CN111398586A - Single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit - Google Patents
Single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit Download PDFInfo
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- CN111398586A CN111398586A CN202010154361.7A CN202010154361A CN111398586A CN 111398586 A CN111398586 A CN 111398586A CN 202010154361 A CN202010154361 A CN 202010154361A CN 111398586 A CN111398586 A CN 111398586A
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- latex
- buffer
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- assay kit
- enhanced immunoturbidimetry
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- 239000004816 latex Substances 0.000 title claims abstract description 66
- 229920000126 latex Polymers 0.000 title claims abstract description 66
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 59
- 238000003149 assay kit Methods 0.000 title claims abstract description 21
- 239000002245 particle Substances 0.000 claims abstract description 29
- 239000000375 suspending agent Substances 0.000 claims abstract description 10
- 238000003556 assay Methods 0.000 claims abstract description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 8
- 239000004094 surface-active agent Substances 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 239000003755 preservative agent Substances 0.000 claims abstract description 6
- 239000003381 stabilizer Substances 0.000 claims abstract description 6
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 5
- 230000002335 preservative effect Effects 0.000 claims abstract description 5
- 238000001514 detection method Methods 0.000 claims description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 235000002639 sodium chloride Nutrition 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 8
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 239000013504 Triton X-100 Substances 0.000 claims description 4
- 229920004890 Triton X-100 Polymers 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- ZNJHFNUEQDVFCJ-UHFFFAOYSA-M sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCS(O)(=O)=O)CC1 ZNJHFNUEQDVFCJ-UHFFFAOYSA-M 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- ZEDAGFBWUVYFQU-UHFFFAOYSA-M sodium;3-morpholin-4-ylpropane-1-sulfonate;hydrate Chemical compound [OH-].[Na+].OS(=O)(=O)CCCN1CCOCC1 ZEDAGFBWUVYFQU-UHFFFAOYSA-M 0.000 claims description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 claims description 2
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 108010076119 Caseins Proteins 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- IYFATESGLOUGBX-YVNJGZBMSA-N Sorbitan monopalmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O IYFATESGLOUGBX-YVNJGZBMSA-N 0.000 claims description 2
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 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 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 229940125717 barbiturate Drugs 0.000 claims description 2
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 210000000601 blood cell Anatomy 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 239000005018 casein Substances 0.000 claims description 2
- 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 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
- 235000011151 potassium sulphates Nutrition 0.000 claims description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims description 2
- 229930182490 saponin Natural products 0.000 claims description 2
- 150000007949 saponins Chemical class 0.000 claims description 2
- KDFYOQXKHPVLKX-UHFFFAOYSA-M sodium 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropane-1-sulfonic acid hydroxide Chemical compound [OH-].[Na+].OCCN(CCO)CC(CS(=O)(=O)O)O KDFYOQXKHPVLKX-UHFFFAOYSA-M 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- 235000011152 sodium sulphate Nutrition 0.000 claims description 2
- VOQNBBBLMXZOET-UHFFFAOYSA-M sodium;3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCCS(O)(=O)=O)CC1 VOQNBBBLMXZOET-UHFFFAOYSA-M 0.000 claims description 2
- QBQVXXQXZXDEHE-UHFFFAOYSA-M sodium;propane-1-sulfonate;hydrate Chemical compound O.[Na+].CCCS([O-])(=O)=O QBQVXXQXZXDEHE-UHFFFAOYSA-M 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 2
- 229940033663 thimerosal Drugs 0.000 claims description 2
- XQSBLCWFZRTIEO-UHFFFAOYSA-N hexadecan-1-amine;hydrobromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[NH3+] XQSBLCWFZRTIEO-UHFFFAOYSA-N 0.000 claims 1
- 239000000523 sample Substances 0.000 description 39
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- 239000000427 antigen Substances 0.000 description 4
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- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- YDNDIAROJYPHGZ-UHFFFAOYSA-N (hexadecylamino)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCN[NH3+] YDNDIAROJYPHGZ-UHFFFAOYSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
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- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 210000002889 endothelial cell Anatomy 0.000 description 1
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- 230000008588 hemolysis Effects 0.000 description 1
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit, which comprises an assay tube, wherein L P-P L A2 latex enhanced immunoturbidimetry assay reagent is contained in the assay tube, and L P-P L A2 latex enhanced immunoturbidimetry assay reagent comprises latex particles, buffer solution, surfactant, inorganic salt, stabilizer, suspending agent, excipient and preservative which are uniformly mixed and marked by L P-P L A2 antibody.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit.
Background
L P-P L A2 is one of the subtypes in the phospholipase superfamily, also known as platelet activating factor acetylhydrolase, secreted by macrophages, T cells and mast cells in the intima of blood vessels, L P-P L A2 is up-regulated in atherosclerotic plaques and strongly expressed in macrophages in the fibrous cap of vulnerable plaques L P-P L A2 can hydrolyze oxidized phospholipids in low density lipoprotein ox-L D L to produce lipid pro-inflammatory substances such as lysolecithin and oxidized free fatty acids, which in turn produce various atherogenic effects including endothelial cell death and endothelial dysfunction, stimulating the production of adhesion factors and cytokines.
L P-P L A2 released into the blood circulation is mainly bound to apolipoprotein (Apo) B rich lipoproteins, low density lipoproteins (L D L) account for 80%, and the remainder is bound to high density lipoproteins (HD L2), lipoprotein a [ L P (a) ] and very low density lipoproteins (V L D L) in atherosclerotic disease patients L P-P L A2 levels are positively correlated with L D L subcomponent levels.
The PETIA method is a method for cross-linking antibodies on the surface of polymer latex microspheres, when the microspheres cross-linked with the antibodies are combined with antigens, the antibodies are rapidly gathered together in a short time, and the light dispersion performance or the light transmittance of a reaction solution is changed, the change of the light dispersion performance or the light transmittance (namely, the absorbance) of the reaction solution has inherent correlation with the concentration of the detected antigens, and the concentration of the detected antigens can be reflected in a certain range, and the L P-P L A2 enhanced latex immunoturbidimetry is a typical test method of the type, and the method is used for testing the antigen, the antibody reaction and the result in a homogeneous reaction system.
The L P-P L A2 latex enhanced immunoturbidimetry kit is used for determining the content of L P-P L A2 in a sample, and the determination sample can be whole blood or a serum or plasma sample, the current L P-P L A2 latex enhanced immunoturbidimetry kit sold in the market is a double reagent and consists of a reaction buffer R1 and a L P-P L A2 antibody labeled latex enhanced particle reagent R2, R1 and R2 which are premixed together easily cause the inactivation of the antibody in R2, the latex particles are easily aggregated, the kit can not be stored for a long time, and particularly for the determination of the whole blood sample, the current market kit needs two steps of hemolysis and post-determination, which is time-consuming and has poor repeatability.
Disclosure of Invention
The invention aims to solve the problems of the conventional kit in the background art and provide a single reagent L P-P L A2 latex enhanced immunoturbidimetry kit capable of solving the problems.
The single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit is characterized by comprising a detection tube, wherein L P-P L A2 latex enhanced immunoturbidimetry assay reagents are contained in the detection tube, and L P-P L A2 latex enhanced immunoturbidimetry assay reagents comprise latex particles, buffer solution, surfactant, inorganic salt, stabilizer, suspending agent, excipient and preservative which are uniformly mixed and marked by L P-P L A2 antibody.
In a further embodiment, the buffer is one of 3- [ N, N-bis (hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid-sodium hydroxide buffer (DIPSO-NaOH), 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid-sodium hydroxide buffer (HEPPS-NaOH), 3- (N-morpholino) propanesulfonic acid-sodium hydroxide buffer (MOPS-NaOH), N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid-NaOH buffer (HEPES-NaOH), Tris (hydroxymethyl) aminomethane-HCl buffer (Tris-HCl), phosphate buffer, glycine-NaOH buffer, and barbiturate buffer, and the pH of the buffer is required to be in the range of 7.0 to 9.0, and the concentration is in the range of 10 to 100 mmol/L.
The further scheme is that the surfactant is a reagent which can quickly dissolve blood cells and can be adsorbed to the surface of latex particles, and the surfactant is one or a combination of more of Tween 20, Tween 40, Tween 80, Triton X-100, span 40, span 60, saponin, hexadecyl-3-methylammonium chloride, hexadecyl amino ammonium bromide and lysophospholipid.
In a further scheme, the inorganic salt is one or a combination of more of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, sodium sulfate and potassium sulfate.
In a further embodiment, the stabilizer is one of bovine serum albumin, gelatin or casein.
The further proposal is that the suspending agent is an agent which can lead the latex particles to be well dispersed and suspended in the solution to lead the latex particles to be gelatinous and not to be precipitated after long-term storage, the suspending agent is one or the combination of more of glycol, glycerol and lactose, and the concentration of the suspending agent is 1 to 20 percent.
The further proposal is that the excipient is a reagent which is beneficial to the long-term stable storage of the antibody, and the excipient is one or the combination of more of sucrose, glucose, maltose, trehalose and mannitol.
In a further scheme, the preservative is one of sodium azide, thimerosal and Proclin-300.
In a further embodiment, the latex particles have a diameter of 50nm to 500 nm; the surface modification group is one of-COOH, -NH2, -SH, -OH and-CHO or physical adsorption type latex particles.
The single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit has the advantages that 1) the single reagent L P-P L A L latex enhanced immunoturbidimetry assay kit is simple in reagent composition, only a single reagent is uniformly mixed and is convenient to transport and store, 2) the single reagent L P-P L A L latex enhanced immunoturbidimetry assay kit is simple to operate, a sample is only required to be directly added into a single reagent L P-P L A L latex enhanced immunoturbidimetry reaction solution, compared with the existing L P-P L A L latex enhanced immunoturbidimetry assay kit, the single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit needs to be added with a reagent R L and then with the sample, the solution added with the reagent R L is simple to operate, the detection efficiency is high, and 3) the single reagent L P-P L A L latex enhanced immunoturbidimetry assay kit can be widely used for storing a sample, the single reagent L P-P L A latex enhanced immunoturbidimetry assay kit can be widely used for detecting a sample after a sample, and a single reagent is widely used for detecting a whole blood sample after a sample, and a single reagent is widely used for a whole blood sample CT detection, and a whole blood sample transmission detection, and a single reagent L, and a single reagent kit can be widely used for detecting a whole blood sample CT (POT) and a whole blood sample CT (POP) and a blood sample CT) and.
Drawings
FIG. 1 is a calibration curve on a biochemical analyzer in example 1 of the present invention.
FIG. 2 is a linear range dependence on a biochemical analyzer of example 1 of the present invention.
FIG. 3 is a calibration curve of the nephelometer of example 2 of the present invention.
FIG. 4 is a plot of the linear range dependence on a nephelometer according to example 2 of the present invention.
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, 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
Firstly, preparation of a determination kit:
1. surface-carboxylated polystyrene latex particles (200 nm) were diluted to 10ml with 50mM MES buffer pH6.0 to a final concentration of 1%.
2. EDC (200 mg) was added to the solution, and L P-P L A2 antibody was added thereto by affinity chromatography (10 mg), followed by mixing at room temperature for 3 hours.
3. 10ml of 100mM glycine was added to the above solution to react for 3 hours, and excess active groups on the surface of the latex particles were blocked, centrifuged at 16000rpm for 30 minutes, and the supernatant was removed.
4. The marked L P-P L A2 antibody latex particles are diluted to a working concentration by the following buffer solution, and a single reagent L P-P L A2 latex enhanced immunoturbidimetry kit is matched with a calibrator, wherein the single reagent comprises 100mM HEPES-NaOH, the pH is 8.0, 0.5% triton X-100, 3% NaCl,0.5% BSA, 3% glycerol, 2% trehalose and 0.1% Proclin-300.
5, L P-P L A2 calibrator, wherein L0P-P L1A 2 pure product is dissolved in 50mM Tris-HCl, pH7.5, 0.85% NaCl,0.5% BSA,0.1% sodium azide and L P-P L A2 at concentrations of 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L respectively.
Secondly, the application of the kit in a biochemical analyzer:
1) 6 calibrators with different concentrations L P-P L A2, wherein the calibrators are 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L matched with a single reagent to calibrate, the wavelength of 546nm is taken as a measurement wavelength, the reagent R, 250ul and 37-degree reaction are added into a detection tube for 120 seconds, 6ul calibrators with different concentrations are respectively added into the detection tube to be mixed uniformly, absorbance values (A1 and A2) of the 15 th and 120 th seconds of the reaction are measured, the difference value delta A = A2-A1 of absorbance is calculated, the absorbance difference delta A of each calibration tube is taken as a vertical coordinate, the corresponding concentration is taken as a horizontal coordinate, and a 'concentration-absorbance difference' calibration curve is prepared, and the 'concentration-absorbance difference' calibration curve is shown in the attached figure 1.
2) The difference in absorbance of the serum sample (6 ul in plasma sample if 10ul of sample is added to the whole blood sample) is determined in the same manner and substituted into the calibration curve to calculate the concentration of L P-P L A2 in the sample.
3) Linear range dependence
L P-P L A2 high concentration sample (2800 ng/m L) is diluted with physiological saline to 1400ng/m L, 700ng/m L, 350ng/m L, 175ng/m L and 88ng/m L, each concentration is measured on a biochemical analyzer with a 546nm wavelength repeatedly for 3 times, the average value of the measured concentrations and the theoretical concentration are subjected to linear regression analysis, and the correlation coefficient is R2=0.9979, see figure 2. the linear range dependence is better in the linear measurement range of 88-1400ng/m L.
4) Sensitivity (lowest detection limit)
The 5% human serum albumin is used as a blank sample, the measurement is repeated for 20 times, the mean value X of the calculation result is 2.15ng/m L, the standard deviation SD is 1.87, and the X +2SD is 5.89ng/m L, so the sensitivity of the kit for measuring L P-P L A2 on a biochemical analyzer can reach 5.89ng/m L.
Example 2
Firstly, preparation of a determination kit:
1. surface-carboxylated polystyrene latex particles (200 nm) were diluted to 10ml with 50mM MES buffer pH6.0 to a final concentration of 1%.
2. EDC (200 mg) was added to the solution, and L P-P L A2 antibody was added thereto by affinity chromatography (10 mg), followed by mixing at room temperature for 3 hours.
3. Adding 10ml of 100mM glycine into the solution, reacting for 3 hours, and blocking redundant active groups on the surface of the latex particles; 16000rpm for 30 minutes, and the supernatant was removed.
4. The marked L P-P L A2 antibody latex particles are diluted to a working concentration by the following buffer solution, and a calibrator, namely a single reagent L P-P L A2 latex enhanced immunoturbidimetric kit, is prepared by using a single reagent formula of 50mM MOPS-NaOH, pH7.5, 0.3% Tween 20, 0.8% NaCl, 1% gelatin, 5% glycerol, 3% trehalose and 0.1% sodium azide.
5, L P-P L A2 calibrator, wherein L0P-P L1A 2 pure product is dissolved in 50mM Tris-HCl, pH7.5, 0.85% NaCl,0.5% BSA,0.1% sodium azide and L P-P L A2 at concentrations of 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L respectively.
Application of kit to nephelometer
1) 6 calibrators with different concentrations L P-P L A2, wherein the calibrators are 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L, are matched with a single reagent to carry out calibration, a reagent R, 250ul and 37-degree reaction are added into a detection tube with the wavelength of 546nm as a measurement wavelength for 120 seconds, 6ul calibrators with different concentrations are respectively added into the detection tube to be mixed uniformly, scattered light signal values (A1 and A2) reacting for 10 seconds and 120 seconds are measured, the difference value of the scattered light signals is calculated to be delta A = A2-A1, the scattered light signal difference value delta A of each calibration tube is taken as a vertical coordinate, and the corresponding concentration is taken as a horizontal coordinate, and a concentration-scattered light difference calibration curve is prepared, and is shown in figure 3.
2) The difference of the scattered light signals of 10ul of whole blood sample (6 ul of plasma sample if 6ul of sample is added for serum sample determination) is determined in the same way, and the difference is substituted into the calibration curve to calculate the concentration of L P-P L A2 in the sample.
3) Linear range dependence
L P-P L A2 high concentration sample (2800 ng/m L) is diluted with physiological saline to 1400ng/m L, 700ng/m L, 350ng/m L, 175ng/m L and 88ng/m L, each concentration is measured on a scatterometer at a wavelength of 546nm, the measurement is repeated 3 times, the average value of the measured concentrations and the theoretical concentration are subjected to linear regression analysis, and the correlation coefficient is R2=0.9972, see figure 4. the linear range dependence is better in the linear measurement range of 88-1400ng/m L.
4) Sensitivity (lowest detection limit)
The measurement is repeated for 20 times by taking 5% human serum albumin as a blank sample, the mean value X of the calculation result is 2.3ng/m L, the standard deviation SD is 1.78, and the X +2SD is 5.86ng/m L, so the sensitivity of the kit for measuring L P-P L A2 on a scatterometer can reach 5.86ng/m L.
Example 3
Firstly, preparation of a determination kit:
1. the surface-aminated polystyrene latex particles (80 nm) were diluted to 10ml with 50mM MES buffer pH6.0 to a final concentration of 1%.
2. EDC (200 mg) was added to the solution, and L P-P L A2 antibody was added thereto by affinity chromatography (10 mg), followed by mixing at room temperature for 3 hours.
3. 10ml of 100mM glycine was added to the above solution to react for 3 hours, and excess active groups on the surface of the latex particles were blocked, centrifuged at 16000rpm for 30 minutes, and the supernatant was removed.
4. The marked L P-P L A2 antibody latex particles are diluted to a working concentration by the following buffer solution, and a calibrator, namely a single reagent L P-P L A2 latex enhanced immunoturbidimetric kit, is prepared, wherein the single reagent comprises 50mM Tris-HCl, pH 8.0, 0.4% triton X-100, 0.9% KCl, 1.5% BSA, 8% glycerol, 5% trehalose and 0.1% sodium azide.
5, L P-P L A2 calibrator, wherein L0P-P L1A 2 pure product is dissolved in 50mM Tris-HCl, pH7.5, 0.85% NaCl,0.5% BSA,0.1% sodium azide and L P-P L A2 at concentrations of 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L respectively.
Application of kit to nephelometer
1) 6 calibrators with different concentrations L P-P L A2, wherein the calibrators are 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L, are matched with a single reagent to carry out calibration, a reagent R, 250ul and 37-degree reaction are added into a detection tube with the wavelength of 546nm as a measurement wavelength for 90 seconds, 6ul calibrators with different concentrations are respectively added into the detection tube to be mixed uniformly, scattered light signal values (A1 and A2) reacting for 10 seconds and 90 seconds are measured, the difference value delta A = A2-A1 of the scattered light signals is calculated, the difference value delta A of the scattered light signals of each calibration tube is taken as a vertical coordinate, and the corresponding concentration is taken as a horizontal coordinate, and a calibration curve of concentration-scattered light difference value is prepared.
2) The difference of the scattered light signals of 10ul of whole blood sample (6 ul of plasma sample if 6ul of sample is added for serum sample determination) is determined in the same way, and the difference is substituted into the calibration curve to calculate the concentration of L P-P L A2 in the sample.
3) Linear range dependence
L P-P L A2 high concentration sample (2800 ng/m L) is diluted with physiological saline to 1400ng/m L, 700ng/m L, 350ng/m L, 175ng/m L and 88ng/m L, each concentration is measured on a scatterometer at a wavelength of 546nm, the measurement is repeated 3 times, the average value of the measured concentrations and the theoretical concentration are subjected to linear regression analysis, and the correlation coefficient is R2=0.9950, and as a result, the linear range correlation was better in the linear measurement range of 88-1400ng/m L.
4) Sensitivity (lowest detection limit)
The measurement is repeated for 20 times by taking 5% human serum albumin as a blank sample, the mean value X of the calculation result is 2.2ng/m L, the standard deviation SD is 1.65, and the X +2SD is 5.5ng/m L, so the sensitivity of the kit for measuring L P-P L A2 on a scatterometer can reach 5.5ng/m L.
Example 4
Firstly, preparation of a determination kit:
1. the surface-aminated polystyrene latex particles (250 nm) were diluted to 10ml with 50mM MES buffer pH6.0 to a final concentration of 1%.
2. EDC (200 mg) was added to the solution, and L P-P L A2 antibody was added thereto by affinity chromatography (10 mg), followed by mixing at room temperature for 3 hours.
3. 10ml of 100mM glycine was added to the above solution to react for 3 hours, and excess active groups on the surface of the latex particles were blocked, centrifuged at 16000rpm for 30 minutes, and the supernatant was removed.
4. The marked L P-P L A2 antibody latex particles are diluted to a working concentration by the following buffer solution, and a single reagent L P-P L A2 latex enhanced immunoturbidimetric kit is prepared by the following calibration products, wherein the single reagent comprises 50mM HEPES-NaOH, the pH value is 8.0, 0.35% Tween 20, 0.85% CaCl2, 2% gelatin, 8% glycerol, 5% trehalose and 0.1% sodium azide.
5, L P-P L A2 calibrator, wherein L0P-P L1A 2 pure product is dissolved in 50mM Tris-HCl, pH7.5, 0.85% NaCl,0.5% BSA,0.1% sodium azide and L P-P L A2 at concentrations of 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L respectively.
Application of kit to nephelometer
1) 6 calibrators with different concentrations L P-P L A2, wherein the calibrators are 0ng/m L, 88ng/m L, 175ng/m L, 350ng/m L, 700ng/m L and 1400ng/m L, are matched with a single reagent to carry out calibration, a reagent R, 250ul and 37-degree reaction are added into a detection tube with the wavelength of 546nm as a measurement wavelength for 90 seconds, 6ul calibrators with different concentrations are respectively added into the detection tube to be mixed uniformly, scattered light signal values (A1 and A2) reacting for 10 seconds and 90 seconds are measured, the difference value delta A = A2-A1 of the scattered light signals is calculated, the difference value delta A of the scattered light signals of each calibration tube is taken as a vertical coordinate, and the corresponding concentration is taken as a horizontal coordinate, and a calibration curve of concentration-scattered light difference value is prepared.
2) The difference of the scattered light signals of 10ul of whole blood sample (6 ul of plasma sample if 6ul of sample is added for serum sample determination) is determined in the same way, and the difference is substituted into the calibration curve to calculate the concentration of L P-P L A2 in the sample.
3) Linear range dependence
L P-P L A2 high concentration samples (2800 ng/m L) were diluted with physiological saline to 1400ng/m L, 700ng/m L, 350ng/m L, 175ng/m L, 88ng/m L, each concentration measured at a wavelength of 546nm on a scatterometerRepeating the determination 3 times, performing linear regression analysis on the average value of the determined concentration and the theoretical concentration, and obtaining a correlation coefficient R2=0.9981, and as a result, the linear range correlation was better in the linear measurement range of 88-1400ng/m L.
4) Sensitivity (lowest detection limit)
The measurement is repeated for 20 times by taking 5% human serum albumin as a blank sample, the mean value X of the calculation result is 2.4ng/m L, the standard deviation SD is 1.82, and the X +2SD is 6.04ng/m L, so the sensitivity of the kit for measuring L P-P L A2 on a scatterometer can reach 6.04ng/m L.
In the above-mentioned embodiments of examples 1-4, although only the optimized combination of several components is illustrated, various agents can be selected from latex particles, buffers, surfactants, inorganic salts, stabilizers, suspending agents, excipients and preservatives, and all of them cannot be illustrated in the embodiments, so that those skilled in the art can reasonably select the aforementioned agents as required to achieve the technical solutions of the present invention based on the embodiments of examples 1-4 to achieve the corresponding effects. Alternative substitutions of these agents are intended to fall within the scope of the invention.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit is characterized by comprising a detection tube, wherein L P-P L A2 latex enhanced immunoturbidimetry assay reagents are contained in the detection tube, and L P-P L A2 latex enhanced immunoturbidimetry assay reagents comprise latex particles, buffer solution, surfactant, inorganic salt, stabilizer, suspending agent, excipient and preservative which are uniformly mixed and marked by L P-P L A2 antibody.
2. The single reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit as claimed in claim 1, wherein the buffer is one of 3- [ N, N-bis (hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid-sodium hydroxide buffer (DIPSO-NaOH), 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid-sodium hydroxide buffer (HEPPS-NaOH), 3- (N-morpholino) propanesulfonic acid-sodium hydroxide buffer (MOPS-NaOH), N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid-NaOH buffer (HEPES-NaOH), Tris-HCl buffer (Tris-HCl), phosphate buffer, glycine-NaOH buffer, and barbiturate buffer, and the buffer has a pH value ranging from 7.0 to 9.0 and a concentration ranging from 10 to 100 mmol/L.
3. The single reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit of claim 1, wherein the surfactant is a reagent which can rapidly dissolve blood cells and can be adsorbed on the surface of latex particles, and the surfactant is one or a combination of more of Tween 20, Tween 40, Tween 80, Triton X-100, span 40, span 60, saponin, hexadecyl-3-methylammonium chloride, hexadecylaminium bromide and lysophospholipid.
4. The single-reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit of claim 1, wherein the inorganic salt is one or a combination of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, sodium sulfate and potassium sulfate.
5. The single reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit of claim 1, wherein the stabilizer is one of bovine serum albumin, gelatin or casein.
6. The single reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit of claim 1, wherein the suspending agent is a reagent which can well disperse and suspend latex particles in a solution, enable the latex particles to be gelatinized and prevent the latex particles from being precipitated after long-term storage, the suspending agent is one or a combination of ethylene glycol, glycerol and lactose, and the concentration of the suspending agent is 1-20%.
7. The single reagent L P-P L A2 latex-enhanced immunoturbidimetric assay kit of claim 1, wherein the excipient is a reagent that facilitates the long-term stable storage of the antibody, and the excipient is one or a combination of sucrose, glucose, maltose, trehalose, and mannitol.
8. The single reagent L P-P L A2 latex-enhanced immunoturbidimetry assay kit of claim 1, wherein the preservative is one of sodium azide, thimerosal and Proclin-300.
9. The single reagent L P-P L A2 latex enhanced immunoturbidimetry assay kit as claimed in claim 1, wherein the diameter of the latex particles is 50nm-500nm, and the surface modification group is one of-COOH, -NH2, -SH, -OH and-CHO or physical adsorption type latex particles.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117517656A (en) * | 2023-09-19 | 2024-02-06 | 深圳上泰生物工程有限公司 | Lp-PLA2 antigen preservation solution |
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Application publication date: 20200710 |