand factor antibody, a standard product (pure von willebrand factor), a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the von willebrand factor standard product is 0,6.25,12.5,25,50,100,200,400 ng/ml;
the biotin labeled antibody is a biotinylated anti-von willebrand factor antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 2.5 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1 hour at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 1 minute each time, adding 200 μ l to each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 8 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
A third detection group comprising: a porous plate coated with fibrinogen antibody, a standard product (pure fibrinogen product), horse radish peroxidase labeled avidin diluent, sample diluent, washing liquid, substrate solution and stop solution, wherein,
the concentration of the fibrinogen standard is 0,125,250,500,1000,2000,4000,8000 ng/ml;
the horseradish peroxidase labeled avidin is streptavidin;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:1000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Adding 50 mul of standard substance or sample to be detected into each hole, immediately adding 50 mul of horseradish peroxidase marker working solution, slightly shaking and uniformly mixing, attaching a plate, and incubating for 45 minutes at 37 ℃;
b) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
c) adding 90 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
d) adding 50 mul of stop solution into each hole to stop the reaction;
e) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
A fourth detection group comprising: a porous plate coated with a collagen antibody, a standard substance (a collagen original pure product), a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the collagen standard substance is 0,0.625,1.25,2.5,5,10,20 and 40 ng/ml;
the biotin labeled antibody is a biotinylated anti-collagen antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 3 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
A fifth detection group comprising: a perforated plate coated with protein-related serine protease 2 antibody, a standard product (protein-related serine protease 2 pure product), a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the protein-related serine protease 2 standard substance is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-protein related serine protease 2 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 10g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:1000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 1.5 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1 hour at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
A sixth detection group comprising: a porous plate coated with a fibronectin antibody, a standard product (a pure fibronectin product), a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the fibronectin standard substance is 0,12.5,25,50,100,200,400 and 800 ng/ml;
the biotin labeled antibody is a biotinylated anti-fibronectin antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 10g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The above-mentioned materials can be prepared by conventional method.
The specific operation method of the content comprises the following steps:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:500 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 2 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.5 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample.
The invention has the beneficial effects that:
the invention uses modern biological technology and bioinformatics analysis to carry out preliminary study on the detection and functions of platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin, the low-concentration platelet factor 4, the von willebrand factor, the fibrinogen, the collagen, the protein-related serine protease 2 and the high-concentration fibronectin have the purpose of predicting the patent ductus arteriosus of congenital heart disease, can be applied to the preparation of targeted drugs for early intervention, and can also be applied to the preparation of a congenital heart disease screening kit, the kit has potential huge application value for preventing the patent ductus arteriosus disease, and lays a foundation for further studying the biological functions of the platelet factor 4, the von willebrand factor, the fibrinogen, the collagen, the protein-related serine protease 2 and the high-concentration fibronectin .
Drawings
FIG. 1 is a graph of the proteomics detected the interaction of networks containing platelet factor 4, von Willebrand factor, fibrinogen, collagen, protein-associated serine protease 2 and fibronectin;
FIG. 2 shows the levels of platelet factor 4, von Willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin in infants with patent ductus arteriosus of congenital heart disease and normal controls measured by ELISA;
FIG. 3 is a graph showing the effects of platelet factor 4, von Willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin on the promotion of arterial vessel occlusion in humans.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings and the detailed description.
Example 1
1. Collection of human plasma samples
Blood samples were collected from all congenital heart disease patients (experimental group) and normal control children (control group) in the morning prior to surgery, fasting for >10 h. Each patient collected 2ml of whole blood from the peripheral vein, immediately centrifuged at 2500rpm for 10 minutes, and the upper plasma was separated. And dividing the separated plasma into a plurality of equal parts, placing the equal parts in a plasma collecting pipe, and placing the equal parts in a refrigerator at the temperature of minus 80 ℃ for freezing storage to be detected.
2. Proteomics detection
1) Plasma proteome detection
Proteins were detected in two sets of each mixed sample using iTRAQ combined with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) (Applied Biosystems, Foster City, CA).
2) Sample preparation
a) A high-abundance protein Removal Kit (ProteExtractTM Albumin/IgG Removal Kit, CALBIOCHEM, USA) was purchased to remove the high-abundance protein.
b) 5 volumes of acetone were added and the precipitate was allowed to settle at-20 ℃ for 1 hour. Centrifuging at 4 ℃ for 10 minutes at 10000g,
and collecting the precipitate.
c) And (3) putting the dried powder solution into a sample lysate, and fully dissolving the protein in a constant-temperature water bath at 30 ℃.
d) The solution was centrifuged at 15000g for 15min at room temperature, the supernatant was collected and centrifuged again to remove the impurities sufficiently.
e) The supernatant is the total protein solution of the tissue, and the protein concentration is measured, subpackaged and stored at-80 ℃ for later use or directly used for iTRAQ analysis.
3) Sample quantification
According to the quantitative principle, the sample: BCA (bicinchoninic acid) and cupric sulfate
And other reagents are mixed together to form the apple green, namely the BCA working reagent. Under alkaline conditions, when the BCA is combined with protein, the protein reduces Cu2+ into Cu +, one Cu + chelates two BCA molecules, the working reagent forms a purple compound from the original apple green, the water-soluble compound shows the maximum light absorption at 562nm, and the light absorption and the protein concentration have good linear relation in a wide range, so the protein concentration can be calculated according to the light absorption value.
Proteins in the sample were quantified according to the BCA method. The experimental procedure was as follows:
a) the standard protein concentration is used as the abscissa and OD562 is used as the ordinate to prepare a standard curve graph, and the standard curve graph is obtained
A regression equation.
b) The OD562 values were used to prepare a standard curve of BSA concentration.
c) Diluting the sample to be detected by a proper volume by 10 times, detecting the absorbance of the sample, and calculating the average value
And substituting OD562 into a regression equation, and finally calculating the measured concentration of the sample to be measured, wherein the measured concentration is multiplied by 10 to obtain the real concentration of the sample.
4) SDS-PAGE electrophoretic sample detection experiment
a) Mu.g of each sample was taken and separated by 12% SDS-PAGE.
b) The separated gel was stained by Coomassie blue staining. The specific operation is as follows: fixing for 2 hours; dyeing for 12 hours; and washing with water until the background is clear.
c) The stained gel was scanned using an ImageScanner scanner in grayscale
Mode, optical density value is 300 dpi.
5) Reductive alkylation and enzymolysis of protein
The method comprises the following specific steps:
a) after the protein is quantified, 100 mu g of each sample is taken, precooled acetone with 5 times of volume is adopted for precipitation, and the solution is placed at the temperature of minus 20 ℃ for 1 hour to fully precipitate the protein.
b) The resulting precipitate was centrifuged at 12,000rpm for 10 minutes at 4 ℃ and vacuum-freeze-dried.
c) The protein precipitate was dissolved well with 50. mu.l of the Dissolution Buffer in the iTRAQ kit, and 4. mu.l of Reducing Reagent was added and reacted at 60 ℃ for 1 hour.
d) Mu.l of Cysteine-Blocking Reagent was added, the reaction was carried out at room temperature for 10 minutes, the protein solution after the reductive alkylation was added to a 10K ultrafiltration tube, the mixture was centrifuged at 12,000rpm for 20 minutes, and the bottom solution of the collection tube was discarded.
e) Add 100. mu.l of Dissolution Buffer, centrifuge at 12,000rpm for 20 minutes, discard the bottom solution of the collection tube, repeat 3 times.
f) The collection tube was replaced with a new one, and 50. mu.l of sequencing grade trypsin solution at a concentration of 50 ng/. mu.l was added to the ultrafiltration tube and reacted at 37 ℃ for 12 hours.
g) Centrifuging at 12,000rpm for 20 min, collecting peptide fragments after enzymolysis, and adding 50 μ l
The precipitation Buffer was centrifuged at 12000rpm for 20 minutes and the bottom of the tube was collected and combined with the previous solution.
6) Protein labeling and mass spectrometry pilot experiments
a) Taking out the iTRAQ reagent from the refrigerator, and balancing to room temperature;
b) centrifuging the iTRAQ reagent to the bottom of the tube;
c) dissolving iTRAQ reagent with 150 μ l isopropanol;
d) transferring 50 mu l of sample (100 mu g of enzymolysis product) into a new centrifuge tube, adding the iTRAQ reagent, and reacting at room temperature for 2 hours;
e) the reaction was stopped by adding 100. mu.l of water;
f) mixing all the marked samples, carrying out vortex oscillation, and centrifuging to the bottom of the tube;
g) and (5) freezing and drying the sample in vacuum, and reserving the sample for iTRAQ separation and identification.
7)2D-LC-MSMS analysis reversed phase chromatographic separation
a) The freeze-dried sample was dissolved with 110. mu.L of mobile phase A solution;
b) peptide fragment separation was performed on an Agilent 1200HPLC, and the column chromatography, Agilent, was purchased with the specific parameters: protecting the core: analytical Guard Column 4.6 x 12.5mm 5-micron separation Column: Narrow-Bore 2.1 × 150mm 5 μm, detection wavelength: ultraviolet 210nm and 280 nm; flow rate: 0.3ml/min, non-linear binary gradient.
The time table of the flow ratio of peptide fragment chromatographic separation is shown in table 1.
TABLE 1
c) Discard 0-5 minutes, collect 1 tube every 4.5 minutes for 6-45 minutes, collect 1 tube for 46-50 minutes, total 10 tubes of sample solution, then freeze dry each tube of solution thoroughly. And obtaining a one-dimensional separation chromatogram.
Reverse chromatography-TripleTOF analysis
a) The lyophilized polypeptide sample after cation exchange separation was redissolved in Nano-RPLC Buffer A.
b) On-line Nano-RPLC liquid chromatography was performed on an eksingent nanoLC-Ultra 2D system (AB SCIEX), and the dissolved sample was loaded onto a C18 pre-column (100m x 3cm,
C18,3μm,
) Then, the flow rate is maintained to flush and desalt for 10 min.
c) The analytical column is a C18 reverse phase chromatographic column (75 μmx15cm C18-3 μm)
ChromXP eksingent), the gradient used in the experiment was such that mobile phase B rose from 5% to 35% within 70 min.
d) The mass spectrum adopts a TripleTOF5600 system (AB SCIEX) combined with a nanoliter spray III ion source (AB SCIEX, USA), the spray voltage is 2.5kV, the air curtain pressure is 30PSI, the atomization pressure is 5PSI, the heater temperature is 150 ℃, the mass spectrum scanning mode is in an Information-Dependent acquisition working mode (IDA, Information Dependent Analysis), the single-spectrum scanning time of the primary TOF-MS is 250MS, at most 35 secondary spectra with charges of 2+ to 5+ and a single-second count greater than 150 are acquired in each IDA cycle, the cycle time of each cycle is fixed to 2.5 seconds, the collision cell energy is set to be suitable for Collision Induced Dissociation (CID) of all precursor ions, the dynamic exclusion is set to be 18 seconds, and is approximately equal to the half-peak width of the chromatogram.
8) Database retrieval
The data processing is carried out by adopting Software of Protein Pilot Software v.5.0(AB SCIEX, USA) containing Paragon algorithm, and the database used in the experiment is a human database which is derived from Uniprot. The protein identification is mainly realized by matching experimental tandem mass spectrum data with theoretical mass spectrum data obtained by database simulation, so that a protein identification result is obtained. The parameters are set as follows:
proteomic mass spectrometry search parameters
Sample Type:iTRAQ 8plex(Peptide Labeled)
Cys.Alkylation:Iodoacetamide
Digestion:Trypsin
Instrument:TripleTOF 5600
Database:Homo Sapiens.fasta
Search Effort:Thorough
User Modified Parameter Files:No
The proteomics detected a network containing platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-associated serine protease 2 and fibronectin as an interaction graph as shown in figure 1.
Example 2
Enzyme-linked immunosorbent assay (ELISA)
Platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-associated serine protease 2 and fibronectin screened from proteomic results were verified in plasma samples of the validation group by ELISA. The experimental principle is as follows: the antibody of the protein to be detected is pre-embedded at the bottom of the 96-well plate, and the protein to be detected in the 96-well plate can be combined with the antibody after the standard substance and the sample are added. After removal of unbound substrate, biotin-conjugated antibody to the protein to be tested is added. Washing, an anti-biotin conjugated horseradish peroxidase labeled antibody (HRP) was added, and unbound HRP was removed by washing. The color developing agent was added, and after the reaction was terminated, the absorbance of the liquid was measured.
A platelet factor 4 protein detection kit comprising:
multi-well plate coated with platelet factor 4 antibody
Standard article
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the platelet factor 4 standard substance is 0,6.25,12.5,25,50,100,200 and 400 ng/ml;
the biotin labeled antibody is a biotinylated anti-platelet factor 4 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, goat serum, Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 20 (0.5%) 5ml, distilled water to 1000 ml.
The substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: sulfuric acid (2 mol/L).
The specific experimental operating method is as follows:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:100 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 3 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a sticking film, and incubating for 1 hour at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
A von willebrand factor detection kit, comprising:
perforated plate coated with von Willebrand factor antibodies
Standard article
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the von willebrand factor standard product is 0,6.25,12.5,25,50,100,200,400 ng/ml;
the biotin labeled antibody is a biotinylated anti-von willebrand factor antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific experimental operating method is as follows:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 2.5 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1 hour at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 1 minute each time, adding 200 μ l to each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 8 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
A fibrinogen detection kit comprising:
porous plate coated with fibrinogen antibody
Standard article
Horse radish peroxidase labeled avidin
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the fibrinogen standard is 0,125,250,500,1000,2000,4000,8000 ng/ml;
the horseradish peroxidase labeled avidin is streptavidin;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific experimental operating method is as follows:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:1000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Adding 50 mul of standard substance or sample to be detected into each hole, immediately adding 50 mul of horseradish peroxidase marker working solution, slightly shaking and uniformly mixing, attaching a plate, and incubating for 45 minutes at 37 ℃;
b) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
c) adding 90 mul of substrate solution into each hole, and developing for 15 minutes at 37 ℃ in a dark place;
d) adding 50 mul of stop solution into each hole to stop the reaction;
e) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
A collagen detection kit comprising:
porous plate coated with collagen antibody
Standard article
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the collagen standard substance is 0,0.625,1.25,2.5,5,10,20 and 40 ng/ml;
the biotin labeled antibody is a biotinylated anti-collagen antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific experimental operating method is as follows:
1) plasma samples were thawed overnight at 4 ℃ and centrifuged at 2500rpm for 10min prior to testing.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 3 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.2 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
A protein-associated serine protease 2 protein detection kit comprising:
multi-well plate coated with protein-associated serine protease 2 antibody
Standard article
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the protein-related serine protease 2 standard substance is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-protein related serine protease 2 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 10g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific experimental operating method is as follows:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:1000 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 1.5 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1 hour at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.2 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 20 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
A fibronectin detection kit comprising:
multi-well plate coated with fibronectin antibody
Standard article
Biotin-labeled antibody
Horse radish peroxidase labeled avidin
Biotin labeled antibody diluent
Horse radish peroxidase labeled avidin diluent
Sample diluent
Cleaning solution
Substrate solution
Stopping liquid
Wherein:
the concentration of the fibronectin standard substance is 0,12.5,25,50,100,200,400 and 800 ng/ml;
the biotin labeled antibody is a biotinylated anti-fibronectin antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M Phosphate Buffered Saline (PBS) ph 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M Phosphate Buffered Saline (PBS) pH7.20.05% thimerosal;
the sample diluent is 85% sodium chloride, 5% disodium hydrogen phosphate, 4% sodium dihydrogen phosphate, 5% goat serum, 1% Proclin-300, and has a pH value of 6-7;
the washing solution comprises: NaCl 10g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid.
The specific experimental operating method is as follows:
1) the plasma sample is melted overnight at 4 ℃ before detection, centrifuged at 2500rpm for 10min, diluted 1:500 times with the sample diluent and detected.
2) Preparing a standard substance: after 2 portions of the standard were centrifuged for 30 seconds, the standard was fully dissolved with 1ml of the sample diluent (S8), repeatedly pipetting at least 5 times using a pipette, taking 7 centrifuge tubes of 1.5ml (S1-S7), adding 250ul of each sample diluent, pipetting 250 μ l of the standard S8 into the first centrifuge tube (S7), gently pipetting and mixing. Aspirate 250. mu.l from S7 into a second EP tube (S6) and gently pipette. And performing dilution of the standard product by times by analogy. S1 is a sample dilution.
3) The method comprises the following operation steps:
a) and setting a standard sample hole and a sample hole to be detected. Respectively adding 100 mul of standard substance or sample to be detected into each hole, slightly shaking and uniformly mixing, pasting a plate paste, and incubating for 2 hours at 37 ℃;
b) discarding liquid in the holes, and spin-drying;
c) adding 100 mul of biotin labeled antibody into each hole, attaching a film, and incubating for 1.5 hours at 37 ℃;
d) discarding liquid in the holes, spin-drying, washing the plate with washing liquid for 3 times, soaking for 2 minutes each time, adding 200 μ l in each hole, and spin-drying;
e) adding 100 mul of horse radish peroxidase labeled avidin working solution into each hole, attaching a pad pasting, and incubating for 1.5 hours at 37 ℃;
f) discarding liquid in the holes, spin-drying, washing the plate for 3 times according to the method of the step 5, and spin-drying;
g) adding 90 mul of substrate solution into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
h) adding 50 mul of stop solution into each hole to stop the reaction;
i) the optical density (OD value) of each well was measured at 450nm with a microplate reader within 5 minutes after the termination of the reaction.
4) Data processing:
and (3) processing the OD value by using CurveExpert (version 1.4) software, drawing a proper standard curve by using the OD value of the standard substance, obtaining a corresponding function, and substituting the OD value of each sample hole into the function to obtain the concentration value of each hole sample. The results are shown in FIG. 2.
To sum up, by example 1, it was found that the concentration of platelet factor 4 in plasma of children with patent ductus arteriosus of congenital heart disease was lower than that of normal children. Platelet factor 4 is a heparin-binding protein with a 70 amino acid composition released from platelet alpha particles. It plays a very important role in chemotaxis, coagulation, inflammation and cell growth. Low platelet factor 4 is associated with patency of the arterial duct, and low amounts thereof may lead to an increased risk of patent ductus arteriosus. The platelet factor 4 has strong adhesion and aggregation capability in the arterial duct cavity, and in the process of dissecting and closing the arterial duct, the platelet factor 4 affects the proliferation of cells and prevents the thrombus from forming in the duct cavity, thereby causing the arterial duct to be incapable of being closed permanently. Von willebrand factor, fibrinogen and collagen are the most viscous matrix and the key proteins in the adhesion and aggregation process of platelets in the aorta. Soluble plasma fibronectin has platelet aggregation and thrombosis inhibiting effects at low concentrations of von willebrand factor and fibrinogen. Protein-associated serine protease 2 produces thrombin and low concentrations of protein-associated serine protease 2 increase the risk of neonatal infection, a factor in patent ductus arteriosus. FIG. 3 is a graph showing the process by which platelet factor 4, von Willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin promote the formation of patent ductus arteriosus.
Aiming at the results of proteomics for screening and diagnosing congenital heart disease in example 1, the ELSIA method is adopted to confirm that the concentrations of platelet factor 4, von Willebrand factor, fibrinogen, collagen and protein-related serine protease 2 in the plasma of patent ductus arteriosus are lower than those of normal children and the concentrations of fibronectin are higher than those of normal children. The results are in accordance with example 1, see FIG. 2.
The above detailed description of the use of the polyprotein composition and the congenital heart disease screening kit with reference to specific embodiments is illustrative and not restrictive, and several examples are given according to the scope of the invention, so that variations and modifications without departing from the general inventive concept are within the scope of the present invention.
Claims (3)
1. The application of a multi-protein composition in the preparation of targeted drugs or devices for patent ductus arteriosus congenital heart disease, wherein the multi-protein composition consists of platelet factor 4, von willebrand factor, fibrinogen, collagen, protein-related serine protease 2 and fibronectin.
2. Use of the multi-protein composition according to claim 1, characterized in that: the apparatus is an arterial duct unclosed congenital heart disease screening kit.
3. An arterial duct unclosed congenital heart disease screening kit is characterized in that: comprises 6 protein detection groups:
a first detection group comprising: a perforated plate coated with a platelet factor 4 antibody, a platelet factor 4 pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the platelet factor 4 standard substance is 0,6.25,12.5,25,50,100,200 and 400 ng/ml;
the biotin labeled antibody is a biotinylated anti-platelet factor 4 antibody;
the horseradish peroxidase labeled avidin is streptavidin;
the biotin labeled antibody diluent is as follows: 0.05% sodium azide, 0.01M phosphate buffer pH 7.2;
the horse radish peroxidase labeled avidin diluent: 0.01M phosphate buffer pH7.20.05% thimerosal;
the sample diluent is 86% of sodium chloride, 4.5% of disodium hydrogen phosphate, 3.5% of sodium dihydrogen phosphate, 5% of goat serum, 1% of Proclin-300 and has a pH value of 6-7;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the substrate solution is 3,3 ', 5, 5' -tetramethyl benzidine;
the stop solution is as follows: 2mol/L sulfuric acid;
a second detection group comprising: a porous plate coated with von willebrand factor antibody, a pure von willebrand factor product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the von willebrand factor standard product is 0,6.25,12.5,25,50,100,200,400 ng/ml;
the biotin labeled antibody is a biotinylated anti-von willebrand factor antibody;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the horse radish peroxidase labeled avidin, the biotin labeled antibody diluent, the horse radish peroxidase labeled avidin diluent, the sample diluent, the substrate solution and the stop solution are the same as the first detection group;
a third detection group comprising: a porous plate coated with fibrinogen antibody, a fibrinogen pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the platelet factor 4 standard substance is 0,125,250,500,1000,2000,4000,8000 ng/ml;
the biotin labeled antibody is a biotinylated anti-fibrinogen antibody;
the washing solution comprises: NaCl 9.0g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the horse radish peroxidase labeled avidin, the biotin labeled antibody diluent, the horse radish peroxidase labeled avidin diluent, the sample diluent, the substrate solution and the stop solution are the same as the first detection group;
a fourth detection group comprising: a porous plate coated with a collagen antibody, a collagen pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the platelet factor 4 standard substance is 0,0.625,1.25,2.5,5,10,20 and 40 ng/ml;
the biotin labeled antibody is a biotinylated anti-collagen antibody;
the washing solution comprises: NaCl 9.0g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the horse radish peroxidase labeled avidin, the biotin labeled antibody diluent, the horse radish peroxidase labeled avidin diluent, the sample diluent, the substrate solution and the stop solution are the same as the first detection group;
a fifth detection group comprising: a porous plate coated with a protein-related serine protease 2 antibody, a protein-related serine protease 2 pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the protein-related serine protease 2 standard substance is 0,23.5,47,94,187.5,375,750,1500 pg/ml;
the biotin labeled antibody is a biotinylated anti-protein related serine protease 2 antibody;
the washing solution comprises: NaCl 10g, Tween 205 ml 0.5%, and distilled water 1000 ml;
the horse radish peroxidase labeled avidin, the biotin labeled antibody diluent, the horse radish peroxidase labeled avidin diluent, the sample diluent, the substrate solution and the stop solution are the same as the first detection group;
a sixth detection group comprising: a porous plate coated with a fibronectin antibody, a fibronectin pure product serving as a standard substance, a biotin-labeled antibody, horseradish peroxidase-labeled avidin, a biotin-labeled antibody diluent, a horseradish peroxidase-labeled avidin diluent, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein,
the concentration of the fibronectin standard substance is 0,12.5,25,50,100,200,400 and 800 ng/ml;
the biotin labeled antibody is a biotinylated anti-fibronectin antibody;
the washing solution comprises: NaCl 10g, Tween 206 ml 0.5%, distilled water to 1000 ml;
the horse radish peroxidase labeled avidin, the biotin labeled antibody diluent, the horse radish peroxidase labeled avidin diluent, the sample diluent, the substrate solution and the stop solution are the same as the first detection group.
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| CN111793131A (en) * | 2020-05-11 | 2020-10-20 | 廊坊天光生物技术有限公司 | Antibody pair for detecting content of PF4 in serum and application thereof |
| CN112213494A (en) * | 2020-08-26 | 2021-01-12 | 泰达国际心血管病医院 | Multi-protein combination biomarker and application thereof and heart valvular disease complicated atrial fibrillation diagnostic kit |
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