CN105738617A - Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof - Google Patents
Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof Download PDFInfo
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- CN105738617A CN105738617A CN201610201530.1A CN201610201530A CN105738617A CN 105738617 A CN105738617 A CN 105738617A CN 201610201530 A CN201610201530 A CN 201610201530A CN 105738617 A CN105738617 A CN 105738617A
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- bladder chalone
- latex
- polystyrene latex
- detection kit
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 70
- 239000002245 particle Substances 0.000 title claims abstract description 69
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 238000004879 turbidimetry Methods 0.000 title abstract description 5
- 102000012192 Cystatin C Human genes 0.000 title abstract 3
- 108010061642 Cystatin C Proteins 0.000 title abstract 3
- 239000004816 latex Substances 0.000 claims abstract description 98
- 229920000126 latex Polymers 0.000 claims abstract description 98
- 239000004793 Polystyrene Substances 0.000 claims abstract description 64
- 229920002223 polystyrene Polymers 0.000 claims abstract description 64
- 239000000203 mixture Substances 0.000 claims abstract description 36
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 20
- 239000003381 stabilizer Substances 0.000 claims abstract description 20
- 239000004094 surface-active agent Substances 0.000 claims abstract description 19
- 239000003755 preservative agent Substances 0.000 claims abstract description 17
- 230000002335 preservative effect Effects 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 230000008030 elimination Effects 0.000 claims abstract 2
- 238000003379 elimination reaction Methods 0.000 claims abstract 2
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- 210000002966 serum Anatomy 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 150000002148 esters Chemical class 0.000 claims description 9
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims description 6
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 6
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 6
- 239000005018 casein Substances 0.000 claims description 5
- 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 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- ZIRURAJAJIQZFG-UHFFFAOYSA-N 1-aminopropane-1-sulfonic acid Chemical compound CCC(N)S(O)(=O)=O ZIRURAJAJIQZFG-UHFFFAOYSA-N 0.000 claims description 3
- UZRZNAWBVOGJAG-UHFFFAOYSA-N 2-hydroxy-1-(methylamino)propane-1-sulfonic acid Chemical compound CNC(C(C)O)S(O)(=O)=O UZRZNAWBVOGJAG-UHFFFAOYSA-N 0.000 claims description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 3
- 108010000912 Egg Proteins Proteins 0.000 claims description 3
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims description 3
- 239000005639 Lauric acid Substances 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 3
- 239000003963 antioxidant agent Substances 0.000 claims description 3
- 230000003078 antioxidant effect Effects 0.000 claims description 3
- 239000002738 chelating agent Substances 0.000 claims description 3
- 210000000991 chicken egg Anatomy 0.000 claims description 3
- JRBPAEWTRLWTQC-UHFFFAOYSA-N dodecylamine Chemical compound CCCCCCCCCCCCN JRBPAEWTRLWTQC-UHFFFAOYSA-N 0.000 claims description 3
- 235000014103 egg white Nutrition 0.000 claims description 3
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 3
- 102000057593 human F8 Human genes 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 229940047431 recombinate Drugs 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 229940033663 thimerosal Drugs 0.000 claims description 3
- 238000003556 assay Methods 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 4
- 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 abstract description 3
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 2
- 239000003795 chemical substances by application Substances 0.000 description 12
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- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000007817 turbidimetric assay Methods 0.000 description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- XCBLFURAFHFFJF-UHFFFAOYSA-N 3-[bis(2-hydroxyethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCCN(CCO)CC(O)CS(O)(=O)=O XCBLFURAFHFFJF-UHFFFAOYSA-N 0.000 description 2
- 239000007996 HEPPS buffer Substances 0.000 description 2
- 235000014683 Hansenula anomala Nutrition 0.000 description 2
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 2
- 241000235063 Wickerhamomyces anomalus Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000010419 fine particle Substances 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 101150076592 CST3 gene Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 230000024924 glomerular filtration Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
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- 102220042509 rs112033303 Human genes 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (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 provides a cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof.The reagent kit comprises a reagent R1 and a reagent R2.The reagent R1 is prepared from a buffer solution, inorganic salt, a surfactant, a preservative, a stabilizer and interference elimination protein.The reagent R2 is prepared from a buffer solution, inorganic salt, a surfactant, a preservative, a stabilizer and a polystyrene latex particle mixture, and the polystyrene latex particle mixture is interlinked with a cystatin C antibody.The reagent kit based on latex-particle-enhanced turbidimetric immunoassay (PETIA) can be generally applied to analysis of various full-automatic biochemical analyzers and is short in assay time, good in specificity, high in precision and good in accuracy when used.
Description
Technical field
The present invention relates to immunologic assay analysis field, particularly to a kind of bladder chalone C latex intensified than turbid detection kit and application thereof.
Background technology
Since 1985, bladder chalone C (cystatinC) has been considered the good sign thing of detection renal function, due to it not by the impact of many physiological and pathological factors, other marks of same glomerular filtration rate (GFR) are compared has numerous superiority.CystatinC also plays in series of physiological and pathological process and acts on, and has important clinical meaning.Bladder chalone C is a kind of protein by CST3 gene code, is the good mark for renal function diagnosis, is present in internal institute's nucleated cell.Bladder chalone C can freely filter mesangium, is the important symbol of instruction glomerule rate filtration (GFR) function.
The development of immunoassay can date back mid-term in 20th century, now measure liquid phase immune agglutination cause optical density change optical technology developed.20 century 70s rise and occur in that skeptophylaxis precipitation assay, i.e. immune transmission turbidimetric assay and immunity scattering turbidimetric assay, and immunoturbidimetry measures and is widely used in the field that in clinical body fluid, protein content detects gradually since then.The immunity turbid determination techniques of transmittance is widely used on automatic clinical chemistry analyzer, and immunity scattering turbidimetric assay then relies on special protein instrument, all achieves high speed, sensitive and automatization.
The more commonly used is colloidal gold immunity chromatography at present, but the method sensitivity is bad, and repeatability is bad, is only used for qualitative detection, and the observation of curative effect for clinical patient has limitation.It is relatively accurate at present that latex particle strengthens turbidimetry, stable detection method.Turbidimetry (Turbidimetry): when Ray Of Light passes through the suspension with fine particle and colloid solution, this solution is subject to light scattering and light absorbs two factor impacts and can make the strength reduction of light, the degree weakened is directly proportional to the content of fine particle in solution, by measuring transmitted intensity, derive the concentration of test substance in solution.To detect antigen, in reactant liquor, amount of antibody is enough, determined antigen is more many, the antigen antibody complex formed is also more many, the strength reduction of transmission light is more many, simultaneously with other physical factor, as closely related in the distance between antigen antibody reaction time, light source power and wavelength, photodetector and reaction cup etc..But current technique there is also the problem that the bad linear dependence of precision is not strong.
Summary of the invention
For the problems referred to above of the prior art, the invention provides a kind of bladder chalone C latex intensified than turbid detection kit and application thereof, described test kit is based on Latex-enhanced immunoturbidimetric assay (PETIA), all kinds of automatic clinical chemistry analyzer analysis can be commonly used to, minute required during use is short, specificity is good, and precision is high, and accuracy is good.
In order to achieve the above object, the present invention adopts the following technical scheme that a kind of bladder chalone C latex intensified is than turbid detection kit, including reagent R1 and reagent R2, described reagent R1 includes buffer, inorganic salt, surfactant, preservative, stabilizer and interference eliminate albumen；Described reagent R2 includes buffer, inorganic salt, surfactant, preservative, stabilizer and polystyrene latex particles mixture；Described polystyrene latex particles mixture is crosslinked with anti-bladder chalone C antibody；
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and the mixture of minor diameter polystyrene latex particles; described major diameter polystyrene latex particles mean diameter is 400nm～600nm; described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
As further preferably, described major diameter polystyrene latex particles and minor diameter polystyrene latex particles are all crosslinked with anti-bladder chalone C polyclonal antibody.In described reagent R2, polystyrene latex particles cross-links the preparation method of anti-bladder chalone C antibody is chemical crosslink technique, is specially the amino residue in anti-bladder chalone C antibody sequence and the polystyrene latex particles surface carboxyl groups after activation carries out chemical crosslinking by EDC.
As further preferably, each component and the content of described reagent R1 is:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Interference eliminates albumen 0.5～1%
Stabilizer 1～10%.
By based on the quality of reagent R1, percentage ratio is mass percent.
As further preferably, each component and the content of described reagent R2 is:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Stabilizer 1～10%
Polystyrene latex particles mixture 0.1-1%.
By based on the quality of reagent R2, percentage ratio is mass percent.
As further preferably, described buffer is one or more in 3-morpholine-2-hydroxy-propanesulfonic acid (MOPSO), 2-(N-morpholine) ethyl sulfonic acid (MES), 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 3-[double; two (2-ethoxy) amino of N-N-]-2-hydroxy-propanesulfonic acid (DIPSO), N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid (TAPSO), N-2-hydroxyethyl piperazine-N'-3-propane sulfonic acid (HEPPS) and 3-(three (methylol) methyl) amino-1-propane sulfonic acid (TAPS)；Described surfactant is fatty alcohol-polyoxyethylene ether, OPEO, polyoxyethylene stearic acid ester, lauric acid polyoxyethylene ester, polyoxyethylene oleic acid ester and one or more in lauryl amine polyoxyethylene ether；Described stabilizer is one or more in protein, inorganic salt, metal chelating agent and antioxidant；Described interference eliminates the HBR blocker that albumen is Scantibodies company；Described inorganic salt is sodium chloride, potassium chloride and one or more in magnesium sulfate；Described preservative is one or more in sodium azide, thimerosal and Proclin-300.
As further preferably, described stabilizer is chicken egg white and casein (casein).
As further preferably, each component and the content of described reagent R1 are respectively as follows:
As further preferably, each component and the content of described reagent R2 are respectively as follows:
As further preferably, the pH value of described 2-(N-morpholine) ethyl sulfonic acid (MES) is 5.5-6.7.
As further preferably, described test kit also includes the reference standard product of bladder chalone C reagent, and described reference standard product are add people to recombinate the human serum of bladder chalone C albumen or the liquid of similar serum substrate.
As further preferably, described people recombinates bladder chalone C albumen by Pichia anomala expression, obtain after affine ion-exchange purification.
As further preferably, the reference standard product of described bladder chalone C reagent have 5 concentration, respectively 0.5mg/L, 1mg/L, 2mg/L, 4mg/L, 8mg/L.
Bladder chalone C latex intensified as above is than turbid detection kit purposes of bladder chalone C content in detection serum.
In sum, beneficial effects of the present invention is as follows:
(1), test kit of the present invention utilize Latex-enhanced immunoturbidimetric assay measure bladder chalone C, be exaggerated detection signal, improve detection sensitivity, shorten the detection time.
(2), the present invention adopt the emulsion reagent of mixing particle diameter, wherein the latex particle ratio of little particle diameter is big, improves linear；Containing the latex particle of a certain proportion of big particle diameter, sensitivity is also higher simultaneously.
(3), test kit of the present invention has been specifically added the material preventing interference, good in anti-interference performance.Meanwhile, the interpolation of surfactant also improves precision.
(4), use automatic clinical chemistry analyzer to measure anti-bladder chalone C antibody, not only make operation more easy, improve automaticity, and also a saving the Financial cost of a small amount of sample detection experiment.A small amount of sample is detected by automatic clinical chemistry analyzer multiple batches of, almost identical with the single batch of cost to a large amount of sample detection；Therefore, automatic clinical chemistry analyzer goes for the detection to a small amount of sample, and does not improve Financial cost.
Detailed description of the invention
The present invention is by providing a kind of bladder chalone C latex intensified than turbid detection kit and application thereof, described test kit is based on Latex-enhanced immunoturbidimetric assay (PETIA), all kinds of automatic clinical chemistry analyzer analysis can be commonly used to, minute required during use is short, specificity is good, precision is high, and accuracy is good.
In order to be better understood from technique scheme, below in conjunction with specific embodiment, technique scheme is described in detail.
The embodiment of the present application bladder chalone C latex intensified, than turbid detection kit, includes buffer, inorganic salt, surfactant, preservative, stabilizer and interference including reagent R1 and reagent R2, described reagent R1 and eliminates albumen；Described reagent R2 includes buffer, inorganic salt, surfactant, preservative, stabilizer and polystyrene latex particles mixture；
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and the mixture of minor diameter polystyrene latex particles; described major diameter polystyrene latex particles mean diameter is 400nm～600nm; described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
Described polystyrene latex particles mixture is crosslinked with anti-bladder chalone C antibody；Described major diameter polystyrene latex particles and minor diameter polystyrene latex particles are all crosslinked with anti-bladder chalone C polyclonal antibody.It is chemical crosslink technique that described polystyrene latex particles cross-links the preparation method of anti-bladder chalone C antibody, is specially the amino residue in anti-bladder chalone C antibody sequence and the polystyrene latex particles surface carboxyl groups after activation carries out chemical crosslinking by EDC.
Each component and the content of described reagent R1 are respectively as follows:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Interference eliminates albumen 0.5～1%
Stabilizer 1～10%.
By based on the quality of reagent R1, percentage ratio is mass percent.
Each component and the content of described reagent R2 is:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Stabilizer 1～10%
Polystyrene latex particles mixture 0.1-1%.
By based on the quality of reagent R2, percentage ratio is mass percent.
Described buffer is 3-morpholine-2-hydroxy-propanesulfonic acid (MOPSO), 2-(N-morpholine) ethyl sulfonic acid (MES), 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 3-[double, two (2-ethoxy) amino of N-N-]-2-hydroxy-propanesulfonic acid (DIPSO), N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid (TAPSO), one or more in N-2-hydroxyethyl piperazine-N'-3-propane sulfonic acid (HEPPS) and 3-(three (methylol) methyl) amino-1-propane sulfonic acid (TAPS)；Described surfactant is fatty alcohol-polyoxyethylene ether, OPEO, polyoxyethylene stearic acid ester, lauric acid polyoxyethylene ester, polyoxyethylene oleic acid ester and one or more in lauryl amine polyoxyethylene ether；Described stabilizer is one or more in protein, inorganic salt, metal chelating agent and antioxidant；Described interference eliminates the HBR blocker that albumen is Scantibodies company；Described inorganic salt is sodium chloride, potassium chloride and one or more in magnesium sulfate；Described preservative is one or more in sodium azide, thimerosal and Proclin-300.
Described stabilizer is preferably chicken egg white and casein (casein).
Concrete, each component and the concentration range of described reagent R1 are respectively as follows:
Concrete, each component and the concentration range of described reagent R2 are respectively as follows:
The pH value of described 2-(N-morpholine) ethyl sulfonic acid (MES) is 5.5-6.7.
Described test kit also includes the reference standard product of bladder chalone C reagent, and described reference standard product are add people to recombinate the human serum of bladder chalone C albumen or the liquid of similar serum substrate.
Described people recombinates bladder chalone C albumen by Pichia anomala expression, obtain after affine ion-exchange purification.
The reference standard product of described bladder chalone C reagent have 5 concentration, respectively 0.5mg/L, 1mg/L, 2mg/L, 4mg/L, 8mg/L.
Embodiment 1
Each component and the concentration range of described reagent R1 be:
Each component and the concentration range of described reagent R2 be:
Reagent R1 is colourless transparent solution, and reagent R2 is homogeneous milky white solution, and the reference calibrations product of bladder chalone C reagent are also colourless transparent solution.
Embodiment 2
Each component and the concentration range of described reagent R1 be:
Each component and the concentration range of described reagent R2 be:
Reagent R1 is colourless transparent solution, and R2 is homogeneous milky white solution, and the reference calibrations product of bladder chalone C reagent are also colourless transparent solution.
Comparative example's 1R1 agent formulations is identical with embodiment 1, outside R2 agent formulations is different with embodiment 1 removing glue breast diameter, all the other compositions are identical, it is 400nm～600nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(7-10): (0-3).
Comparative example's 2R1 agent formulations is identical with embodiment 2, outside R2 agent formulations is different with embodiment 2 removing glue breast diameter, all the other compositions are identical, it is 400nm～600nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 10nm～12nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
Comparative example's 3R1 agent formulations is identical with embodiment 1, outside R2 agent formulations is different with embodiment 1 removing glue breast diameter, all the other compositions are identical, it is 400nm～600nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 40nm～100nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
Comparative example's 4R1 agent formulations is identical with embodiment 2, outside R2 agent formulations is different with embodiment 2 removing glue breast diameter, all the other compositions are identical, it is 200nm～300nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
Comparative example's 5R1 agent formulations is identical with embodiment 1, outside R2 agent formulations is different with embodiment 1 removing glue breast diameter, all the other compositions are identical, it is 700nm～1000nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
Comparative example's 6R1 agent formulations is identical with embodiment 2, outside R2 agent formulations is different with embodiment 2 removing glue breast diameter, all the other compositions are identical, it is 400nm～600nm that R2 latex diameter is respectively as follows: described major diameter polystyrene latex particles mean diameter, described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(5-6): (4-5).
The dependency of the embodiment of the present application bladder chalone C test kit, sensitivity, precision test:
Selected clinical sample is the patients serum that hospital collects, in the embodiment of the present application detection kit detection sample, the flow process of bladder chalone C content is as follows: to 5uL sample to be detected, (calibration pipe makees sample with calibration object, blank with distilled water for sample, calibration object is purchased from Landau company of Britain) in add 150uL reagent R1 fully mix, in 37 DEG C of constant temperature 5 minutes, 50uL reagent R2 is added again in mixed system, mixing, 37 DEG C of constant temperature are after 1 minute, blank tube returns to zero, wavelength 540nm, measure each pipe absorbance A 1, each pipe absorbance A 2 is measured after 4 minutes, calculate Δ A=A2-A1.According to multiple spot calibration object concentration and corresponding absorbance changing value Δ A, adopting multiple spot gamma correction pattern to determine working curve, sample absorbance changes concentration value corresponding on working curve and is mensuration concentration.Following contrast agents is that the bladder chalone C latex intensified of double; two emulsion reagent methods of Meikang, Ningbo is than turbid test kit.
1 dependency experiment
The two kinds of reagent and the contrast agents that use the embodiment of the present invention 1 and embodiment 2 respectively adopt Hitachi 7180 automatic clinical chemistry analyzer that 50 parts of human serums (comprising normal and monstrosity) are measured by each autoregressive parameter simultaneously.Measured value is carried out linear regression, obtaining regression equation is: y1=1.014x+1.8423, y2=1.024x+1.6553 correlation coefficient is: R21=0.9965, correlation coefficient is: (y1 is the embodiment of the present invention 1 reagent to R22=0.9947, y2 is the embodiment of the present invention 2 reagent, x is contrast agents), result shows that reagent of the present invention is fine with contrast agents dependency, further relates to reagent of the present invention and has good specificity and accuracy.In addition this reagent is applicable not only to Hitachi 7180 automatic clinical chemistry analyzer, it may also be used for the full-automatic and semi-automatic biochemical analyzer of other series such as Hitachi, Beckman, design parameter can make the appropriate adjustments according to major parameter.Comparative example 1 of the present invention is used to adopt Hitachi 7180 automatic clinical chemistry analyzer that 50 parts of human serums (comprising normal and monstrosity) are measured by each autoregressive parameter simultaneously to two kinds of reagent and the contrast agents of comparative example 6 respectively.Measured value is carried out linear regression, and obtaining regression equation is: y1=1.113x+2.8526, y2=1.210x+2.0417, y3=1.035x+1.9591, y4=1.073x+1.7660, y5=1.014x+1.9873, y6=1.009x+1.7860.R12=0.9878, R12=0.9274, R12=0.9782, R12=0.9679, R12=0.9769, R12=0.9790, R12=0.9567,
2 sensitivity experiments
The appraisal procedure of the detection sensitivity standard deviation of 10-20 dummy signal strength, and the standard deviation of 3-15 least significant non-zero sample signal strength, calculate gained with statistical software EPEvaluatorrelease6.Experimental result shows that reagent of the present invention has good sensitivity, can comply fully with the improvement of U.S. clinical laboratory and amend legislation.Sensitivity is respectively as follows: bladder chalone C respectively 0.26mg/L and the 0.32mg/L of the present invention, is better than contrast agents 0.37mg/L.
Table 1 reagent detection sensitivity
3. precision test
Use the human serum sample of 2 kinds of different bladder chalone C content, measure the withinrun precision of detection kit of the present invention.It is shown that the withinrun precision of the detection kit of the embodiment of the present invention 1 and 2 is 1.47% and 2.86%；2.2% and 3.295%, the scheme provided according to NCCLSEP15-A file carries out the performance verification of precision.Measure these five kinds of serum specimens 4 times every day with three kinds of reagent of different manufacturers, measure 5d altogether, calculate batch interior percentage variation (CV) of different manufacturers blood fat reagent detection, it may be judged whether less than the data that producer provides.
Table 2
Same method measures the comparative example 1 reagent to comparative example 6, and result is following table such as, it is seen that Data Representation is substantially not as good as embodiments of the invention 1 and 2:
Table 3
4. the comparison of the interference free performance of two kinds of test kits:
Adding four kinds of chaff interferences in Roche quality-control product (sign value 8.2mg/l) and form pooled serum, measurement result is as follows: total bilirubin determination reagent kit of the present invention and the contrast of existing kit measurement interference free performance:
Table 4
Technical scheme in above-mentioned the embodiment of the present application, at least has the following technical effect that or advantage:
(1), test kit of the present invention utilize Latex-enhanced immunoturbidimetric assay measure bladder chalone C, be exaggerated detection signal, improve detection sensitivity, shorten the detection time.
(2), the present invention adopt the emulsion reagent of mixing particle diameter, wherein the latex particle ratio of little particle diameter is big, improves linear；Containing the latex particle of a certain proportion of big particle diameter, sensitivity is also higher simultaneously.
(3), test kit of the present invention has been specifically added the material preventing interference, good in anti-interference performance.Meanwhile, the interpolation of surfactant also improves precision.
(4), use automatic clinical chemistry analyzer to measure anti-bladder chalone C antibody, not only make operation more easy, improve automaticity, and also a saving the Financial cost of a small amount of sample detection experiment.A small amount of sample is detected by automatic clinical chemistry analyzer multiple batches of, almost identical with the single batch of cost to a large amount of sample detection；Therefore, automatic clinical chemistry analyzer goes for the detection to a small amount of sample, and does not improve Financial cost.
Although preferred embodiments of the present invention have been described, but those skilled in the art are once know basic creative concept, then these embodiments can be made other change and amendment.So, claims are intended to be construed to include preferred embodiment and fall into all changes and the amendment of the scope of the invention.Obviously, the present invention can be carried out various change and modification without deviating from the spirit and scope of the present invention by those skilled in the art.So, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.
Claims (10)
1. a bladder chalone C latex intensified is than turbid detection kit, it is characterised in that: include reagent R1 and reagent R2, described reagent R1 and include buffer, inorganic salt, surfactant, preservative, stabilizer and interference elimination albumen；Described reagent R2 includes buffer, inorganic salt, surfactant, preservative, stabilizer and polystyrene latex particles mixture；Described polystyrene latex particles mixture is crosslinked with anti-bladder chalone C antibody；
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and the mixture of minor diameter polystyrene latex particles; described major diameter polystyrene latex particles mean diameter is 400nm～600nm; described minor diameter polystyrene latex mean diameter is 16nm～32nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=(1-3): (7-9).
2. bladder chalone C latex intensified according to claim 1 is than turbid detection kit, it is characterised in that:
Each component and the content of described reagent R1 is:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Stabilizer 1～10%
Interference eliminates albumen 0.5～1%.
3. bladder chalone C latex intensified according to claim 1 is than turbid detection kit, it is characterised in that: each component and the content of described reagent R2 is:
Buffer 30～120mmol/L
Inorganic salt 80～300mmol/L
Surfactant 3～10mmol/L
Preservative 0.01%～1.0%
Stabilizer 1～10%
Polystyrene latex particles mixture 0.1-1%.
4. the bladder chalone C latex intensified according to Claims 2 or 3 is than turbid detection kit, it is characterised in that: described buffer is one or more in 3-morpholine-2-hydroxy-propanesulfonic acid, 2-(N-morpholine) ethyl sulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, 3-[double; two (2-ethoxy) amino of N-N-]-2-hydroxy-propanesulfonic acid, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid, N-2-hydroxyethyl piperazine-N '-3-N-morpholinopropanesulfonic acid and 3-(three (methylol) methyl) amino-1-propane sulfonic acid；Described surfactant is fatty alcohol-polyoxyethylene ether, OPEO, polyoxyethylene stearic acid ester, lauric acid polyoxyethylene ester, polyoxyethylene oleic acid ester and one or more in lauryl amine polyoxyethylene ether；Described stabilizer is one or more in protein, inorganic salt, metal chelating agent and antioxidant；Described interference eliminates the HBR blocker that albumen is Scantibodies company；Described inorganic salt is sodium chloride, potassium chloride and one or more in magnesium sulfate；Described preservative is one or more in sodium azide, thimerosal and Proclin300.
5. bladder chalone C latex intensified according to claim 4 is than turbid detection kit, it is characterised in that: described stabilizer is chicken egg white and casein.
6. bladder chalone C latex intensified according to claim 5 is than turbid detection kit, it is characterised in that: each component and the content of described reagent R1 are respectively as follows:
7. bladder chalone C latex intensified according to claim 5 is than turbid detection kit, it is characterised in that: each component and the content of described reagent R2 are respectively as follows:
8. bladder chalone C latex intensified according to claim 1 is than turbid detection kit, it is characterized in that:, described test kit also includes the reference standard product of bladder chalone C reagent, and described reference standard product are add people to recombinate the human serum of bladder chalone C albumen or the liquid of similar serum substrate.
9. bladder chalone C latex intensified according to claim 8 is than turbid detection kit, it is characterised in that: the reference standard product of described bladder chalone C reagent have 5 concentration, respectively 0.5mg/L, 1mg/L, 2mg/L, 4mg/L and 8mg/L.
10. the bladder chalone C latex intensified as described in any one of claim 1-9 is than the purposes of turbid detection kit, it is characterised in that: described bladder chalone C latex intensified is applied to the bladder chalone C content in detection serum than turbid detection kit.
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