CN111856004A - Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof - Google Patents
Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof Download PDFInfo
- Publication number
- CN111856004A CN111856004A CN202010218736.1A CN202010218736A CN111856004A CN 111856004 A CN111856004 A CN 111856004A CN 202010218736 A CN202010218736 A CN 202010218736A CN 111856004 A CN111856004 A CN 111856004A
- Authority
- CN
- China
- Prior art keywords
- solution
- magnetic bead
- covid
- buffer
- coating tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000025721 COVID-19 Diseases 0.000 title claims abstract description 58
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000003018 immunoassay Methods 0.000 title claims abstract description 21
- 239000011248 coating agent Substances 0.000 claims abstract description 94
- 238000000576 coating method Methods 0.000 claims abstract description 94
- 239000007788 liquid Substances 0.000 claims abstract description 47
- 238000002372 labelling Methods 0.000 claims abstract description 44
- 239000004005 microsphere Substances 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- -1 acridine ester Chemical class 0.000 claims abstract description 18
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 6
- 239000011324 bead Substances 0.000 claims description 102
- 239000000243 solution Substances 0.000 claims description 89
- 238000002156 mixing Methods 0.000 claims description 84
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 81
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 66
- 239000000872 buffer Substances 0.000 claims description 55
- 238000000502 dialysis Methods 0.000 claims description 52
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 36
- 239000011780 sodium chloride Substances 0.000 claims description 36
- 239000003761 preservation solution Substances 0.000 claims description 33
- 238000007885 magnetic separation Methods 0.000 claims description 27
- 238000005406 washing Methods 0.000 claims description 23
- 239000012224 working solution Substances 0.000 claims description 23
- 239000011734 sodium Substances 0.000 claims description 22
- 239000012154 double-distilled water Substances 0.000 claims description 20
- 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 20
- 239000000758 substrate Substances 0.000 claims description 19
- 238000007865 diluting Methods 0.000 claims description 18
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 17
- 239000012452 mother liquor Substances 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 12
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 12
- 238000003908 quality control method Methods 0.000 claims description 12
- 229960004793 sucrose Drugs 0.000 claims description 12
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 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 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 9
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004472 Lysine Substances 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000010413 mother solution Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 5
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 229910019145 PO4.2H2O Inorganic materials 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000005057 refrigeration Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 3
- 231100000956 nontoxicity Toxicity 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract description 2
- 230000005284 excitation Effects 0.000 abstract description 2
- 230000036632 reaction speed Effects 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 4
- 230000003252 repetitive effect Effects 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002966 serum Anatomy 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a reagent for detecting COVID-19 by chemiluminescence immunoassay and a detection method thereof, wherein an indirect method is adopted, COVID-19 antigen is coated on magnetic microspheres, acridine ester is used for labeling anti-human IgG/IgM, and excitation liquid is used for exciting the acridine ester to emit light, so that the detection of a COVID-19 antibody is carried out. The superparamagnetic nano microsphere marking technology is a technology for coating antigen or antibody on nano microspheres, and can enlarge the reaction surface area by 10-50 times and accelerate the reaction efficiency; the reaction process is suspended in the reaction liquid, belongs to a similar homogeneous reaction and can accelerate the reaction speed; the connection belongs to chemical connection of bioactive substances, so that the coating efficiency is higher, and the coating is more stable in a liquid environment. The detection reagent has the advantages of high specificity, high sensitivity, simple and convenient separation, rapidness, no toxicity, safety and stability, short detection time by adopting the reagent, high efficiency and good stability.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis immunological assay, in particular to a reagent for detecting COVID-19 by chemiluminescence immunoassay and a detection method thereof.
Background
The detection method for the COVID-19 clinically at present comprises the following steps: PCR amplification method, gold labeling method and enzyme immunoassay method, but the PCR amplification method has complex operation and long detection time, and the retest needs to be as long as 12 hours in total; the gold labeling method and the enzyme immunoassay method have low sensitivity and narrow detection range, and have the danger of missing detection to a great extent.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a reagent for detecting COVID-19 by chemiluminescence immunoassay, which has the advantages of high specificity, high sensitivity, simple and convenient separation, rapidness, no toxicity, safety and stability, and also provide a detection method adopting the reagent, and the reagent can be used for automatically detecting COVID-19 on a chemiluminescence analyzer.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a reagent for detecting COVID-19 by chemiluminescence immunoassay, which is characterized in that: the preparation method of the reagent comprises the following steps:
(1) preparing COVID-19 superparamagnetic nano microsphere working solution
1) Magnetic bead washing
Uniformly mixing the reagent bottle filled with the superparamagnetic nano microspheres in a vortex manner for 1min at room temperature until the magnetic bead suspension is uniformly mixed, sucking 0.24mL of magnetic bead labeled buffer solution C1 into a coating tube, adding 10 mu L of nano microsphere mother solution, screwing down a tube cover, and uniformly mixing in a vortex manner for 1 min;
2) magnetic bead coating
Under the condition of room temperature, placing the coating tube on a magnetic separation frame for 1min, completely absorbing liquid, taking the coating tube off the magnetic separation frame, adding 0.08mL of magnetic bead labeled buffer C1 into the coating tube, screwing down a tube cover, carrying out vortex mixing for 1min, adding 0.02mg of COVID-19 antigen into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, adding 0.16mL of magnetic bead labeled buffer C2 into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, placing the uniformly mixed coating tube in a constant temperature incubator, setting the temperature to be 37 ℃, setting the rotating speed to be 150rpm/min, and reacting for 16 hours;
3) magnetic bead washing
Taking out the coating tube from the constant-temperature incubator in the step 2, placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing in a vortex manner; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely sucking up liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃ and the rotation speed at 150rpm/min, and reacting for 7 hours;
4) Magnetic bead sealing
Taking out the coating tube from the constant-temperature incubator in the step 3), placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing by vortex; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃ and the rotation speed at 150 rpm/min, and reacting for 16 hours;
5) cleaning of
After the sealing is finished, taking down the coating tube from the constant-temperature incubator in the step 4), and placing the coating tube on a magnetic separation frame for 1min at room temperature to suck up liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
Adding 0.4mL of magnetic bead labeled buffer solution FF into the coating tube after three times of vortex mixing, wherein the temperature of the solution needs to be balanced to room temperature, screwing a tube cover, carrying out vortex mixing for 1min, and finishing coating of the COVID-19 superparamagnetic nano microsphere mother solution;
6) preparation of magnetic microsphere working solution
Diluting the COVID-19 superparamagnetic nano microsphere mother liquor by using a magnetic bead preservation solution FF, wherein the ratio of the magnetic bead preservation solution FF to the COVID-19 superparamagnetic nano microsphere mother liquor is 9: 1, and preparing a COVID-19 superparamagnetic nano microsphere working solution;
(2) preparation of acridinium ester labeled anti-human IgG/IgM working solution
1) Dialysis bag treatment
Shearing the dialysis bag by scissors; boiling in dialysis bag treatment liquid A for 10 min; after natural cooling, thoroughly washing with pure water; boiling in dialysis bag treatment liquid B for 10 min; naturally cooling, and storing in a refrigerator at 4 deg.C for use;
2) raw material treatment
Centrifuging and mixing the antihuman IgG/IgM, taking 1.0mg of the antibody after mixing, diluting the antibody to 1.0mg/ml by using a labeling buffer solution, and oscillating and mixing the antibody uniformly; washing the dialysis bag in the step 1) by pure water, then filling the uniformly mixed antibody into the dialysis bag, and fixing the two ends of the dialysis bag by a dialysis clamp; putting the mixture into 1000 mL of large-volume labeling buffer solution, and performing rotary dialysis on the mixture for 24 hours at the temperature of 4 ℃ by using a magnetic stirrer at the still/min;
3) Marking
Dissolving 0.05mg acridinium ester with 20.0uL DMF/DMSO, and mixing; taking out the antibody dialyzed in the step 2) into a centrifuge tube; adding the dissolved acridinium ester, oscillating and uniformly mixing, and then rotating and uniformly mixing at 37 ℃ for reaction for 1 h; adding 0.07mL of reaction stopping solution, oscillating, uniformly mixing, rotating and uniformly mixing at 37 ℃ and reacting for 30 min;
4) purification of
Putting the uniformly mixed antibody obtained in the step 3) into a dialysis bag cleaned by pure water, and fixing the two ends of the dialysis bag by a dialysis clamp; putting into 1000 mL of large-volume purified buffer solution, performing rotary dialysis for 24-48h at 4 ℃ by using a magnetic stirrer at 200 still/min, wherein the solution is changed once within 6-8h, and monitoring the concentration of free acridinium ester in the dialysate before the solution is changed; when monitoring RLU is less than 10000, taking out the marker in a centrifuge tube; 1:1, adding glycerol to make the final concentration of the glycerol be 0.2 mg/mL, and uniformly mixing the glycerol and the glycerol by oscillation; labeling the acridinium ester labeled anti-human IgG/IgM mother liquor;
5) preparation of acridinium ester labeled anti-human IgG/IgM working solution
Diluting the acridine ester marked anti-human IgG/IgM mother liquor by using a magnetic bead preservation liquor FF, wherein the ratio of the magnetic bead preservation liquor FF to the acridine ester marked anti-human IgG/IgM mother liquor is 1000: 1, and preparing an anti-human IgG/IgM acridine ester working solution;
(3) preparing COVID-19 calibrator and quality control product
Diluting COVID-19 standard substance with magnetic bead preservation solution FF according to a certain proportion to obtain a series of calibrators, wherein the concentrations of the calibrators are 0AU/ml, 5 AU/ml, 20AU/ml, 50 AU/ml, 200 AU/ml and 500 AU/ml respectively; the concentration of the quality control product is 20AU/ml and 200 AU/ml;
(4) preparing concentrated washing liquid
Preparing according to the condition that each 1LpH7.5 Tris-HCl buffer solution contains 7g of NaCl, 0.2ml of Tween-20 and 6ml of Proclin-300;
(5) preparing substrate solution A and substrate solution B
Substrate solution A: preparing a solution containing hydrogen peroxide with the concentration of 0.01mol/L and nitric acid with the concentration of 0.2mol/L by using a Tris-HCl solution with the concentration of 0.01mol/L pH7.5;
substrate solution B: preparing a sodium hydroxide solution with the concentration of 0.1g/L by using 0.01mol/LpH7.5 of Tris-HCl solution;
(6) subpackaging the COVID-19 superparamagnetic nano microsphere working solution, the acridine ester labeled anti-human IgG/IgM working solution, the COVID-19 calibrator and quality control material, the concentrated washing solution, the substrate solution A and the substrate solution B prepared in the steps (1) to (5), and then placing at 2-8 ℃ for refrigeration and preservation.
As a further improvement of the above technical solution:
the magnetic bead labeling buffer C1 comprises Na2HPO4、NaH2PO4And ddH 2O;
the magnetic bead labeling buffer C2 contained (NH)4)SO4、Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O;
The magnetic bead labeling buffer WB comprises Tris, NaCl, BSA, 50% HCI and ddH 2O;
the magnetic bead labeling buffer BB comprises Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O.
As a further improvement of the above technical solution:
na in the magnetic bead labeling buffer C12HPO433.90g of NaH2PO40.83g, and the volume is 1L from ddH 2O;
magnetic bead labeling buffer C2 (NH)4)SO439.64g of Na2HPO429.00g of NaH2PO42.96g of NaCl, 9.00g of NaCl, 5.00g of cane sugar and 5.00g of BSA, and the volume is determined to be 100 mL by ddH 2O;
adjusting the pH value to 8.0 +/-0.1 by using 1.21g of Tris, 9.00g of NaCl, 5.00g of BSA and 50% HCI in the magnetic bead labeling buffer WB, and fixing the volume to 1L by using ddH 2O;
labeling Na in buffer BB with magnetic beads2HPO429.00g of NaH2PO42.96g, 9.00g NaCl, 5.00g sucrose and 5.00g BSA were added to make up 1L from ddH 2O.
As a further improvement of the above technical solution:
the magnetic bead preservation solution FF comprises Tris, NaCl, PC300, BSA, 50% HCI and ddH2O, wherein the pH value of Tris in the magnetic bead preservation solution FF is 1.21g, the pH value of NaCl in the magnetic bead preservation solution FF is 9.00g, the pH value of PC300 in the magnetic bead preservation solution is 0.50g, the pH value of BSA in the magnetic bead preservation solution is 1.00g, and the volume of the solution is adjusted to 1L from ddH 2O.
As a further improvement of the above technical solution:
the dialysis bag treatment fluid A comprises NaHCO3、EDTA-Na21M NaOH/1M HCl and ddH 2O;
the dialysis bag treatment liquid B comprises EDTA-Na 21M NaOH/1M HCl and ddH 2O;
NaHCO in dialysis bag treatment liquid A320.0g of EDTA-Na2372.24mg, regulating pH to 8.4 + -0.1 with 1M NaOH/1M HCl, and diluting to 1L with ddH 2O;
EDTA-Na in the dialysis bag treatment liquid B2372.24mg, 1M NaOH/1M HCl adjusted pH 5.3. + -. 0.1, ddH2O to 1L.
As a further improvement of the above technical solution:
the labeling buffer comprises NaHCO31M NaOH/1M HCl and ddH2O, 8.4g NaHCO in the labeling buffer3The pH of the mixture was adjusted to 8.4. + -. 0.1 with 1M NaOH/1M HCl, and the volume was adjusted to 1L from ddH 2O.
As a further improvement of the above technical solution:
the termination reaction solution comprises lysine and ddH2O, wherein the lysine in the termination reaction solution is 0.007g, and the ddH2O is 70 uL.
As a further improvement of the above technical solution:
the purification buffer comprises Na2HPO4.12H2O、NaH2PO4.2H2O, NaCl, 1M NaOH/1M HCl and ddH2O, Na in the purification buffer2HPO4.12H2O is 2.58g, NaH2PO4.2H2O0.44 g, NaCl 8.2g, 1M NaOH/1M HCl pH 7.3. + -. 0.1, ddH2O to 1L.
A detection method for detecting COVID-19 by chemiluminescence immunoassay, the detection method comprising the following steps:
removing the reagent of any one of claims 1 to 8 from the refrigerated environment and allowing the reagent to equilibrate for more than 15 minutes at room temperature;
And (3) mixing the concentrated washing solution according to the proportion of 1: diluting at the ratio of 40, and shaking uniformly for later use as an application washing solution;
and (3) placing the COVID-19 calibrator, the quality control material and the sample to be detected on a chemiluminescence analyzer to complete the setting of relevant measurement parameters, and then detecting.
As a further improvement of the above technical solution:
the determination parameters are as follows:
the fitting type is as follows: four parameter Logistic fitting
And (3) selecting coordinates: X-Y.
By adopting the technical scheme, the invention has the following advantages:
the invention adopts an indirect method, COVID-19 antigen is coated on magnetic microspheres, acridine ester is used for labeling anti-human IgG/IgM, and excitation liquid is used for exciting the acridine ester to emit light, so that the detection of the COVID-19 antibody is carried out.
The acridinium ester or acridinium sulfonamide compound is used as a common chemiluminescent marker, emits light with the wavelength of 470nm when being oxidized by H2O2 under the alkaline condition, has high luminous efficiency, emits light in a flash type, can reach the maximum emission light intensity about 0.4 s after a luminous starting reagent is added, and the luminous efficiency in the luminous reaction is basically not influenced by a substituent structure.
The acridinium ester or acridinium sulfonamide compound chemiluminescence does not need a catalyst, and can emit light in a dilute alkaline solution with H2O2, so that the application of the compound in chemiluminescence detection has many advantages, and the advantages mainly comprise: the background luminescence is low, and the signal-to-noise ratio is high; secondly, the interference factors of the luminescence reaction are few; the light release is fast and centralized, the luminous efficiency is high, and the luminous intensity is large; fourthly, the protein is easy to be connected and the photon yield is not reduced after the connection; the marker is stable (can be stored for several months at 2-8 ℃).
The superparamagnetic nano microsphere marking technology is a technology for coating antigen or antibody on nano microspheres, and can enlarge the reaction surface area by 10-50 times and accelerate the reaction efficiency; the reaction process is suspended in the reaction liquid, belongs to a similar homogeneous reaction and can accelerate the reaction speed; the connection belongs to chemical connection of bioactive substances, so that the coating efficiency is higher, and the coating is more stable in a liquid environment.
The detection reagent has the advantages of high specificity, high sensitivity, simple and convenient separation, rapidness, no toxicity, safety and stability, short detection time by adopting the reagent, high efficiency and good stability.
The present invention will be further described with reference to the following examples.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: a reagent for detecting COVID-19 by chemiluminescence immunoassay, which is prepared by the following steps:
(1) preparing COVID-19 superparamagnetic nano microsphere working solution
1) Magnetic bead washing
And (3) uniformly mixing the reagent bottle filled with the superparamagnetic nano microspheres in a vortex manner at room temperature for 1min until the magnetic bead suspension is uniformly mixed, sucking 0.24mL of magnetic bead labeled buffer solution C1 into a coating tube, adding 10 mu L of nano microsphere mother solution, screwing down a tube cover, and uniformly mixing in a vortex manner for 1 min.
2) Magnetic bead coating
Under the condition of room temperature, placing the coating tube on a magnetic separation frame for 1min, completely absorbing liquid, taking the coating tube off the magnetic separation frame, adding 0.08mL of magnetic bead labeled buffer C1 into the coating tube, screwing down a tube cover, carrying out vortex mixing for 1min, adding 0.02mg of COVID-19 antigen into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, adding 0.16mL of magnetic bead labeled buffer C2 into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, placing the uniformly mixed coating tube in a constant temperature incubator, setting the temperature to be 37 ℃, setting the rotation speed to be 150rpm/min, and reacting for 16 hours.
3) Magnetic bead washing
Taking out the coating tube from the constant-temperature incubator in the step 2, placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing in a vortex manner; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely sucking up liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; and (3) placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃, setting the rotation speed at 150 rpm/min, and reacting for 7 hours.
4) Magnetic bead sealing
Taking out the coating tube from the constant-temperature incubator in the step 3), placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing by vortex; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; and (3) placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃, setting the rotating speed at 150 rpm/min, and reacting for 16 hours.
5) Cleaning of
After the sealing is finished, taking down the coating tube from the constant-temperature incubator in the step 4), and placing the coating tube on a magnetic separation frame for 1min at room temperature to suck up liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, keeping the solution temperature to be room temperature, screwing down the tube cover, and uniformly mixing for 1min by vortex.
Placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, keeping the solution temperature to be room temperature, screwing down the tube cover, and uniformly mixing for 1min by vortex.
Placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, keeping the solution temperature to be room temperature, screwing down the tube cover, and uniformly mixing for 1min by vortex.
Adding 0.4mL of magnetic bead labeling buffer solution FF into the coating tube after three times of vortex mixing, balancing the solution temperature to room temperature, screwing down the tube cover, vortex mixing for 1min, finishing coating of the COVID-19 superparamagnetic nano microsphere mother solution, and marking the concentration of the magnetic beads, the product name, the production date, the storage condition and the effective period on the coating tube label.
7) Preparation of magnetic microsphere working solution
And (3) diluting the COVID-19 superparamagnetic nano microsphere mother liquor by using a magnetic bead preservation solution FF, wherein the ratio of the magnetic bead preservation solution FF to the COVID-19 superparamagnetic nano microsphere mother liquor is 9: 1, so as to prepare the COVID-19 superparamagnetic nano microsphere working solution.
(3) Preparation of acridinium ester labeled anti-human IgG/IgM working solution
1) Dialysis bag treatment
Cutting the dialysis bag with scissors at 15cm x 2(22 mm); boiling in dialysis bag treatment liquid A for 10 min; after natural cooling, thoroughly washing with pure water; boiling in dialysis bag treatment liquid B for 10 min; naturally cooling, and storing in a refrigerator at 4 deg.C for use; remarking: the dialysis bag should be kept immersed in the liquid and rinsed with pure water before use.
2) Raw material treatment
Centrifuging and mixing the antihuman IgG/IgM, taking 1.0mg of the antibody after mixing, diluting the antibody to 1.0mg/ml by using a labeling buffer solution, and oscillating and mixing the antibody uniformly; washing the dialysis bag in the step 1) by pure water, then filling the uniformly mixed antibody into the dialysis bag, and fixing the two ends of the dialysis bag by a dialysis clamp; put into 1000 mL of large-volume labeling buffer, and the magnetic stirrer is put at 200 still/min for rotary dialysis at 4 ℃ for 24 h.
3) Marking
Dissolving 0.05mg acridinium ester with 20.0uL DMF/DMSO, and mixing; taking out the antibody dialyzed in the step 2) into a centrifuge tube; adding the dissolved acridinium ester, oscillating and uniformly mixing, and then rotating and uniformly mixing at 37 ℃ for reaction for 1 h; adding 0.07mL of reaction stopping solution, oscillating, uniformly mixing, rotating and uniformly mixing at 37 ℃ and reacting for 30 min;
4) purification of
Putting the uniformly mixed antibody obtained in the step 3) into a dialysis bag cleaned by pure water, and fixing the two ends of the dialysis bag by a dialysis clamp; putting into 1000 mL of large-volume purified buffer solution, performing rotary dialysis for 24-48h at 4 ℃ by using a magnetic stirrer at 200 still/min, wherein the solution is changed once within 6-8h, and monitoring the concentration of free acridinium ester in the dialysate before the solution is changed; when monitoring RLU is less than 10000, taking out the marker in a centrifuge tube; 1:1, adding glycerol to make the final concentration of the glycerol be 0.2 mg/mL, and uniformly mixing the glycerol and the glycerol by oscillation; labeling the acridinium ester labeled anti-human IgG/IgM mother liquor; the label of the labeling tube is marked with the antibody concentration, product name, production date, storage condition, and expiration date.
5) Preparation of acridinium ester labeled anti-human IgG/IgM working solution
Diluting the acridine ester marked anti-human IgG/IgM mother liquor by using a magnetic bead preservation liquor FF, wherein the ratio of the magnetic bead preservation liquor FF to the acridine ester marked anti-human IgG/IgM mother liquor is 1000: 1, and thus obtaining the anti-human IgG/IgM acridine ester working liquor.
(3) Preparing COVID-19 calibrator and quality control product
Diluting COVID-19 standard (high concentration serum) with magnetic bead preservation solution FF according to a certain proportion to obtain a series of calibrators, wherein the concentrations of the calibrators are 0 AU/ml, 5 AU/ml, 20 AU/ml, 50 AU/ml, 200 AU/ml and 500 AU/ml respectively; the concentration of the quality control product is 20 AU/ml and 200 AU/ml.
(4) Preparing concentrated washing liquid
The composition was prepared in a Tris-HCl buffer solution (1 LpH7.5) containing 7g of NaCl, 0.2ml of Tween-20 and 6ml of Proclin-300.
(5) Preparing substrate solution A and substrate solution B
Substrate solution A: Tris-HCl solution of 0.01mol/LpH7.5 is used to prepare hydrogen peroxide solution of 0.01mol/L and nitric acid solution of 0.2 mol/L.
Substrate solution B: a solution containing 0.1g/L sodium hydroxide was prepared using 0.01mol/L of Tris-HCl solution, pH 7.5.
(6) Subpackaging the COVID-19 superparamagnetic nano microsphere working solution, the acridine ester labeled anti-human IgG/IgM working solution, the COVID-19 calibrator and quality control material, the concentrated washing solution, the substrate solution A and the substrate solution B prepared in the steps (1) to (5), and then placing at 2-8 ℃ for refrigeration and preservation.
The magnetic bead labeling buffer C1 comprises Na2HPO4、NaH2PO4And ddH 2O;
the magnetic bead labeling buffer C2 contained (NH)4)SO4、Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O;
the magnetic bead labeling buffer WB comprises Tris, NaCl, BSA, 50% HCI and ddH 2O;
magnetic beadLabeling buffer BB comprises Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O.
Na in the magnetic bead labeling buffer C12HPO433.90g of NaH2PO40.83g, and the volume is 1L from ddH 2O;
magnetic bead labeling buffer C2 (NH)4)SO439.64g of Na2HPO429.00g of NaH2PO42.96g of NaCl, 9.00g of NaCl, 5.00g of cane sugar and 5.00g of BSA, and the volume is determined to be 100 mL by ddH 2O;
adjusting the pH value to 8.0 +/-0.1 by using 1.21g of Tris, 9.00g of NaCl, 5.00g of BSA and 50% HCI in the magnetic bead labeling buffer WB, and fixing the volume to 1L by using ddH 2O;
labeling Na in buffer BB with magnetic beads2HPO429.00g of NaH2PO42.96g, 9.00g NaCl, 5.00g sucrose and 5.00g BSA were added to make up 1L from ddH 2O.
The magnetic bead preservation solution FF comprises Tris, NaCl, PC300, BSA, 50% HCI and ddH2O, wherein the pH value of Tris in the magnetic bead preservation solution FF is 1.21g, the pH value of NaCl in the magnetic bead preservation solution FF is 9.00g, the pH value of PC300 in the magnetic bead preservation solution is 0.50g, the pH value of BSA in the magnetic bead preservation solution is 1.00g, and the volume of the solution is adjusted to 1L from ddH 2O.
The dialysis bag treatment fluid A comprises NaHCO3、EDTA-Na21M NaOH/1M HCl and ddH 2O;
the dialysis bag treatment liquid B comprises EDTA-Na 21M NaOH/1M HCl and ddH 2O;
NaHCO in dialysis bag treatment liquid A320.0g of EDTA-Na2372.24mg, regulating pH to 8.4 + -0.1 with 1M NaOH/1M HCl, and diluting to 1L with ddH 2O;
EDTA-Na in the dialysis bag treatment liquid B2372.24mg, 1M NaOH/1M HCl adjusted pH 5.3. + -. 0.1, ddH2O to 1L.
The labeling buffer comprises NaHCO31M NaOH/1M HCl and ddH2O, 8.4g NaHCO in the labeling buffer3The pH of the mixture was adjusted to 8.4. + -. 0.1 with 1M NaOH/1M HCl, and the volume was adjusted to 1L from ddH 2O.
The reaction terminating solution includes: lysine and ddH2O, wherein the lysine in the termination reaction liquid is 0.007g, and the ddH2O is 70 uL.
The purification buffer comprises Na2HPO4.12H2O、NaH2PO4.2H2O, NaCl, 1M NaOH/1M HCl and ddH2O, Na in the purification buffer2HPO4.12H2O is 2.58g, NaH2PO4.2H2O0.44 g, NaCl 8.2g, and 1M NaOH/1MHCl at pH 7.3. + -. 0.1, to a constant volume of 1L from ddH 2O.
Example 2: a detection method for detecting COVID-19 by chemiluminescence immunoassay, the detection method comprising the following steps:
the reagent prepared in example 1 was taken out from the refrigerated environment and the reagent was allowed to equilibrate at room temperature for more than 15 minutes.
And (3) mixing the concentrated washing solution according to the proportion of 1: diluted at a ratio of 40, shaken well for use as the application wash.
And (3) placing the COVID-19 calibrator, the quality control material and the sample to be detected on a chemiluminescence analyzer to complete the setting of relevant measurement parameters, and then detecting.
The determination parameters are as follows:
the fitting type is as follows: four parameter Logistic fitting
And (3) selecting coordinates: X-Y.
Performance index of reagent kit
1) Minimum limit of detection
The sample is tested by using a zero-concentration calibrator or a sample diluent, the test is repeated for 20 times, the RLU value (relative luminescence value) of 20 measurement results is obtained, and the average value (M) and the Standard Deviation (SD) are calculated, so that M-2SD is obtained. And substituting the RLU value of the M-2SD into a calibration curve to obtain a corresponding concentration value, namely the lowest detection limit, wherein the result is less than or equal to 1.0 AU/ml.
2) Precision degree
The kit is used for detecting repetitive reference products with the concentrations of 20 AU/ml (+ -20%) and 200 AU/ml (+ -20%), the repetitive detection is carried out for 10 times respectively, the average value M and the standard deviation SD of 10 times of measurement results are calculated, the variation coefficient CV is obtained according to the formula CV = SD/M multiplied by 100%, the result is less than or equal to 10.0%, and the range difference of the measurement concentrations (the difference between the maximum measurement concentration value and the minimum measurement concentration value) of the high-concentration repetitive reference products and the low-concentration repetitive reference products is less than or equal to 20.0% of the average value M of the 10 times of measurement concentrations.
3) Dilution line
And (3) diluting the high-value sample with the low-value sample in sequence according to a proportion, so that the theoretical concentration of the obtained linear reference substance basically covers the linear range of the item, and the concentrations are related in a multiple ratio. And (3) detecting each linear reference substance for 3 times, calculating the average value of the concentration of each linear reference substance, performing linear fitting on the result average value and the theoretical concentration by using a least square method, and calculating a linear correlation coefficient r, wherein the result is more than or equal to 0.9900, and the deviation of the tested concentration average value of each linear reference substance 2-5 and the theoretical concentration is within +/-10%.
Linear reference theoretical concentration = [ (last linear reference theoretical concentration × last linear reference added volume) + (low value sample actual concentration × low value sample added volume) ]/(high concentration sample added volume + low value sample added volume).
4) Accuracy of
The recovery rate was in the range of 85.0% to 115.0% when 50ul of the high concentration sample at 50.0 AU/ml was added to 950ul of the blood substrate at a concentration of about 2.0 AU/ml.
5) Rate of agreement
More than or equal to 80 clinical samples distributed in the detection range of the comparison reagent or the reagent to be detected are randomly selected (the clinical samples which exceed the detection range and are diluted according to the requirements of respective specifications are tested), the number of normal samples and the number of abnormal samples are approximately equally divided (about 40 samples respectively), the comparison reagent and the reagent to be detected are respectively used for testing, the comparison reagent is used for single-hole testing, and the reagent to be detected is used for multi-hole testing to obtain the average value (if the test result of the comparison reagent can be obtained from a hospital, and a project responsible for the test is not used, only the test of the reagent. And comparing the detection results of the two reagents by taking the test result of the comparison reagent as a reference. The normal compliance rate is more than or equal to 90 percent, the abnormal compliance rate is more than or equal to 90 percent, and the total compliance rate is more than or equal to 90 percent.
6) Stability of
Storing the reagent at 37 deg.C, after 7 days, respectively crossing with accelerating reagent component and 4 deg.C contrast reagent component, and repeating the test product calibrator for 3 times, wherein the variation amplitude of the same calibrator/sample is less than or equal to 30%.
Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and the embodiments are examples, wherein the details that are not described are all the common general knowledge of those skilled in the art, and the technical solutions described in the foregoing embodiments can be modified or some technical features can be equivalently replaced by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A reagent for detecting COVID-19 by chemiluminescence immunoassay, which is characterized in that: the preparation method of the reagent comprises the following steps:
(1) preparing COVID-19 superparamagnetic nano microsphere working solution
1) Magnetic bead washing
Uniformly mixing the reagent bottle filled with the superparamagnetic nano microspheres in a vortex manner for 1min at room temperature until the magnetic bead suspension is uniformly mixed, sucking 0.24mL of magnetic bead labeled buffer solution C1 into a coating tube, adding 10 mu L of nano microsphere mother solution, screwing down a tube cover, and uniformly mixing in a vortex manner for 1 min;
2) magnetic bead coating
Under the condition of room temperature, placing the coating tube on a magnetic separation frame for 1min, completely absorbing liquid, taking the coating tube off the magnetic separation frame, adding 0.08mL of magnetic bead labeled buffer C1 into the coating tube, screwing down a tube cover, carrying out vortex mixing for 1min, adding 0.02mg of COVID-19 antigen into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, adding 0.16mL of magnetic bead labeled buffer C2 into the coating tube, screwing down the tube cover, carrying out vortex mixing for 1min, placing the uniformly mixed coating tube in a constant temperature incubator, setting the temperature to be 37 ℃, setting the rotating speed to be 150rpm/min, and reacting for 16 hours;
3) Magnetic bead washing
Taking out the coating tube from the constant-temperature incubator in the step 2, placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing in a vortex manner; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely sucking up liquid, adding 0.4mL of magnetic bead marking buffer WB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃ and the rotation speed at 150 rpm/min, and reacting for 7 hours;
4) magnetic bead sealing
Taking out the coating tube from the constant-temperature incubator in the step 3), placing the coating tube on a magnetic separation frame for 1min at room temperature, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and uniformly mixing by vortex; placing the coating tube after vortex mixing on a magnetic separation frame for 1min, completely absorbing liquid, adding 0.4mL of magnetic bead labeled buffer BB into the coating tube, balancing the temperature of the solution to room temperature, screwing a tube cover, and vortex mixing; placing the uniformly mixed coating tube in a constant-temperature incubator, setting the temperature at 37 ℃ and the rotation speed at 150 rpm/min, and reacting for 16 hours;
5) Cleaning of
After the sealing is finished, taking down the coating tube from the constant-temperature incubator in the step 4), and placing the coating tube on a magnetic separation frame for 1min at room temperature to suck up liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
placing the coating tube after vortex mixing on a magnetic separation frame for 1min, and completely absorbing liquid; adding 0.4mL of magnetic bead preservation solution FF into the coating tube, wherein the temperature of the solution needs to be balanced to room temperature, screwing down a tube cover, and uniformly mixing for 1min in a vortex manner;
adding 0.4mL of magnetic bead labeled buffer solution FF into the coating tube after three times of vortex mixing, wherein the temperature of the solution needs to be balanced to room temperature, screwing a tube cover, carrying out vortex mixing for 1min, and finishing coating of the COVID-19 superparamagnetic nano microsphere mother solution;
6) preparation of magnetic microsphere working solution
Diluting the COVID-19 superparamagnetic nano microsphere mother liquor by using a magnetic bead preservation solution FF, wherein the ratio of the magnetic bead preservation solution FF to the COVID-19 superparamagnetic nano microsphere mother liquor is 9: 1, and preparing a COVID-19 superparamagnetic nano microsphere working solution;
(2) Preparation of acridinium ester labeled anti-human IgG/IgM working solution
1) Dialysis bag treatment
Shearing the dialysis bag by scissors; boiling in dialysis bag treatment liquid A for 10 min; after natural cooling, thoroughly washing with pure water; boiling in dialysis bag treatment liquid B for 10 min; naturally cooling, and storing in a refrigerator at 4 deg.C for use;
2) raw material treatment
Centrifuging and mixing the antihuman IgG/IgM, taking 1.0mg of the antibody after mixing, diluting the antibody to 1.0mg/ml by using a labeling buffer solution, and oscillating and mixing the antibody uniformly; washing the dialysis bag in the step 1) by pure water, then filling the uniformly mixed antibody into the dialysis bag, and fixing the two ends of the dialysis bag by a dialysis clamp; putting the mixture into 1000 mL of large-volume labeling buffer solution, and performing rotary dialysis on the mixture for 24 hours at the temperature of 4 ℃ by using a magnetic stirrer at the still/min;
3) marking
Dissolving 0.05mg acridinium ester with 20.0uL DMF/DMSO, and mixing; taking out the antibody dialyzed in the step 2) into a centrifuge tube; adding the dissolved acridinium ester, oscillating and uniformly mixing, and then rotating and uniformly mixing at 37 ℃ for reaction for 1 h; adding 0.07mL of reaction stopping solution, oscillating, uniformly mixing, rotating and uniformly mixing at 37 ℃ and reacting for 30 min;
4) purification of
Putting the uniformly mixed antibody obtained in the step 3) into a dialysis bag cleaned by pure water, and fixing the two ends of the dialysis bag by a dialysis clamp; putting into 1000 mL of large-volume purified buffer solution, performing rotary dialysis for 24-48h at 4 ℃ by using a magnetic stirrer at 200 still/min, wherein the solution is changed once within 6-8h, and monitoring the concentration of free acridinium ester in the dialysate before the solution is changed; when monitoring RLU is less than 10000, taking out the marker in a centrifuge tube; 1:1, adding glycerol to make the final concentration of the glycerol be 0.2 mg/mL, and uniformly mixing the glycerol and the glycerol by oscillation; labeling the acridinium ester labeled anti-human IgG/IgM mother liquor;
5) Preparation of acridinium ester labeled anti-human IgG/IgM working solution
Diluting the acridine ester marked anti-human IgG/IgM mother liquor by using a magnetic bead preservation liquor FF, wherein the ratio of the magnetic bead preservation liquor FF to the acridine ester marked anti-human IgG/IgM mother liquor is 1000: 1, and preparing an anti-human IgG/IgM acridine ester working solution;
(3) preparing COVID-19 calibrator and quality control product
Diluting COVID-19 standard substance with magnetic bead preservation solution FF according to a certain proportion to obtain a series of calibrators, wherein the concentrations of the calibrators are 0AU/ml, 5 AU/ml, 20AU/ml, 50 AU/ml, 200 AU/ml and 500 AU/ml respectively; the concentration of the quality control product is 20AU/ml and 200 AU/ml;
(4) preparing concentrated washing liquid
Preparing according to the condition that each 1LpH7.5 Tris-HCl buffer solution contains 7g of NaCl, 0.2ml of Tween-20 and 6ml of Proclin-300;
(5) preparing substrate solution A and substrate solution B
Substrate solution A: preparing a solution containing hydrogen peroxide with the concentration of 0.01mol/L and nitric acid with the concentration of 0.2mol/L by using a Tris-HCl solution with the concentration of 0.01mol/L pH7.5;
substrate solution B: preparing a sodium hydroxide solution with the concentration of 0.1g/L by using 0.01mol/L Tris-HCl solution with the pH value of 7.5;
(6) subpackaging the COVID-19 superparamagnetic nano microsphere working solution, the acridine ester labeled anti-human IgG/IgM working solution, the COVID-19 calibrator and quality control material, the concentrated washing solution, the substrate solution A and the substrate solution B prepared in the steps (1) to (5), and then placing at 2-8 ℃ for refrigeration and preservation.
2. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein: the magnetic bead labeling buffer C1 comprises Na2HPO4、NaH2PO4And ddH 2O;
the magnetic bead labeling buffer C2 contained (NH)4)SO4、Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O;
the magnetic bead labeling buffer WB comprises Tris, NaCl, BSA, 50% HCI and ddH 2O;
the magnetic bead labeling buffer BB comprises Na2HPO4、NaH2PO4NaCl, sucrose, BSA, and ddH 2O.
3. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 2, wherein: na in the magnetic bead labeling buffer C12HPO433.90g of NaH2PO40.83g, and the volume is 1L from ddH 2O;
magnetic bead labeling buffer C2 (NH)4)SO439.64g of Na2HPO429.00g of NaH2PO42.96g of NaCl, 9.00g of NaCl, 5.00g of cane sugar and 5.00g of BSA, and the volume is determined to be 100 mL by ddH 2O;
adjusting the pH value to 8.0 +/-0.1 by using 1.21g of Tris, 9.00g of NaCl, 5.00g of BSA and 50% HCI in the magnetic bead labeling buffer WB, and fixing the volume to 1L by using ddH 2O;
labeling Na in buffer BB with magnetic beads2HPO429.00g of NaH2PO42.96g, 9.00g NaCl, 5.00g sucrose and 5.00g BSA were added to make up 1L from ddH 2O.
4. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein:
The magnetic bead preservation solution FF comprises Tris, NaCl, PC300, BSA, 50% HCI and ddH2O, wherein the pH value of Tris in the magnetic bead preservation solution FF is 1.21g, the pH value of NaCl in the magnetic bead preservation solution FF is 9.00g, the pH value of PC300 in the magnetic bead preservation solution is 0.50g, the pH value of BSA in the magnetic bead preservation solution is 1.00g, and the volume of the solution is adjusted to 1L from ddH 2O.
5. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein:
the dialysis bag treatment fluid A comprises NaHCO3、EDTA-Na21M NaOH/1M HCl and ddH 2O;
the dialysis bag treatment liquid B comprises EDTA-Na21M NaOH/1M HCl and ddH 2O;
NaHCO in dialysis bag treatment liquid A3Is 20.0g、EDTA-Na2372.24mg, regulating pH to 8.4 + -0.1 with 1M NaOH/1M HCl, and diluting to 1L with ddH 2O;
EDTA-Na in the dialysis bag treatment liquid B2372.24mg, 1M NaOH/1M HCl adjusted pH 5.3. + -. 0.1, ddH2O to 1L.
6. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein: the labeling buffer comprises NaHCO31M NaOH/1M HCl and ddH2O, 8.4g NaHCO in the labeling buffer31M NaOH/1M HCl was adjusted to pH 8.4. + -. 0.1, and the volume was adjusted to 1L from ddH 2O.
7. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein: the termination reaction solution comprises lysine and ddH2O, wherein the lysine in the termination reaction solution is 0.007g, and the ddH2O is 70 uL.
8. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 1 wherein: the purification buffer comprises Na2HPO4.12H2O、NaH2PO4.2H2O, NaCl, 1M NaOH/1M HCl and ddH2O, Na in the purification buffer2HPO4.12H2O is 2.58g, NaH2PO4.2H2O0.44 g, NaCl 8.2g, 1M NaOH/1M HCl pH 7.3. + -. 0.1, ddH2O to 1L.
9. A detection method for detecting COVID-19 by chemiluminescence immunoassay is characterized in that: the detection method comprises the following steps:
removing the reagent of any one of claims 1 to 8 from the refrigerated environment and allowing the reagent to equilibrate for more than 15 minutes at room temperature;
and (3) mixing the concentrated washing solution according to the proportion of 1: diluting at the ratio of 40, and shaking uniformly for later use as an application washing solution;
and (3) placing the COVID-19 calibrator, the quality control material and the sample to be detected on a chemiluminescence analyzer to complete the setting of relevant measurement parameters, and then detecting.
10. The reagent for detecting COVID-19 in a chemiluminescent immunoassay according to claim 9 wherein: the determination parameters are as follows:
the fitting type is as follows: four parameter Logistic fitting
And (3) selecting coordinates: X-Y.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010218736.1A CN111856004A (en) | 2020-03-25 | 2020-03-25 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010218736.1A CN111856004A (en) | 2020-03-25 | 2020-03-25 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111856004A true CN111856004A (en) | 2020-10-30 |
Family
ID=72985949
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010218736.1A Pending CN111856004A (en) | 2020-03-25 | 2020-03-25 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111856004A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112269021A (en) * | 2020-12-21 | 2021-01-26 | 北京中关村水木医疗科技有限公司 | IgM quality control product and preparation method thereof |
| CN113109558A (en) * | 2021-04-21 | 2021-07-13 | 山东康华生物医疗科技股份有限公司 | Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof |
| CN113759109A (en) * | 2021-08-20 | 2021-12-07 | 捷和泰（北京）生物科技有限公司 | Chemiluminescence immunoassay kit for detecting anti-SS-B antibody by double-antigen sandwich method |
| WO2022121151A1 (en) * | 2020-12-10 | 2022-06-16 | 丹娜(天津)生物科技股份有限公司 | Magnetic particle chemiluminescence-based novel coronavirus antibody detection kit |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120329050A1 (en) * | 2009-12-22 | 2012-12-27 | Becton, Dickinson And Company | Methods for the detection of microorganisms |
| US20150072889A1 (en) * | 2013-09-06 | 2015-03-12 | Theranos, Inc. | Systems and methods for detecting infectious diseases |
| CN107490574A (en) * | 2017-08-11 | 2017-12-19 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of melamine and preparation method thereof |
| WO2018000898A1 (en) * | 2016-06-30 | 2018-01-04 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transporter protein 8 antibody chemiluminescence immunoassay kit and preparation method therefor |
| CN108872595A (en) * | 2018-07-05 | 2018-11-23 | 潍坊市康华生物技术有限公司 | A kind of carcinomebryonic antigen detection kit and preparation method thereof |
| CN109100516A (en) * | 2018-09-19 | 2018-12-28 | 迪瑞医疗科技股份有限公司 | A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit and preparation method thereof |
| CN110568198A (en) * | 2019-09-12 | 2019-12-13 | 潍坊市康华生物技术有限公司 | N-terminal B-type brain natriuretic peptide precursor magnetic particle chemiluminescence detection kit |
| CN110672850A (en) * | 2019-10-31 | 2020-01-10 | 迪瑞医疗科技股份有限公司 | Hepatitis A virus antibody IgM detection kit and preparation method thereof |
| WO2020028559A1 (en) * | 2018-07-31 | 2020-02-06 | University Of Florida Research Foundation | Apparatus and method for performing microorganism detection |
| WO2020056110A1 (en) * | 2018-09-13 | 2020-03-19 | The Trustees Of The University Of Pennsylvania | Microbubbling and indicator material displacement systems and methods |
-
2020
- 2020-03-25 CN CN202010218736.1A patent/CN111856004A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120329050A1 (en) * | 2009-12-22 | 2012-12-27 | Becton, Dickinson And Company | Methods for the detection of microorganisms |
| US20150072889A1 (en) * | 2013-09-06 | 2015-03-12 | Theranos, Inc. | Systems and methods for detecting infectious diseases |
| WO2018000898A1 (en) * | 2016-06-30 | 2018-01-04 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transporter protein 8 antibody chemiluminescence immunoassay kit and preparation method therefor |
| CN107490574A (en) * | 2017-08-11 | 2017-12-19 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of melamine and preparation method thereof |
| CN108872595A (en) * | 2018-07-05 | 2018-11-23 | 潍坊市康华生物技术有限公司 | A kind of carcinomebryonic antigen detection kit and preparation method thereof |
| WO2020028559A1 (en) * | 2018-07-31 | 2020-02-06 | University Of Florida Research Foundation | Apparatus and method for performing microorganism detection |
| WO2020056110A1 (en) * | 2018-09-13 | 2020-03-19 | The Trustees Of The University Of Pennsylvania | Microbubbling and indicator material displacement systems and methods |
| CN109100516A (en) * | 2018-09-19 | 2018-12-28 | 迪瑞医疗科技股份有限公司 | A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit and preparation method thereof |
| CN110568198A (en) * | 2019-09-12 | 2019-12-13 | 潍坊市康华生物技术有限公司 | N-terminal B-type brain natriuretic peptide precursor magnetic particle chemiluminescence detection kit |
| CN110672850A (en) * | 2019-10-31 | 2020-01-10 | 迪瑞医疗科技股份有限公司 | Hepatitis A virus antibody IgM detection kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| BO DIAO 等: "Diagnosis of Acute Respiratory Syndrome Coronavirus 2 Infection by Detection of Nucleocapsid Protein" * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121151A1 (en) * | 2020-12-10 | 2022-06-16 | 丹娜(天津)生物科技股份有限公司 | Magnetic particle chemiluminescence-based novel coronavirus antibody detection kit |
| CN112269021A (en) * | 2020-12-21 | 2021-01-26 | 北京中关村水木医疗科技有限公司 | IgM quality control product and preparation method thereof |
| CN112269021B (en) * | 2020-12-21 | 2021-04-06 | 北京中关村水木医疗科技有限公司 | IgM quality control product and preparation method thereof |
| CN113109558A (en) * | 2021-04-21 | 2021-07-13 | 山东康华生物医疗科技股份有限公司 | Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof |
| CN113759109A (en) * | 2021-08-20 | 2021-12-07 | 捷和泰（北京）生物科技有限公司 | Chemiluminescence immunoassay kit for detecting anti-SS-B antibody by double-antigen sandwich method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111856004A (en) | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof | |
| CN110618263B (en) | Method for detecting C-reactive protein in whole process and corresponding kit | |
| CN103278623A (en) | Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) nano magnetic particle and preparation method of kit | |
| CN113009153A (en) | Magnetic particle chemiluminescence-based detection kit for neutralizing antibody of novel coronavirus and application thereof | |
| CN101377514A (en) | Insulin autoantibody chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN103698535A (en) | Quantitative detection kit of lipoprotein associated phospholipase A2, as well as preparation and operation method of quantitative detection kit | |
| CN115469105B (en) | One-step method rapid detection kit for African swine fever ELISA antibody | |
| CN108089007A (en) | A kind of kit and preparation method for quantitatively detecting c reactive protein | |
| CN111323577A (en) | Anti-cardiolipin antibody IgG magnetic particle chemiluminescence immunoassay kit | |
| CN110672850A (en) | Hepatitis A virus antibody IgM detection kit and preparation method thereof | |
| CN110988333A (en) | Serum tissue metalloproteinase inhibitor-1 chemiluminescence immunoassay kit and preparation method thereof | |
| CN111474340A (en) | Enzyme-labeled antigen for novel coronavirus detection, preparation method, kit and application | |
| JP5426661B2 (en) | Immunoassay method and reagent therefor | |
| CN103344635A (en) | Clara cell protein nano-magnetic particle quantitative chemiluminescence immunoassay test kit and preparation method thereof | |
| CN111504959A (en) | C-peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof | |
| CN110554193A (en) | Preparation method of colloidal gold immunochromatographic test strip for paraquat in urine | |
| CN108872609A (en) | It is a kind of for detecting the time-resolved fluoroimmunoassay kit of canine parvovirus antibody | |
| CN106568961A (en) | Enzyme linked immunosorbent assay kit for detection of paraquat and detection method thereof | |
| CN112326954A (en) | Homogeneous fluoroimmunoassay reagent for rapidly and quantitatively detecting D-dimer | |
| CN113552372A (en) | Magnetic particle chemiluminescence immunoassay kit for SAA detection and preparation method thereof | |
| CN111548416B (en) | Kit and composition for detecting anti-double-stranded DNA antibody and application | |
| Saleem et al. | Improved micromethod for plasma fibrinogen unaffected by heparin therapy | |
| CN113391065A (en) | Receptor reagent for detecting novel coronavirus and application thereof | |
| CN112505332A (en) | High-sensitivity cTnI detection method and kit thereof | |
| JP3228791B2 (en) | Measuring method of antigen or antibody in sample |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201030 |
|
| RJ01 | Rejection of invention patent application after publication |