CN104459115A - GP73 and PIVKA-II combined detection kit - Google Patents
GP73 and PIVKA-II combined detection kit Download PDFInfo
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- CN104459115A CN104459115A CN201410735789.5A CN201410735789A CN104459115A CN 104459115 A CN104459115 A CN 104459115A CN 201410735789 A CN201410735789 A CN 201410735789A CN 104459115 A CN104459115 A CN 104459115A
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- pivka
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- antagonist
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to the field of biological detection and in particular relates to a detection kit for quantitatively detecting a Golgi apparatus glycoprotein-73 GP73 and a protein PIVKA-II produced by lacking of vitamin K or induction of an antagonist-II as well as a preparation method and a purpose of the detection kit. The detection kit comprises a test paper card for the Golgi apparatus glycoprotein-73 GP73 and a test paper card for the protein PIVKA-II produced by lacking of the vitamin K or induction of the antagonist-II, wherein the test paper cards are mutually independent and respectively comprise a bottom plate, a sample pad, a colloidal-gold pad, a nitrocellulose membrane and a water absorption pad, and each sample pad, each colloidal-gold pad, each nitrocellulose membrane and each water absorption pad are sequentially arranged on the surface of the corresponding bottom plate starting from the sample feeding end. The detection kit provided by the invention firstly detects GP73 and PIVKA-II by virtue of a fluorescent microsphere immunity-chromatography technology, has both the sensitivity and the specificity and has the advantages of rapidness, convenience and simpleness in operation, accurate result, economy, suitability and the like.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of GP73, PIVKA-II combined detection kit and its production and use.
Background technology
Golgiosome glycoprotein-73 (GolgiProtein 73, GP73) also known as II type golgiosome memebrane protein (Golgiphosphoprotein2, GOLPH 2), its concentration in the patients serum such as chronic hepatitis, cirrhosis raises, and raise more obvious in hepatocellular carcinoma patients serum, with in significant difference between normal person or optimum hepatopathy patients.There are some researches show, serum GP73 is used for the diagnosis of liver cancer, is better than Serum AFP and detects.According to the literature, the sensitivity that GP73 detects liver cancer is 69%, and specificity is 75%.For the diagnosis of early liver cancer, the susceptibility of GP73 is 62%, and AFP susceptibility is only 25%.In the liver cancer patient of AFP level lower than 20 μ g/L, the crowd GP73 level of 57% is had significantly to raise.This shows, the application of GP73 can improve the recall rate of the liver cancer patient to AFP negative greatly.
Protein (the Protein Induced by Vitamin K Absence orAntagonist-II that vitamin K deficiency or antagonist-II induce, PIVKA-II), be again de--γ-carboxyl factor (Des-gamma-carboxy prothrombin, DCP) or abnormal prothrombin, the mark of primary carcinoma of liver within 1984, is identified as first.In patients with hepatocellular carcinoma serum, PIVKA-II concentration level is far above cirrhosis and metastatic hepatic carcinoma patient, and the dynamic change of the change of its serum-concentration and Patients ' Hepatocytes cancer (operation, treatment and recurrence etc.) is relevant.Research afterwards confirms, PIVKA-II and AFP detects and can complement one another, and the diagnosis of hepatocellular carcinoma can be brought up to 84% by the joint-detection of the two, and only the patients with hepatocellular carcinoma of about 16% is both negative result.Current PIVKA-II detects and is put in " Japanese liver cancer association liver cancer diagnosis and treatment specification version in 2009 ", for the examination of hepatocellular carcinoma people at highest risk and the auxiliary diagnosis of primary carcinoma of liver; Mark for hepatocellular carcinoma auxiliary diagnosis in " primary carcinoma of liver diagnosis and treatment specification (version in 2011) " that the Ministry of Public Health of China issues also comprises PIVKA-II.
To sum up, GP73 and PIVKA-II is the Typical Representative of novel liver cancer marker.Comparatively speaking, the susceptibility of GP73 is higher, and the specificity of PIVKA-II is better, false negative to a certain degree and false positive is all there is in two kinds of indexs when applying separately, if by appropriate mode and means, and joint-detection two indexs simultaneously, complement one another, to contribute to the false negative and the false positive that reduce hepatocellular carcinoma clinical diagnosis further, improve the diagnosis performance of these marks to hepatocellular carcinoma, is clinical and patient service better.
Summary of the invention
The shortcoming of prior art in view of the above, a kind of quantitative detection golgiosome glycoprotein-73GP73, vitamin K deficiency or antagonist-II is the object of the present invention is to provide to induce detection kit of the albumen PIVKA-II produced and its production and use, for solving the problems of the prior art.
For achieving the above object and other relevant objects, the invention provides a kind of quantitatively detection golgiosome glycoprotein-73GP73, vitamin K deficiency or antagonist-II induce the detection kit of the albumen PIVKA-II produced, comprise the albumen PIVKA-II Test paper card of independently golgiosome glycoprotein-73GP73 Test paper card and vitamin K deficiency or antagonist-II induction generation mutually, described Test paper card comprises base plate all separately, and be positioned at the sample pad be arranged in order from application of sample end of backplate surface, gold mark pad, nitrocellulose filter and adsorptive pads, the gold mark pad of described golgiosome glycoprotein-73GP73 Test paper card comprises golgiosome glycoprotein-73GP73 antibody, described vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody gold mark pad of the albumen PIVKA-II Test paper card produced comprising vitamin K deficiency or antagonist-II induction generation, described each nitrocellulose filter is coated with detection line and nature controlling line, golgiosome glycoprotein-73GP73 antibody on described gold mark pad and albumen PIVKA-II antibody of vitamin K deficiency or antagonist-II induction generation adopt fluorescent microsphere mark.
Preferably, the antibody on each gold mark pad adopts 180nm fluorescent microsphere mark, EX (nm)=650/Em (nm)=670, and have signal little by background interference, detection sensitivity is high, the advantage that result is reproducible.
Preferably, described base plate is PVC base plate.
Preferably, on described each nitrocellulose filter, detection line is positioned at side close to application of sample end, and nature controlling line is positioned at side away from application of sample end.
Preferably, the detection line of described golgiosome glycoprotein-73GP73 Test paper card is coated with golgiosome glycoprotein-73GP73 antibody; Described vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody detection line of the albumen PIVKA-II Test paper card produced being coated with vitamin K deficiency or antagonist-II induction generation.
Antibody in each test card on gold mark pad can be identical antibody with the antibody on detection line, also can be different antibody.
Preferably, each nature controlling line wraps by sheep anti-mouse antibody.
Preferably, described each sample pad adopts damping fluid process, described damping fluid is selected from one or more the combination in PBS damping fluid, Tris-HCl damping fluid, glycine buffer, borate buffer solution and citrate-phosphate salt buffer, and the concentration of damping fluid is 20-200mM.
Preferably, each gold mark of the present invention is paid somebody's debt and expected repayment later through pre-service, and the pre-treatment buffer used during pre-service is selected from one or more combination of glycine buffer, Tris-HCl damping fluid, borate buffer solution, and the concentration of damping fluid is 5-50mM.
Preferably, described damping fluid also comprises increased response agent, described increased response agent be selected from PEG4000, PEG6000, PEG8000 and PEG20000 any one, the concentration of described increased response agent is 10 ~ 50g/L.
Preferably, described buffer solution also comprises surfactant, described surfactant is selected from any one or multiple combination in S-19TWEEN 20, S-20TWEEN 80, S-13TRITON X-45, S-14TRITON X-100, S-15TRITON X305, and the concentration of described surfactant is 10 ~ 50g/L.
Preferably, in order to make kit, there is better sensitivity and color developing effect, each gold mark of the present invention pads the pre-treatment buffer used when pre-service and comprises following component: stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate and glycocoll, and the total concentration of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate, glycocoll is 3.5-7.5g/L, the pH value of damping fluid is 7.2-7.6.
Preferably, the concentration of each component in damping fluid is:
The solvent of described pre-treatment buffer is water.
Described pretreated concrete steps are: each gold mark pad is soaked 1.5 ~ 2h in pretreatment fluid, takes out and be put in 36 ~ 38 DEG C of oven dry.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the adjustment of pH value.
Preferably, described kit also comprises and getting stuck, described getting stuck comprises back of the body card and upper cover, the described back of the body is arranged with two parallel test card draw-in grooves, described test card is embedded in described test card draw-in groove, be covered with two testing windows and two wells on described, matching with the detection line of two test cards and the position of nature controlling line respectively in the position of described two testing windows, matches with the position of the sample pad of two test cards in the position of described two wells.
Preferred, described in get stuck for plastics get stuck.
Preferred, be also provided with the translot of connection two wells between described two wells.
Preferably, described detection kit is used for quantitatively detecting the content of the albumen PIVKA-II of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation in serum or blood plasma.
The detection kit of the albumen PIVKA-II that quantitative detection golgiosome glycoprotein-73GP73 provided by the present invention, vitamin K deficiency or antagonist-II induction produces, can supporting immune quantitative analytical instrument use.Immunoassay instrument, by acquisition testing line (T) and nature controlling line (C) band fluorescence signal, calculates T/C signal value.First various criterion product are added drop-wise on test card before using, analyzing and processing sets up calibration curve (relation of T/C signal value and standard items actual value), again the T/C value that obtains when detecting sample is compared with typical curve, albumen PIVKA-II content detecting golgiosome glycoprotein-73GP73 in sample and vitamin K deficiency or antagonist-II induction generation can be obtained.
Second aspect present invention provides described quantitative detection golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II to induce the preparation method of the detection kit of the albumen PIVKA-II produced, and comprises the steps:
1) spray each self-corresponding gold mark pad respectively with the golgiosome glycoprotein-73GP73 antibody of fluorescent microsphere mark and albumen PIVKA-II antibody-solutions of vitamin K deficiency or antagonist-II induction generation, obtain the gold mark pad comprising albumen PIVKA-II antibody that golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction produce respectively;
2) on the detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card, golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody is sprayed respectively; Albumen PIVKA-II antibody and sheep anti-mouse antibody that the detection line of the nitrocellulose filter of the albumen PIVKA-II Test paper card produced and nature controlling line spray respectively vitamin K deficiency or antagonist-II induction generation is induced at vitamin K deficiency or antagonist-II;
3) by two cover sample pad, steps 1) prepare two cover gold mark pad, step 2) prepare two cover nitrocellulose filters, two cover adsorptive pads be pasted onto successively on base plate, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
Preferably, each gold mark of the present invention is paid somebody's debt and expected repayment later through pre-service, and the pre-treatment buffer used during pre-service is selected from one or more combination of glycine buffer, Tris-HCl damping fluid, borate buffer solution, and the concentration of damping fluid is 5-50mM.
Preferably, in order to make kit, there is better sensitivity and color developing effect, each gold mark of the present invention pads the pre-treatment buffer used when pre-service and comprises following component: stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate and glycocoll, and the total concentration of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate, glycocoll is 3.5-7.5g/L, the pH value of damping fluid is 7.2-7.6.
Preferably, the concentration of each component in damping fluid is:
The solvent of described pre-treatment buffer is water.
Described pretreated concrete steps are: gold mark pad is soaked 1.5 ~ 2h in pretreatment fluid, take out and are put in 36 ~ 38 DEG C of oven dry.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the adjustment of pH value.
Third aspect present invention provides described quantitative detection golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II to induce the detection kit of the albumen PIVKA-II produced to induce the purposes of albumen PIVKA-II detection field produced at golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II.
Beneficial effect of the present invention is:
The albumen PIVKA-II of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation is detected by fluorescent micro-ball immune chromatography technology by the detection kit of the albumen PIVKA-II of quantitative detection golgiosome glycoprotein-73GP73 provided by the present invention and vitamin K deficiency or antagonist-II induction generation first, have high sensitivity and high specific concurrently, the content that golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induce the albumen PIVKA-II produced can be detected fast.In addition, described detection kit has the advantages such as operation is fast and convenient, result is accurate, economic and practical, disturb little by the serious blood fat of serum (or blood plasma), haemolysis, as serum (or blood plasma) haemoglobin≤500mg/L, triglyceride≤30mg/dL, variation < 10% is affected on accuracy.
Embodiment
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this instructions can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this instructions also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention; In instructions of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, all technology used in the present invention are identical with the meaning that those skilled in the art of the present technique understand usually with scientific terminology.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell chulture, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Embodiment 1
The preparation of test card of the present invention:
1) pre-treatment buffer is used to carry out pre-service to gold mark pad, pre-treatment buffer is: stachyose 2g/L, alum 0.5g/L, Fructose Diphosphate 1.5g/L, sodium hexametaphosphate 0.3g/L, the aqueous solution of glycocoll 1.88g/L, pH=7.4, pretreated concrete steps are: gold mark pad is soaked 2h in pretreatment fluid, take out and are put in 37 DEG C of oven dry; Then spray each self-corresponding pretreated gold mark pad respectively with the golgiosome glycoprotein-73GP73 antibody of fluorescent microsphere mark and albumen PIVKA-II antibody-solutions of vitamin K deficiency or antagonist-II induction generation, obtain the gold mark pad comprising albumen PIVKA-II antibody that golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction produce respectively; In solution, the mass ratio of fluorescent microsphere and antibody is 5:1, and the concentration of solution is 10mg/ml, and quantity for spray is 4ul/cm;
2) on the detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card, golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody is sprayed respectively; Albumen PIVKA-II antibody and sheep anti-mouse antibody that the detection line of the nitrocellulose filter of the albumen PIVKA-II Test paper card produced and nature controlling line spray respectively vitamin K deficiency or antagonist-II induction generation is induced at vitamin K deficiency or antagonist-II; Obtained two kinds of bags by after nitrocellulose filter, the concentration of spray solution is 1mg/ml, and quantity for spray is 1ul/cm;
3) by two cover sample pad, steps 1) prepare two cover gold mark pad, step 2) prepare two cover nitrocellulose filters, two cover adsorptive pads be pasted onto successively on base plate, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
Typical curve:
Be 0 by concentration respectively, 2, 5, 20, 60, 100, 200, 400, 600, 800, golgiosome glycoprotein-73GP73 the buffer solution of 1000ng/ml drips in sample pad, each concentration establishes 5 repetitions (testing result gets 5 mean values repeated), after rete is analysed 10 minutes, use immunoassay instrument by acquisition testing line (T) and nature controlling line (C) band fluorescence signal, the sensing range of analyser to fluorescence signal is AD value 0-10000, calculate T/C signal value, set up calibration curve, wherein Y-axis is T/C signal value, X-axis is standard items actual value.
Be 0 by concentration respectively, 1.0, 1.5, 2.0, 5.0, 20, 100, 200, 500, 800, the vitamin K deficiency of 1000ng/ml or antagonist-II induce albumen PIVKA-II buffer solution produced to drip in sample pad, each concentration establishes 5 repetitions (testing result gets 5 mean values repeated), after rete is analysed 10 minutes, use immunoassay instrument by acquisition testing line (T) and nature controlling line (C) band fluorescence signal, the sensing range of analyser to fluorescence signal is AD value 0-10000, calculate T/C signal value, set up calibration curve, wherein Y-axis is T/C signal value, X-axis is standard items actual value.
Albumen PIVKA-II anti-interference of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation and specific detection:
Detection sample drop is added in sample pad, 5 repetitions (testing result gets 5 mean values repeated) established by each sample, after rete is analysed 10 minutes, the T/C value obtained when detecting sample is compared with typical curve, the detection data of albumen PIVKA-II content of the golgiosome glycoprotein-73GP73 in acquisition detection sample and vitamin K deficiency or antagonist-II induction generation, albumen PIVKA-II content data and true golgiosome glycoprotein-73GP73 and vitamin K deficiency that produce or antagonist-II is induced to induce albumen PIVKA-II content data produced to contrast by detecting the golgiosome glycoprotein-73GP73 that obtains and vitamin K deficiency or antagonist-II again, obtain accuracy and affect deviate.
Albumen PIVKA-II, 50mg/L haemoglobin, 50mg/dL triglyceride that sample 1:1ng/ml golgiosome glycoprotein-73GP73,1.5ng/ml vitamin K deficiency or antagonist-II induction produces;
Albumen PIVKA-II, 500mg/L haemoglobin, 10mg/dL triglyceride that sample 2:3ng/ml golgiosome glycoprotein-73GP73,3ng/ml vitamin K deficiency or antagonist-II induction produces;
Albumen PIVKA-II, 100mg/L haemoglobin, 20mg/dL triglyceride that sample 3:6ng/ml golgiosome glycoprotein-73GP73,6ng/ml vitamin K deficiency or antagonist-II induction produces;
Albumen PIVKA-II, 150mg/L haemoglobin, 30mg/dL triglyceride that sample 4:150ng/ml golgiosome glycoprotein-73GP73,12ng/ml vitamin K deficiency or antagonist-II induction produces;
Albumen PIVKA-II, 200mg/L haemoglobin, 40mg/dL triglyceride that sample 5:500ng/ml golgiosome glycoprotein-73GP73,18ng/ml vitamin K deficiency or antagonist-II induction produces;
Blank: 50mg/L haemoglobin, 50mg/dL triglyceride;
Golgiosome glycoprotein-73GP73 the content data of the detection that sample 1-5 obtains is respectively 1.01ng/ml, 2.99ng/ml, 6.02ng/ml, 149.99ng/ml, 500.03ng/ml; Vitamin K deficiency or antagonist-II induce albumen PIVKA-II content data produced to be respectively 1.49ng/ml, 3.01ng/ml, 5.99ng/ml, 11.95ng/ml, 18.02ng/ml; Accuracy affect variation < 10%, do not find when detecting in blank that obvious fluorescence signal changes.
Embodiment 2
The preparation of comparative example test card: adopt 25mM glycine buffer pre-service gold mark pad, other reagent and experimental technique are all with embodiment 1.
1) 25mM glycine buffer pre-service gold mark pad is adopted, then spray each self-corresponding pretreated gold mark pad respectively with the golgiosome glycoprotein-73GP73 antibody of fluorescent microsphere mark and albumen PIVKA-II antibody-solutions of vitamin K deficiency or antagonist-II induction generation, obtain the gold mark pad comprising albumen PIVKA-II antibody that golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction produce respectively; In solution, the mass ratio of fluorescent microsphere and antibody is 5:1, and the concentration of solution is 10mg/ml, and quantity for spray is 4ul/cm;
2) on the detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card, golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody is sprayed respectively; Albumen PIVKA-II antibody and sheep anti-mouse antibody that the detection line of the nitrocellulose filter of the albumen PIVKA-II Test paper card produced and nature controlling line spray respectively vitamin K deficiency or antagonist-II induction generation is induced at vitamin K deficiency or antagonist-II; Obtained two kinds of bags by after nitrocellulose filter, the concentration of spray solution is 1mg/ml, and quantity for spray is 1ul/cm;
3) by two cover sample pad, steps 1) prepare two cover gold mark pad, step 2) prepare two cover nitrocellulose filters, two cover adsorptive pads be pasted onto successively on base plate, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
The sensitivity of the albumen PIVKA-II of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation and detectability contrast experiment:
Judge with fluorescent instrument, analyser be AD value 0-10000 to the sensing range of fluorescence signal, according to the performance of instrument, CUTOFF value is 50, under certain concentration, more than 90% test example AD value >=50, namely think that kit can be used in the detection under this concentration.
Adopt 5%BSA normal saline solution as dummy, dummy should not contain measured object.Golgiosome glycoprotein-73GP73 the known sample getting 1 ~ 20pg/ml gradient concentration carries out sensitivity detection, and arrange a gradient at interval of 0.5pg/ml, each gradient arranges 40 samples, record testing result.The lowest detection of the kit of result display prepared by embodiment 1 is limited to 2.5pg/ml; And the lowest detectable limit of kit prepared by embodiment 2 is higher than 20pg/ml.
Adopt 5%BSA normal saline solution as dummy, dummy should not contain measured object.The vitamin K deficiency or the antagonist-II that get 0.1 ~ 60ug/L gradient concentration induce albumen PIVKA-II known sample produced to carry out sensitivity detection, and arrange a gradient at interval of 0.2ng/ml, each gradient arranges 300 samples, record testing result.The lowest detection of the kit of result display prepared by embodiment 1 is limited to 0.2ng/ml; And the lowest detectable limit of kit prepared by embodiment 2 is higher than 60ug/L.
In sum, detection kit provided by the present invention has good anti-interference and specificity, and has good sensitivity, and negative background is lower, effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (10)
1. one kind is quantitatively detected golgiosome glycoprotein-73 GP73, vitamin K deficiency or antagonist-II induce the detection kit of the albumen PIVKA-II produced, comprise the albumen PIVKA-II Test paper card of independently golgiosome glycoprotein-73 GP73 Test paper card and vitamin K deficiency or antagonist-II induction generation mutually, described Test paper card comprises base plate all separately, and be positioned at the sample pad be arranged in order from application of sample end of backplate surface, gold mark pad, nitrocellulose filter and adsorptive pads, the gold mark pad of described golgiosome glycoprotein-73 GP73 Test paper card comprises golgiosome glycoprotein-73 GP73 antibody, described vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody gold mark pad of the albumen PIVKA-II Test paper card produced comprising vitamin K deficiency or antagonist-II induction generation, described each nitrocellulose filter is coated with detection line and nature controlling line, golgiosome glycoprotein-73 GP73 antibody on described gold mark pad and albumen PIVKA-II antibody of vitamin K deficiency or antagonist-II induction generation adopt fluorescent microsphere mark.
2. detection kit as claimed in claim 1, it is characterized in that, on described each nitrocellulose filter, detection line is positioned at side close to application of sample end, and nature controlling line is positioned at side away from application of sample end.
3. detection kit as claimed in claim 1, is characterized in that, the detection line of described golgiosome glycoprotein-73 GP73 Test paper card is coated with golgiosome glycoprotein-73 GP73 antibody; Described vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody detection line of the albumen PIVKA-II Test paper card produced being coated with vitamin K deficiency or antagonist-II induction generation.
4. detection kit as claimed in claim 1, is characterized in that, each nature controlling line wraps by sheep anti-mouse antibody.
5. detection kit as claimed in claim 1, it is characterized in that, described each sample pad adopts damping fluid process, described damping fluid is selected from one or more the combination in PBS damping fluid, Tris-HCl damping fluid, glycine buffer, borate buffer solution and citrate-phosphate salt buffer, and the concentration of damping fluid is 20-200mM.
6. detection kit as claimed in claim 1, it is characterized in that, described each gold mark pad adopts damping fluid process, and described damping fluid is selected from one or more combination of glycine buffer, Tris-HCl damping fluid, borate buffer solution, and the concentration of damping fluid is 5-50mM.
7. detection kit according to claim 6, it is characterized in that, also comprise increased response agent in described damping fluid, described increased response agent be selected from PEG4000, PEG6000, PEG8000 and PEG20000 any one, the concentration of described increased response agent is 10 ~ 50g/L.
8. detection kit as claimed in claim 1, it is characterized in that, also comprise and getting stuck, described getting stuck comprises back of the body card and upper cover, the described back of the body is arranged with two parallel test card draw-in grooves, and described test card is embedded in described test card draw-in groove, is covered with two testing windows and two wells on described, matching with the detection line of two test cards and the position of nature controlling line respectively in the position of described two testing windows, matches with the position of the sample pad of two test cards in the position of described two wells.
9. detection kit as claimed in claim 8, is characterized in that, be also provided with the translot of connection two wells between described two wells.
10. the preparation method of the detection kit according to the arbitrary claim of claim 1 ~ 9, specifically comprises the steps:
1) spray each self-corresponding gold mark pad respectively with golgiosome glycoprotein-73 GP73 antibody of fluorescent microsphere mark and albumen PIVKA-II antibody-solutions of vitamin K deficiency or antagonist-II induction generation, obtain the gold mark pad comprising albumen PIVKA-II antibody that golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction produce respectively;
2) on the detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73 GP73 Test paper card, golgiosome glycoprotein-73 GP73 antibody and sheep anti-mouse antibody is sprayed respectively; Albumen PIVKA-II antibody and sheep anti-mouse antibody that the detection line of the nitrocellulose filter of the albumen PIVKA-II Test paper card produced and nature controlling line spray respectively vitamin K deficiency or antagonist-II induction generation is induced at vitamin K deficiency or antagonist-II;
3) by two cover sample pad, steps 1) prepare two cover gold mark pad, step 2) prepare two cover nitrocellulose filters, two cover adsorptive pads be pasted onto successively on base plate, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
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