CN116298277A - Sample treatment fluid and application thereof - Google Patents
Sample treatment fluid and application thereof Download PDFInfo
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- CN116298277A CN116298277A CN202310358223.4A CN202310358223A CN116298277A CN 116298277 A CN116298277 A CN 116298277A CN 202310358223 A CN202310358223 A CN 202310358223A CN 116298277 A CN116298277 A CN 116298277A
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- 239000012530 fluid Substances 0.000 title claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 103
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 11
- 238000012545 processing Methods 0.000 claims description 32
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- 159000000003 magnesium salts Chemical class 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 239000013504 Triton X-100 Substances 0.000 claims description 12
- 229920004890 Triton X-100 Polymers 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 241000711573 Coronaviridae Species 0.000 claims description 9
- 241000700605 Viruses Species 0.000 claims description 9
- 238000003018 immunoassay Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 229920000136 polysorbate Polymers 0.000 claims description 5
- 241000712461 unidentified influenza virus Species 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 229920004896 Triton X-405 Polymers 0.000 claims description 4
- 229920001992 poloxamer 407 Polymers 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 241001678559 COVID-19 virus Species 0.000 claims 1
- 238000012286 ELISA Assay Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 18
- 150000003839 salts Chemical class 0.000 abstract description 3
- 239000002280 amphoteric surfactant Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 90
- 238000001514 detection method Methods 0.000 description 21
- 239000000203 mixture Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- 238000009472 formulation Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 238000003908 quality control method Methods 0.000 description 11
- 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 7
- 235000018102 proteins Nutrition 0.000 description 7
- 239000004094 surface-active agent Substances 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000003317 immunochromatography Methods 0.000 description 5
- 230000027455 binding Effects 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 241001493065 dsRNA viruses Species 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 241000713196 Influenza B virus Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- -1 BB (borate buffer) Chemical compound 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 208000025721 COVID-19 Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- 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
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a sample treatment fluid and application thereof. According to the invention, the amphoteric surfactant and the nonionic surfactant for salt parallel connection are added into the sample treatment liquid, so that the sensitivity of immunodetection can be effectively improved.
Description
Technical Field
The present invention relates to the field of biotechnology. In particular, the invention discloses a sample treatment fluid and application thereof.
Background
RNA enveloped viruses are a large family of RNA viruses, and the RNA virus particles are provided with lipid envelopes, so that the viruses can enter and exit target cells through the envelopes, and the RNA enveloped viruses have strong infection capacity. Common influenza viruses and coronaviruses all belong to RNA-enveloped viruses. 2019 novel coronavirus (2019-nCoV) is the 7 th currently known coronavirus that can infect humans.
The immunological detection is based on the immunological principle of antigen-antibody specific binding, and common methods mainly include immunochromatography detection, ELISA detection or chemiluminescence detection. Different samples are complicated in composition and large in variation, and if no corresponding treatment is performed, nonspecific reactions other than antigen-antibody binding easily occur, resulting in generation of adverse cross reactions, reduction in detection sensitivity, and the like.
Taking new coronavirus detection as an example, the currently marketed immunological detection products have low sensitivity and risk of missed detection. The present invention has been proposed to further improve the detection sensitivity.
Disclosure of Invention
In order to improve the sensitivity of the existing immunodetection products, the invention discloses a method for improving the detection sensitivity by contacting a sample to be detected with a sample treatment liquid containing a soluble magnesium salt in immunodetection of antigen-antibody reaction with a component to be detected.
The invention includes the following embodiments:
a sample processing method in immunodetection is to make a sample to be detected contact with a sample processing liquid containing soluble magnesium salt.
In some embodiments, the sample may be any sample suspected of containing the component to be tested, such as, without limitation, a pharyngeal swab collection sample, an oropharyngeal swab collection sample, a nasopharyngeal swab collection sample, a sputum sample, an alveolar lavage sample, a peripheral blood sample, a plasma sample, or a serum sample; in some embodiments, the sample is, but not limited to, a pharyngeal swab or a nasopharyngeal swab, for example.
In some embodiments, the sample is a sample containing a component to be tested; in some embodiments, the component to be detected is an RNA virus; in some embodiments, the RNA virus is an RNA enveloped virus; in some embodiments, the RNA-enveloped virus is at least any one of a coronavirus or an influenza virus; in some embodiments, the RNA enveloped virus is, for example, 2019-nCoV, such as SARS, or such as influenza virus, but is not limited thereto.
In the present invention, contacting the sample to be detected with the sample processing liquid includes, for example, adding the sample to be detected to the sample processing liquid, mixing the sample to be detected with the sample processing liquid, adding the sample to be detected to an immunoassay tool (for example, a sample pad in immunochromatography), adding the sample to be detected and the sample processing liquid to the immunoassay tool (for example, a sample pad in immunochromatography), respectively, fixing the sample processing liquid to the immunoassay tool (for example, a sample pad in immunochromatography), adding the sample to be detected, or a combination of the above forms.
In some embodiments, the final concentration of soluble magnesium salt in the sample treatment fluid is 10 to 400mM; in some embodiments, the final concentration of soluble magnesium salt in the sample treatment fluid is 40-160mM; in some embodiments, the final concentration of soluble magnesium salt in the sample treatment fluid is 80-160mM; in some embodiments, the final concentration of the soluble magnesium salt in the sample treatment fluid includes, but is not limited to, e.g., 10mM, e.g., 40mM, e.g., 80mM, e.g., 160mM, e.g., 200mM, e.g., 260mM, e.g., 320mM, e.g., 380mM, e.g., 400 mM.
In some embodiments, the final concentration of the amphoteric surfactant S9 in the sample treatment fluid is 0.1-1.5 (w/v); in some embodiments, the final concentration of S9 in the sample processing fluid is 0.25-1 (w/v)%; in some embodiments, the final concentration of S9 in the sample processing fluid is 0.5-1 (w/v)%; in some embodiments, the final concentration of S9 in the sample processing liquid includes, for example, 0.1 (w/v)%, such as 0.25 (w/v)%, such as 0.5 (w/v)%, such as 1 (w/v)%, such as 1.2 (w/v)%, such as 1.5 (w/v)%, but is not limited thereto.
In some embodiments, the sample processing fluid further comprises a buffer; in some embodiments, the buffer includes, for example, tris-HCl, such as PB (phosphate buffer), such as BB (borate buffer), but is not limited thereto. In some embodiments, the buffer component is used at a concentration of 10-50mM; in some embodiments, the buffer component is used at a concentration including, but not limited to, e.g., 10mM, e.g., 15mM, e.g., 25mM, e.g., 30mM, e.g., 35mM, e.g., 40mM, e.g., 50 mM. In some embodiments, the pH of the buffer is 8.0-9.5; in some embodiments, the pH of the buffer includes, but is not limited to, e.g., 8.0, e.g., 8.2, e.g., 8.5, e.g., 8.8, e.g., 9.0, e.g., 9.2, e.g., 9.5.
In some embodiments, the sample processing fluid further comprises an inert protein; in some embodiments, the inert protein includes, for example, casein, such as BSA, but is not limited thereto. In some embodiments, the final concentration of inert protein in the sample processing fluid is 0.3-1 (w/v)%; in some embodiments, the final concentration of the inert protein in the sample processing fluid includes, for example, 0.3 (w/v)%, such as 0.5 (w/v)%, such as 0.8 (w/v)%, such as 1 (w/v)%, but is not limited thereto.
In some embodiments, the sample processing fluid further comprises sodium chloride; in some embodiments, the final concentration of sodium chloride in the sample treatment fluid is 50-200mM; in some embodiments, the final concentration of sodium chloride in the sample processing fluid includes, for example, 50mM, such as 100mM, such as 150mM, such as 200mM, but is not limited thereto.
In some embodiments, the sample processing fluid further comprises a nonionic surfactant; the nonionic surfactant can improve the hydrophilicity and the wettability, reduce the surface tension of the liquid, and is beneficial to improving the detection signal of the component to be detected, and the specific effect is required to depend on the compatibility between the surfactant and other matrixes; in some embodiments, the nonionic surfactant includes, but is not limited to, for example, tween 20 (tween 20), for example tween 40 (tween 40), for example Pluronic F-127 (plarnike F127), for example Triton X-405 (Triton 405), for example Triton X-100 (Triton 100).
In some embodiments, the final concentration of nonionic surfactant in the sample treatment fluid is 0.1-1.5 (w/v); in some embodiments, the final concentration of the nonionic surfactant in the sample treatment fluid is 0.25-1 (w/v)%; in some embodiments, the final concentration of the nonionic surfactant in the sample treatment fluid is 0.5-1 (w/v)%; in some embodiments, the final concentration of the nonionic surfactant in the sample processing liquid includes, for example, 0.1 (w/v)%, such as 0.25 (w/v)%, such as 0.5 (w/v)%, such as 1 (w/v)%, such as 1.2 (w/v)%, such as 1.5 (w/v)%, but is not limited thereto.
In some embodiments, the sample processing fluids of the present invention are used in immunoassays. The term "immunoassay" as used herein is to be understood in a broad sense and refers to a detection method by specific binding of an antigen-antibody, including, but not limited to, immunochromatographic detection, e.g., ELISA detection, e.g., chemiluminescent detection. It will be appreciated by those skilled in the art that the sample processing fluid of the present invention may be used in an immunoassay for an antigen or antibody. In some embodiments, for example, a sample containing the novel coronavirus is contacted with the sample treatment solution of the invention, and then subjected to antigen-antibody reaction with the labeled, coated end protein, thereby realizing the effect of detecting the novel coronavirus.
In some embodiments, the present disclosure encompasses the sample processing fluids of any of the preceding embodiments.
In some embodiments, the test kits or test strips of the invention comprise a sample processing fluid of any of the preceding embodiments. The detection kit of the invention is to be understood in a broad sense and mainly refers to a tool for carrying reagents related to the immunological detection. In some embodiments, the detection kit may further comprise a number of kit-of-parts. The detection test strip comprises absorbent paper, a marker binding pad, a coating film, a sample pad and a bottom plate.
Detailed Description
The present invention will be described in further detail with reference to the following examples. The following examples are provided to illustrate embodiments of the invention and are not intended to limit the invention. The invention may optionally include embodiments not shown by way of example.
Methods not described in detail in the detailed description may refer to conventional methods or manufacturer's instructions in the art; reagents not described in the detailed description are available commercially.
In the present invention, the definition of soluble magnesium salt means that for 1g (ml) of magnesium salt, the volume of solvent required for dissolution is <100ml, i.e. soluble magnesium salt.
In the present invention, the concentration unit mM means mmol/L, and M means mol/L.
Marking
And (3) diluting protein capable of reacting with antigen and antibody of the component to be detected, marking on colloidal gold particles with the particle size of 30-80nm, adding BSA after marking, centrifuging, removing supernatant, redissolving colloidal gold precipitate by using a protective solution, dripping the redissolved colloidal gold into a glass cellulose membrane, and drying to obtain the marker binding pad.
Coating
Protein which can react with antigen and antibody of the detected component is diluted and then coated on a nitrocellulose membrane, and the membrane dividing parameter is 1.0 mu l/cm. And (3) after coating, drying in a forced air drying oven at 37 ℃ for 2 hours to obtain the coating film.
Assembly
And assembling the marker binding pad and the coating film with other materials (a bottom plate, absorbent paper and a sample pad), cutting into 2.5mm pieces, and preparing the corresponding immunodetection test strip.
Example 1
(base liquid 1) formula of base liquid 1: 0.5 (w/v)% casein, 150mM NaCl,25mM Tris-HCl, pH 9.0;
(base liquid 1-1) formula of base liquid 1-1: 1.0 (w/v)% anhydrous sodium sulfate was added to the base liquid 1;
(base liquid 1-2) formula of base liquid 1-2: 1.0 (w/v)% trisodium citrate is added to the base liquid 1;
(base liquid 1-3) formula of base liquid 1-3: 1.0 (w/v)% lithium chloride was added to the base liquid 1;
(base liquid 1-4) formula of base liquid 1-4: 1.0 (w/v)% magnesium chloride was added to the base liquid 1;
(base liquid 1-5) formula of base liquid 1-5: 1.0 (w/v)% magnesium sulfate was added to the base liquid 1;
to explore the best salt composition in the sample treatment fluid, the performance of several salts was compared.
And (3) dripping the components serving as a sample treatment liquid to a blank sample pad, drying, assembling to an immunochromatography device, and dripping a new coronavirus quality control product subjected to gradient dilution onto the treated sample pad during detection.
The result is recorded as the read value of the colloidal gold color card, and the smaller the value is, the stronger the color development is, which indicates that the higher the sensitivity is.
As a result, as shown in Table 1, when a soluble magnesium salt was added to the base liquid 1, the sensitivity was improved.
Table 1, test results of example 1
Quality control product | 1μg/ml | 0.1μg/ml | 25ng/ml | 0.5ng/ml |
Base liquid 1 | C3 | C5 | C8 | B |
Base liquid 1-1 | C2 | C4+ | C7 | C9 |
Base liquid 1-2 | C3+ | C4 | C7 | C9 |
Base liquid 1-3 | C3+ | C4- | C7+ | B |
Base liquid 1-4 | C2+ | C3- | C7+ | C9 |
Base liquid 1-5 | C2+ | C3 | C6 | C9 |
Example 2
(base liquid 2) formula of base liquid 2: 0.5 (w/v)% casein, 150mM NaCl,80mM magnesium sulfate, 25mM Tris-HCl, pH 9.0;
(base liquid 2-1) formula of base liquid 2-1: adding 0.5 (w/v)% tween 20 to the base liquid 2;
(base liquid 2-2) formula of base liquid 2-2: adding 0.5 (w/v)% tween 40 to the base liquid 2;
(base liquid 2-3) formula of base liquid 2-3: 0.5 (w/v)% Pluronic F-127 was added to base liquid 2;
(base liquid 2-4) formula of base liquid 2-4: adding 0.5 (w/v)% Triton X-405 to base liquid 2;
(base liquid 2-5) formula of base liquid 2-5: adding 0.5 (w/v)% Triton X-100 to base liquid 2;
(base liquid 2-6) formula of base liquid 2-6: 0.5 (w/v)% S9 was added to the base liquid 2;
to further explore the optimal surfactant component in the treatment fluid, the performance of several surfactants was compared. The method of using the base liquid as a sample treatment liquid was the same as in example 1.
As a result, as shown in Table 2, when S9 was added to the base liquid 2, the sensitivity was higher than that of the other surfactants.
Table 2, test results of example 2
Quality control product | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
Base liquid 2 | C3 | C6 | C8 | C9 | B |
Base liquid 2-1 | C3 | C6+ | C8+ | C9+ | C9- |
Base liquid 2-2 | C3 | C6+ | C8+ | C9+ | C9- |
2-3 parts of base liquid | C3+ | C5 | C8 | C9+ | C9- |
2-4 parts of base liquid | C3+ | C5 | C8 | C9+ | C9- |
2-5 parts of base liquid | C3+ | C5 | C8 | C9+ | C9- |
2-6 parts of base liquid | C2 | C4 | C7 | C8 | C9 |
Example 3
(base liquid 3) formula of base liquid 3: 0.5 (w/v)% casein, 150mM NaCl,80mM magnesium sulfate, 0.5 (w/v)% S9, 25mM Tris-HCl, pH 9.0;
(base liquid 3-1) formula of base liquid 3-1: 0.5 (w/v)% tween 20 was added to the base liquid 3;
(base liquid 3-2) formula of base liquid 3-2: adding 0.5 (w/v)% tween 40 to the base liquid 3;
(base liquid 3-3) formula of base liquid 3-3: 0.5 (w/v)% Pluronic F-127 was added to base liquid 3;
(base liquid 3-4) formula of base liquid 3-4: adding 0.5 (w/v)% Triton X-405 to the base liquid 3;
(base liquid 3-5) formula of base liquid 3-5: adding 0.5 (w/v)% Triton X-100 to the base liquid 3;
to further increase the sensitivity, the dominant surfactant S9 obtained in example 2 was used in combination with other surfactants. The method of using the base liquid as a sample treatment liquid was the same as in example 1.
As shown in Table 3, since the sensitivity was further improved when S9 and Triton X-100 were combined, the sample treatment solution obtained by introducing magnesium sulfate and combining S9 and Triton X-100 was designated as COV-SP and was subjected to the subsequent optimization.
Table 3, test results of example 3
Quality control product | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
Base liquid 3 | C2 | C4 | C7 | C8 | C9 |
Base liquid 3-1 | C2 | C4 | C7 | C8 | C9 |
Base liquid 3-2 | C2 | C4 | C7 | C8 | C9 |
Base liquid 3-3 | C2 | C4 | C7 | C8 | C9 |
Base liquid 3-4 | C2 | C4+ | C7 | C8 | C9 |
3-5 parts of base liquid | C2+ | C3 | C6 | C8 | C9 |
Example 4
(S1) formulation of S1 sample treatment solution: consistent with COV-SP except that no magnesium sulfate was added;
(S2) formulation of S2 sample treatment solution: consistent with COV-SP except that the magnesium sulfate concentration therein was changed to 40mM;
(S3) formulation of S3 sample treatment solution: consistent with COV-SP except that the magnesium sulfate concentration therein was changed to 160mM;
COV-SP was compared with the formulations of examples S1-S3. The sample treatment liquid was used in the same manner as in example 1.
The results are shown in Table 4, and the sensitivity of the group (COV-SP) into which magnesium sulfate was introduced was about 1C higher than that of the group (S1) into which magnesium sulfate was not introduced; the magnesium sulfate dosage is reduced to 40mM or increased to 160mM, and the sensitivity is consistent with COV-SP; 100 samples of the throat swab of the healthy person are taken for specificity verification, and no false positive is detected.
Table 4, test results of example 4
Quality control product | 1μg/ml | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
S1 | C1 | C3 | C4 | C7 | C9 | B+ |
S2 | C1+ | C2+ | C3 | C6 | C8 | C9 |
S3 | C1+ | C2+ | C3 | C6 | C8 | C9 |
COV-SP | C1+ | C2+ | C3 | C6 | C8 | C9 |
Example 5
(S4) formulation of S4 sample treatment solution: consistent with COV-SP except that the 0.5 (w/v)% S9+0.5 (w/v)% Triton X-100 combination group was replaced with 1 (w/v)% S9;
(S5) formulation of S5 sample treatment solution: consistent with COV-SP except that the 0.5 (w/v)% S9+0.5 (w/v)% Triton X-100 combination group was replaced with 1 (w/v)% Triton X-100;
(S6) formulation of S6 sample treatment solution: consistent with COV-SP except that the S9 concentration therein was changed to 0.25 (w/v)%;
(S7) formulation of S7 sample treatment solution: consistent with COV-SP except that the S9 concentration therein was changed to 1 (w/v)%;
(S8) formulation of S8 sample treatment solution: consistent with COV-SP except that Triton X-100 concentration therein was changed to 0.25 (w/v)%;
(S9) formulation of S9 sample treatment solution: consistent with COV-SP except that Triton X-100 concentration therein was changed to 1 (w/v)%;
COV-SP was compared with the formulations of examples S4-S9. The sample treatment liquid was used in the same manner as in example 1.
As a result, as shown in Table 5, it was found that either S9 (S4) alone or Triton X-100 (S5) alone was less sensitive than the combination of both. 100 samples of the throat swab of the healthy person are taken for specificity verification, and no false positive is detected.
Table 5, test results of example 5
Quality control product | 1μg/ml | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
S4 | C1 | C3+ | C4 | C7 | C8- | B+ |
S5 | C1 | C2 | C4+ | C7+ | C8 | C9 |
S6 | C1+ | C2+ | C3 | C6 | C8 | C9 |
S7 | C1+ | C2+ | C3 | C6 | C8 | C9 |
S8 | C1 | C2 | C4+ | C7+ | C8- | C9- |
S9 | C1+ | C2+ | C3 | C6 | C8 | C9 |
COV-SP | C1+ | C2+ | C3 | C6 | C8 | C9 |
Example 6
(COV-SP-B) formulation of COV-SP-B sample treatment fluid: consistent with COV-SP except that 25mM Tris-HCl, pH 9.0 was changed to 25mM PB, pH 8.0;
(COV-SP-C) formulation of COV-SP-C sample treatment fluid: consistent with COV-SP except that 25mM Tris-HCl, pH 9.0 was changed to 25mM BB, pH 9.0;
COV-SP was compared with the COV-SP-B, COV-SP-C formulation of this example. The sample treatment liquid was used in the same manner as in example 1.
The results of comparing COV-SP-B, COV-SP-C with COV-SP are shown in Table 6, and it can be obtained that the sensitivity can reach the same level as Tris-HCl by using other buffer systems such as phosphate buffer and boric acid buffer instead.
Table 6, test results of example 6
Quality control product | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
COV-SP-B | C2+ | C3 | C6 | C8 | C9 |
COV-SP-C | C2+ | C3 | C6 | C8 | C9 |
COV-SP | C2+ | C3 | C6 | C8 | C9 |
Example 7
Mode 1: mixing COV-SP as sample diluent with the quality control product of the novel coronavirus, and dripping the mixture to a blank sample pad;
mode 2: COV-SP was used as both the sample diluent (same as in example 1) and the sample pad treatment solution (same as in example 1);
the results are shown in Table 7, and the sensitivity can be greatly improved by adding COV-SP after being uniformly mixed with the sample into an immunoassay tool; the two treatment modes are overlapped and used, and the sensitivity is equivalent.
100 samples of the throat swab of a healthy person are taken for specificity verification, and no false positive is detected.
Table 7, test results of example 7
Quality control product | 1μg/ml | 0.1μg/ml | 25ng/ml | 5ng/ml | 0.5ng/ml | 0.1ng/ml |
Mode 1 | C1+ | C2+ | C3 | C6 | C8 | C9 |
Mode 2 | C1+ | C2+ | C3 | C6 | C8 | C9 |
Example 8
COV-SP was used as a sample treatment solution for detecting influenza virus, and was compared with base solution 1. The sample treatment liquid was used in the same manner as in example 1.
As shown in Table 8, the application of COV-SP to influenza items also greatly improved the sensitivity of detection.
Table 8, test results of example 8
Quality control product | Influenza B virus/1K | Influenza B virus/5K | Influenza B virus/10K |
Base liquid 1 | C4 | C9 | B |
COV-SP | C2+ | C7 | C8 |
Example 9
COV-SP was used as a sample treatment solution for detecting SARS virus, and compared with base solution 1. The sample treatment liquid was used in the same manner as in example 1.
As shown in Table 9, the application of COV-SP to SARS item also greatly improves the sensitivity of detection.
Table 9, test results of example 9
Quality control product | 5μg/ml | 1μg/ml | 0.5μg/ml | 50ng/ml | 10ng/ml | 5ng/ml |
Base liquid 1 | C2 | C3 | C4 | C8 | C9- | B |
COV-SP | C1+ | C1 | C2- | C6 | C8 | C9 |
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (9)
1. A method for treating a sample in an immunoassay for RNA-enveloped viruses, characterized in that the sample to be examined is brought into contact with a sample treatment liquid containing a soluble magnesium salt, the sample treatment liquid further containing S9.
2. The method of claim 1, wherein the soluble magnesium salt in the sample processing fluid comprises at least any one of magnesium sulfate and magnesium chloride.
3. The method of claim 1, wherein the final concentration of soluble magnesium salt in the sample processing fluid is 10-400mM;
optionally, the final concentration of the soluble magnesium salt in the sample processing fluid is 40-160mM;
optionally, the final concentration of the soluble magnesium salt in the sample processing fluid is 80-160mM.
4. The method of claim 1, wherein the final concentration of S9 in the sample processing fluid is 0.1-1.5 (w/v)%;
optionally, the final concentration of S9 in the sample processing liquid is 0.25-1 (w/v)%;
optionally, the final concentration of S9 in the sample processing liquid is 0.5-1 (w/v)%.
5. The method of any one of claims 1-4, wherein the sample processing fluid further comprises at least any one of a buffer, an inert protein, sodium chloride, a nonionic surfactant; for example, the nonionic surfactant is tween 20, tween 40, pluronic F-127, triton X-405 or Triton X-100; for example, the final concentration of the nonionic surfactant in the sample processing liquid is 0.1 to 1.5 (w/v)%; further 0.25-1 (w/v)%; further 0.5-1 (w/v)%.
6. The method of claim 1, wherein the sample is a sample containing a component to be tested;
alternatively, the RNA-enveloped virus is at least any one of coronavirus or influenza virus, such as 2019-nCoV, e.g., SARS.
7. The method of claim 1, wherein the immunoassay is an immunochromatographic assay, an ELISA assay, or a chemiluminescent assay.
8. The sample processing fluid of any one of claims 1-7.
9. A test kit or test strip comprising the sample processing fluid of claim 8.
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CN114015806B (en) * | 2021-10-29 | 2024-01-30 | 杭州金域医学检验所有限公司 | Coronavirus quality control product diluent and preparation method and application thereof |
CN113985028A (en) * | 2021-11-25 | 2022-01-28 | 济南百博生物技术股份有限公司 | Diluent for colloidal gold method antigen detection |
CN114675017A (en) * | 2022-03-25 | 2022-06-28 | 艾康生物技术(杭州)有限公司 | Biological sample extracting solution and application and using method thereof |
CN115047187A (en) * | 2022-07-26 | 2022-09-13 | 石家庄希宝生物科技有限公司 | Test strip sample pad treatment solution, preparation method and application thereof |
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