CN106198959A - Serum hyaluronic acid chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
Serum hyaluronic acid chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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- CN106198959A CN106198959A CN201610516569.2A CN201610516569A CN106198959A CN 106198959 A CN106198959 A CN 106198959A CN 201610516569 A CN201610516569 A CN 201610516569A CN 106198959 A CN106198959 A CN 106198959A
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- hyaluronic acid
- serum
- reagent kit
- detection reagent
- immune detection
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 109
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 109
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 109
- 210000002966 serum Anatomy 0.000 title claims abstract description 84
- 238000001514 detection method Methods 0.000 title claims abstract description 74
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000006249 magnetic particle Substances 0.000 claims abstract description 31
- 238000002372 labelling Methods 0.000 claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 16
- 102000018866 Hyaluronan Receptors Human genes 0.000 claims description 14
- 108010013214 Hyaluronan Receptors Proteins 0.000 claims description 14
- 239000000047 product Substances 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 13
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical group O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 7
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- BZSVVCFHMVMYCR-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1 BZSVVCFHMVMYCR-UHFFFAOYSA-N 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 4
- 239000007987 MES buffer Substances 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 238000003018 immunoassay Methods 0.000 abstract description 12
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000000034 method Methods 0.000 description 16
- 239000000126 substance Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- 239000000872 buffer Substances 0.000 description 8
- 238000004020 luminiscence type Methods 0.000 description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000000504 luminescence detection Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000009738 saturating Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 230000002300 anti-fibrosis Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- BPYKTIZUTYGOLE-UHFFFAOYSA-N billirubin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(C=C3C(=C(C=C)C(=O)N3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-UHFFFAOYSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000003725 endotheliocyte Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000005213 imbibition Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000004531 microgranule Substances 0.000 description 1
- -1 pH 8.0) Chemical compound 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000003904 radioactive pollution Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
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
- G01N33/543—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
- 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
- G01N21/763—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of Serum hyaluronic acid chemiluminescence immune detection reagent kit and preparation method thereof, Serum hyaluronic acid chemiluminescence immune detection reagent kit includes: the coated carboxylated magnetic particle of Serum hyaluronic acid associated proteins and the chemiluminescent labels of hyaluronic acid labelling.This Serum hyaluronic acid chemiluminescence immune detection reagent kit can complete the detection of hyaluronic acid with Full-automatic chemiluminescence immunoassay analysis meter for detection instrument.This Serum hyaluronic acid chemiluminescence immune detection reagent kit, through experiment, its detection sensitivity reaches 2.0ng/mL, at least improve 10 times relative to the detection method sensitivity of traditional Serum hyaluronic acid, the accuracy of detection of this Serum hyaluronic acid chemiluminescence immune detection reagent kit is higher.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of Serum hyaluronic acid chemiluminescence immune detection reagent kit
And preparation method thereof.
Background technology
Hyaluronic acid (Hyaluronic acid, hereinafter abbreviated as HA) is a kind of macromole aminopolysaccharide, by N-acetyl ammonia
The repetitive structure composition linear polysaccharide structure of base glucose and D-Glucose aldehydic acid, is synthesized by Interstitial cell.HA is mainly at liver
Intracellular metabolite, when cirrhosis, liver damages, and endotheliocyte is the most impaired, and picked-up declines with the ability of decomposing H A, causes HA to increase, because of
This, HA is a good serological index of liver cirrhosis.It addition, HA additionally aids the diagnosis and differential diagnosis of hepatopathy, the state of an illness is sentenced
Break and prognosis evaluation, or a very important dynamic Observations Means of anti-fibrosis medicine treatment.
The common methods of Clinical detection hyaluronic acid has enzyme linked immunosorbent assay, radio immunoassay, enzymatic at present
Learn luminescence method, but in place of these methods all also exist some shortcomings.
One, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antibody be coated apparatus and
Reaction vessel, can only be divided into 12 batches, 6 batches, 8 batches or imposite first use in use, it is impossible to carry out independent, single
The detection of part；
(2) reagent type used by quantitative determination is more, and each detectable will contain with reagent bottle, and often
Being required for changing imbibition nozzle when using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, adds
The operation of note reagent is the most loaded down with trivial details；
(3) lack the corresponding mark to detection information, just can only be will appreciate that by the mark checking test kit external packing box
Or know product batch number and the effect duration information of detectable, and the information known is uncontrolled during detection, has
The biggest randomness；
(4) detectable is in open space during detection, easily causes the cross-contamination between various reagent
And affect the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, operating process the most loaded down with trivial details and
Complexity, is susceptible to bust, and accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, the need to
Detect 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if the most a sample needs to detect 10
Individual different project, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, the shortcoming that there is inadequate economical rationality.
Two, radio immunoassay
Radioimmunoassay method radioactive pollution is big, load procedure is loaded down with trivial details, operation is complicated, operating time length, mensuration knot
The weak points such as fruit is unstable, the reagent holding time is short, test kit operating automation degree is low, necessary instrument is expensive.
Two, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase (HRP) and alkali phosphatase two kinds, but has certain office
Sex-limited, horseradish peroxidase major defect is: luminol, also can be by H2O2 in the case of not having horseradish peroxidase
Aoxidizing self luminous, background is of a relatively high, affects signal to noise ratio, and kinetics is complicated, and influence factor is many, and result is not sufficiently stable,
Obtain highly sensitive and plateau length substrate to be not easy.Alkali phosphatase major defect is: substrate reach plateau time
Between long, substrate cost is high, causes testing cost high, patient burden's weight.
Acridinium ester is compared enzyme-catalyzed chemical luminescence as the direct chemiluminescence of label and is had clear superiority, mainly shows
: reaction need not catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, and signal to noise ratio is high, disturb because of
Element is few, and reagent stability is good, can be with two-point calibration, and system is simple, exciting liquid low cost, acridinium ester easily and protein bind, and
After connection, photon productivity does not reduces.
Summary of the invention
Based on this, it is necessary to provide the Serum hyaluronic acid chemiluminescence immunoassay detectable that a kind of detection sensitivity is higher
Box and preparation method thereof.
A kind of Serum hyaluronic acid chemiluminescence immune detection reagent kit, including: Serum hyaluronic acid associated proteins is coated
Carboxylated magnetic particle and the chemiluminescent labels of hyaluronic acid labelling.
In the coated carboxylated magnetic particle of described Serum hyaluronic acid associated proteins, described Serum hyaluronic acid combines egg
White and described carboxylated magnetic particle ratio is 1:25～35.
In the chemiluminescent labels of described hyaluronic acid labelling, described hyaluronic acid and described chemiluminescent labels
Ratio is 50:1～10.
The particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Described Serum hyaluronic acid chemiluminescence immune detection reagent kit, also includes Chemoluminescent substrate, describedization
Learn luminous substrate liquid and include A liquid and B liquid.
Described A liquid is H2O2Solution, described B liquid is NaOH solution.
Described Serum hyaluronic acid chemiluminescence immune detection reagent kit, also includes that Serum hyaluronic acid calibrates product.
In one embodiment, described Serum hyaluronic acid calibration product are that concentration is respectively
The Serum hyaluronic acid of 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL
Solution.
The preparation method of a kind of above-mentioned Serum hyaluronic acid chemiluminescence immune detection reagent kit, comprises the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC water
Solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into hyaluronic acid binding protein, suspendible 2h～10h under room temperature,
Magneto separate is resuspended with Tris buffer after removing supernatant, obtains the coated carboxylated magnetic particle of hyaluronic acid binding protein；With
And
Take hyaluronic acid, mix after adding carbonate buffer solution, mix, under room temperature after being subsequently adding chemiluminescent labels
Remove impurity after lucifuge reaction 1h～2h, obtains the chemiluminescent labels of hyaluronic acid labelling.
This Serum hyaluronic acid chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter
For detection instrument, complete the detection of Serum hyaluronic acid.This Serum hyaluronic acid chemiluminescence immune detection reagent kit, passes through
Experiment, its detection sensitivity reaches 2.0ng/mL, at least carries relative to the detection method sensitivity of traditional Serum hyaluronic acid
High 10 times, the accuracy of detection of this Serum hyaluronic acid chemiluminescence immune detection reagent kit is higher.
Accompanying drawing explanation
Fig. 1 is the flow process of the preparation method of the Serum hyaluronic acid chemiluminescence immune detection reagent kit of an embodiment
Figure；
Fig. 2 is the Serum hyaluronic acid canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
The Serum hyaluronic acid chemiluminescence immune detection reagent kit of one embodiment, including: hyaluronic acid binding protein
Coated carboxylated magnetic particle and the chemiluminescent labels of hyaluronic acid labelling.
Preferably, in the coated carboxylated magnetic particle of hyaluronic acid binding protein, hyaluronic acid binding protein and carboxyl
The ratio of the magnetic particle changed is 1:25～35.
Preferably, in the chemiluminescent labels of Serum hyaluronic acid labelling, hyaluronic acid and chemiluminescent labels
Ratio is 50:1～10.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
In other examples, above-mentioned Serum hyaluronic acid chemiluminescence immune detection reagent kit also includes chemiluminescence
Substrate solution.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
In other examples, above-mentioned Serum hyaluronic acid chemiluminescence immune detection reagent kit also includes that serum is transparent
Matter acid cut mark product.
Serum hyaluronic acid calibration product are that concentration is respectively 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL,
The solution of the hyaluronic acid of 1500ng/mL, 2000ng/mL.
Concrete, hyaluronic acid calibration product can use standard substance buffer that hyaluronic acid is configured to concentration and be respectively
The solution of the hyaluronic acid of 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL.
This Serum hyaluronic acid chemiluminescence immune detection reagent kit, when Serum hyaluronic acid detects, utilizes the most certainly
Hyaluronic acid calibration product are detected by dynamic chemical illumination immunity analysis instrument, draw standard curve, are built in computer software；Then
Test actual sample, calculates concentration of specimens according to sample luminous value；Finally to the immunity of Serum hyaluronic acid Full-automatic chemiluminescence
Analysis system carries out the evaluation of performance (sensitivity, linear, precision, interference).
This Serum hyaluronic acid chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter
For detection instrument, complete the detection this Serum hyaluronic acid chemiluminescence immune detection reagent kit of Serum hyaluronic acid, pass through
Experiment, its detection sensitivity reaches 2.0ng/mL, at least carries relative to the detection method sensitivity of traditional Serum hyaluronic acid
High 10 times, the accuracy of detection of this Serum hyaluronic acid chemiluminescence immune detection reagent kit is higher.
Additionally, this Serum hyaluronic acid chemiluminescence immune detection reagent kit also has the advantage that
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, and this luminescence system is straight
Connecing chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more cost-effective；
2, select the chemiluminescence immunoassay system range of linearity width of acridinium ester, can reach
2.0ng/mL-2000ng/mL., and the inspection range of linearity of the detection method of traditional Serum hyaluronic acid is 20ng/
ML～1000ng/mL；
3, acridinium ester chemiluminescent immunoassay system repeatability is high, and in batch and difference between batch is all within 5%, this is other
Chemiluminescence immunoassay system is unapproachable；
4, chemiluminescence immunoassay system has realized the quantitative of sample, by built-in standard curve to test software, only
Need test sample just can directly obtain the concentration value of sample；
5, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample has instrument to complete entirely,
Operate easier, decrease artificial error.
The preparation method of above-mentioned Serum hyaluronic acid chemiluminescence immune detection reagent kit as shown in Figure 1, including as follows
Step:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC water
Solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into hyaluronic acid binding protein, suspendible 2h～10h under room temperature,
Magneto separate is resuspended with Tris buffer after removing supernatant, obtains the coated carboxylated magnetic particle of hyaluronic acid binding protein.
The concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer be 0.02M, pH be 5.5.
The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH.
The concentration of EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) aqueous solution is 10mg/mL～20mg/
ML, EDC are 0.05:0.1～1 with the ratio of carboxylated magnetic particle.
Preferably, in the coated carboxylated magnetic particle of hyaluronic acid binding protein, hyaluronic acid binding protein and carboxyl
The ratio of the magnetic particle changed is 1:25～35.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Take hyaluronic acid, mix after adding carbonate buffer solution, mix, under room temperature after being subsequently adding chemiluminescent labels
Remove impurity after lucifuge reaction 1h～2h, obtains the chemiluminescent labels of Serum hyaluronic acid labelling.
Carbonate buffer solution concentration be 0.1M, pH be 9.0～9.5,
The operation of remove impurity is centrifugal desalting column desalination, and concrete operations are: the most respectively with pure water and TBS buffer (40mM
Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) process centrifugal desalting column, it is eventually adding the hyaluronic acid obtained and combines egg
The solution of white coated carboxylated magnetic particle, finally collects the liquid in centrifuge tube.
Preferably, in the chemiluminescent labels of hyaluronic acid labelling, hyaluronic acid and the ratio of chemiluminescent labels
For 50:1～10.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
The coated carboxylated magnetic particle of hyaluronic acid binding protein obtained and the chemiluminescence mark of hyaluronic acid labelling
Note thing combination i.e. can get above-mentioned Serum hyaluronic acid chemiluminescence immune detection reagent kit.
This Serum hyaluronic acid chemiluminescence immune detection reagent kit is in use, in addition it is also necessary to Chemoluminescent substrate and
Serum hyaluronic acid calibration product.
Chemoluminescent substrate and Serum hyaluronic acid calibration product can be prepared voluntarily and obtain.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
Concrete, Serum hyaluronic acid calibration product can use standard substance buffer hyaluronic acid to be configured to concentration respectively
For 0ng/mL, the solution of the hyaluronic acid of 100ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL.
The preparation method of this Serum hyaluronic acid chemiluminescence immune detection reagent kit is simple and convenient, and the serum prepared is saturating
The detection sensitivity of bright matter acid chemiluminescence immune detection reagent kit is higher, has a good application prospect.
It it is below specific embodiment.
Embodiment 1: the preparation of Serum hyaluronic acid chemiluminescence immune detection reagent kit
(1) preparation of the coated carboxylated magnetic particle of Serum hyaluronic acid associated proteins:
Take and hang containing the carboxylated magnetic particle (MagnaBindTM, article No. 21353) that 50mg particle diameter is 0.05 μm～1 μm
Supernatant liquid, Magneto separate removes supernatant, is that 5.5MES buffer is resuspended with 0.02M, pH, adds the EDC water of 10mg/mL newly configured for 1mL
Solution, activated magnetic beads surface carboxyl groups, add 4mg hyaluronic acid binding protein (biorbyt, article No. orb48780), mixed under room temperature
Outstanding 6h, Magneto separate, remove supernatant, be resuspended to 1mg/mL with the Tris buffer that the 0.1M containing 2%BSA, pH are 8.0, obtain
The coated carboxylated magnetic particle of bright matter acid binding protein, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of Serum hyaluronic acid labelling:
Taking 50 μ L concentration is the hyaluronic acid of 25mg/mL, add 150 μ L concentration be 0.1M, pH be the carbonic acid of 9.0～9.5
Salt buffer, mixing, it is subsequently adding the acridinium ester solution mixing that 1.5 μ L concentration are 5mg/mL, lucifuge reaction under room temperature, after 1.5h
Take out, be centrifuged desalting column desalting processing with the zeba of 2mL, desalination processes is carried out with pure water and TBS buffer the most respectively
Processing, be eventually adding the acridinium ester solution of the hyaluronic acid labelling obtained, the liquid collected in centrifuge tube is in control blood to preservation
The acridinium ester of clear hyalomitome acidity scale note, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(3) preparation of Serum hyaluronic acid calibration product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0), hyaluronic acid is configured to
Concentration is 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL, and every bottle of 0.5mL subpackage freezes
Dry, 4 DEG C save backup.
Embodiment 2: Serum hyaluronic acid chemical luminous immune detection method
It is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter (YHLO, article No. iFlash3000), methodology pattern
The coated carboxylated magnetic of Serum hyaluronic acid associated proteins of the sample of 50 μ L, 50 μ L it is sequentially added into for competition law, i.e. instrument
The coated acridinium ester of hyaluronic acid of microgranule and 50 μ L, after reaction 20min, carries out Magneto separate, and reactant mixture is sent into by instrument
Darkroom, is sequentially added into luminous substrate A liquid (H2O2) and B liquid (NaOH) carry out luminescence-producing reaction, finally record luminous value.
Embodiment 3: Serum hyaluronic acid chemiluminescence immune detection reagent kit performance evaluation
Use the method in embodiment 2 that Serum hyaluronic acid calibration product are detected, obtain drawing standard curve such as Fig. 2
Shown in.
Then to then testing actual sample, concentration of specimens is calculated according to sample luminous value.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate Serum hyaluronic acid chemiluminescence immunoassay detectable
The sensitivity of box, the sensitivity tried to achieve is 0.68ng/mL.
Linear detection:
It is 0ng/mL to concentration, 100ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL standard substance
Do linear analysis, calculate linearly dependent coefficient, r=0.9996, it addition, the line that this test kit is to Serum hyaluronic acid sample detection
Property scope is 20ng/mL～1000ng/mL.
Precision measures:
Taking concentration is 500ng/mL and two Serum hyaluronic acid samples of 1000ng/mL, and each concentration of each sample respectively does 3
Individual parallel, detect with three batches of test kits, calculate test kit criticize interior and difference between batch, result show this test kit criticize interior and batch between
Difference is respectively less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid,
Glyceride, adding proportion is carried out according to 1:20, the survey of pooled serum after measuring pooled serum respectively and with the addition of various chaff interference
Value, calculates deviation therebetween, with ± 10% as tolerance interval.Result shows, interference all reaches the files-designated of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's Serum hyaluronic acid situation.
Embodiment 4, the contrast experiment of Serum hyaluronic acid chemiluminescence immune detection reagent kit
By chemical luminescence detection method and traditional enzyme linked immunosorbent assay, concentration is about the serum of 0ng/mL respectively saturating
Bright matter acid sample detects, and two kinds of method detection sensitivities are compared, and data are as shown in the table:
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves about 20 times.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a Serum hyaluronic acid chemiluminescence immune detection reagent kit, it is characterised in that including: Serum hyaluronic acid combines
The coated carboxylated magnetic particle of albumen and the chemiluminescent labels of hyaluronic acid labelling.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described blood
In the clear coated carboxylated magnetic particle of hyaluronic acid binding protein, described Serum hyaluronic acid associated proteins is carboxylated with described
The ratio of magnetic particle be 1:25～35.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described
In the chemiluminescent labels of bright matter acidity scale note, described Serum hyaluronic acid is 50:1 with the ratio of described chemiluminescent labels
～10.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described carboxylic
The particle diameter of the magnetic particle of base is 0.05 μm～1 μm.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that describedization
Luminous marker is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that also include
Chemoluminescent substrate, described Chemoluminescent substrate includes A liquid and B liquid.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 6, it is characterised in that described A
Liquid is H2O2Solution, described B liquid is NaOH solution.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that include blood
Clear hyalomitome acid cut mark product.
Serum hyaluronic acid chemiluminescence immune detection reagent kit the most according to claim 8, it is characterised in that described blood
Clear hyalomitome acid cut mark product be concentration be respectively 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and
The solution of the Serum hyaluronic acid of 1600pg/mL.
10. one kind according to the Serum hyaluronic acid chemiluminescence immune detection reagent kit according to any one of claim 1～9
Preparation method, it is characterised in that comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution,
The surface carboxyl groups of the magnetic particle of activated carboxyl, is subsequently added into hyaluronic acid binding protein, suspendible 2h～10h under room temperature, and magnetic divides
Leave away except resuspended with Tris buffer after supernatant, obtain the coated carboxylated magnetic particle of hyaluronic acid binding protein；And
Take hyaluronic acid, mix after adding carbonate buffer solution, mix after being subsequently adding chemiluminescent labels, lucifuge under room temperature
Remove impurity after reaction 1h～2h, obtains the chemiluminescent labels of hyaluronic acid labelling.
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| CN110907641A (en) * | 2019-12-18 | 2020-03-24 | 郑州安图生物工程股份有限公司 | Hyaluronic acid detection kit and detection method |
| CN112146955A (en) * | 2020-09-23 | 2020-12-29 | 深圳市亚辉龙生物科技股份有限公司 | Method for preparing serum |
| CN112213490A (en) * | 2020-09-16 | 2021-01-12 | 迪瑞医疗科技股份有限公司 | Hyaluronic acid chemiluminescence immunoassay kit and preparation method thereof |
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Application publication date: 20161207 |