CN104407155B - A kind of AFP, GP73, PIVKA-II combined detection kit - Google Patents
A kind of AFP, GP73, PIVKA-II combined detection kit Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention relates to field of biological detection, particularly relate to one and quantitatively detect AFP, golgiosome glycoprotein-73? detection kit of the albumen PIVKA-II that GP73, vitamin K deficiency or antagonist-II induction produces and its production and use.Detection kit of the present invention, comprise mutual independently AFP Test paper card, golgiosome glycoprotein-73? the albumen PIVKA-II Test paper card of GP73 Test paper card and vitamin K deficiency or antagonist-II induction generation, described Test paper card comprises base plate all separately and is 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.AFP, GP73 and PIVKA-II is detected by fluorescent micro-ball immune chromatography technology by kit provided by the present invention first, has sensitivity and specificity concurrently, has the advantages such as operation is fast and convenient, result is accurate, economic and practical.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of AFP, GP73, PIVKA-II combined detection kit and its production and use.
Background technology
Alpha-fetoprotein (alphafetoprotein, AFP) be a kind of glycoprotein that development of fetus is synthesized by liver and yolk bag in early days, along with the growth at age after development of fetus and birth, in blood, AFP level declines gradually, and the reference interval of HAS AFP concentration is 1-20 μ g/L.AFP is the special mark of the sensitivity of hepatocellular carcinoma (hepatocellularcarcinoma, HCC), serum concentration of AFP more than lasting more than 5 weeks of lasting 4 weeks of 400 μ g/L or 200-400 μ g/L, strong suspicion hepatocellular carcinoma; Serum afp also has the size judgement of available liver cancer, the curative effect evaluation of liver cancer and Index for diagnosis simultaneously.But according to statistics, the patients with hepatocellular carcinoma serum afp of about 20%-30% is not high; The optimum hepatopathy of part, comprises oxyhepatitis, chronic hepatitis and large-area hepatonecrosis, also has the rising of Serum AFP.Therefore, there is false positive to a certain degree and false negative by Serum AFP as the mark of hepatocellular carcinoma.
Golgiosome glycoprotein-73 (GolgiProtein73, GP73) also known as II type golgiosome memebrane protein (Golgiphosphoprotein2, GOLPH2), 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 ProteinInducedbyVitaminKAbsenceorAntagonist-II that vitamin K deficiency or antagonist-II induce, PIVKA-II), be again de--γ-carboxyl factor (Des-gamma-carboxyprothrombin, 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, AFP is the biochemical marker of classical hepatocellular carcinoma, 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 three kinds of indexs when applying separately, if by appropriate mode and means, and joint-detection three 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 AFP, 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 one and quantitatively detect AFP, golgiosome glycoprotein-73GP73, vitamin K deficiency or antagonist-II induce the detection kit of the albumen PIVKA-II produced, comprise mutual independently AFP Test paper card, the albumen PIVKA-II Test paper card of golgiosome glycoprotein-73GP73 Test paper card and vitamin K deficiency or antagonist-II induction generation, 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 AFP Test paper card comprises AFP antibody, 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, AFP antibody on described gold mark pad, albumen PIVKA-II antibody of golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction generation adopts 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 AFP Test paper card is coated with AFP antibody; 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-19TWEEN20, S-20TWEEN80, S-13TRITONX-45, S-14TRITONX-100, S-15TRITONX305, 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 three parallel test card draw-in grooves, described test card is embedded in described test card draw-in groove, be covered with three testing windows and three wells on described, matching with the detection line of three test cards and the position of nature controlling line respectively in the position of described three testing windows, matches with the position of the sample pad of three test cards in the position of described three wells.
Preferred, described in get stuck for plastics get stuck.
Preferred, be also provided with the translot of connection three wells between described three wells.
Preferably, described detection kit is used for quantitatively detecting the content of the albumen PIVKA-II that AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction produce in serum or blood plasma.
The detection kit of the albumen PIVKA-II that quantitative detection AFP provided by the present invention, golgiosome glycoprotein-73GP73, 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 AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency in sample or antagonist-II induction generation can be obtained.
Second aspect present invention provides described quantitative detection lipoprotein AFP, 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) induce albumen PIVKA-II antibody-solutions produced to spray each self-corresponding gold mark pad respectively with the AFP antibody of fluorescent microsphere mark, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II, obtainedly comprise the gold mark pad that AFP antibody, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody produced respectively;
2) on the detection line and nature controlling line of the nitrocellulose filter of AFP Test paper card, AFP antibody and sheep anti-mouse antibody is sprayed respectively; The detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card spray golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody 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 three cover sample pad, steps 1) prepare three cover gold mark pad, step 2) prepare three cover nitrocellulose filters, three 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 provide described quantitative detection lipoprotein AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induce the detection kit of the albumen PIVKA-II produced AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induce the purposes of albumen PIVKA-II detection field produced.
Beneficial effect of the present invention is:
Quantitative detection AFP provided by the present invention, the detection kit of the albumen PIVKA-II of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation is first by AFP, 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, have high sensitivity and high specific concurrently, AFP can be detected fast, the content of the albumen PIVKA-II of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation.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 MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & Sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, 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 induce albumen PIVKA-II antibody-solutions produced to spray each self-corresponding pretreated gold mark pad respectively with the AFP antibody of fluorescent microsphere mark, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II, obtainedly comprise the gold mark pad that AFP antibody, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody produced 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 AFP Test paper card, AFP antibody and sheep anti-mouse antibody is sprayed respectively; The detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card spray golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody 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 three kinds of bags by after nitrocellulose filter, the concentration of spray solution is 1mg/ml, and quantity for spray is 1ul/cm;
3) by three cover sample pad, steps 1) prepare three cover gold mark pad, step 2) prepare three cover nitrocellulose filters, three 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 respectively by concentration, 5,20,100,200,400,500,600,800, the AFP buffer solution of 1000ug/L 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, and X-axis is standard items actual value.
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.
AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induce albumen PIVKA-II anti-interference and specific detection that produce:
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, obtain the AFP detected in sample, the detection data of albumen PIVKA-II content of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation, the AFP obtained will be detected again, albumen PIVKA-II content data of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation and true AFP, albumen PIVKA-II content data of golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induction generation contrasts, obtain accuracy and affect deviate.
Albumen PIVKA-II, 50mg/L haemoglobin, 50mg/dL triglyceride that sample 1:20ug/L AFP, 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 2:50ug/L AFP, 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 3:100ug/L AFP, 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 4:400ug/L AFP, 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 5:800ug/L AFP, 500ng/ml golgiosome glycoprotein-73GP73,18ng/ml vitamin K deficiency or antagonist-II induction produces;
Blank: 50mg/L haemoglobin, 50mg/dL triglyceride;
The AFP content data of the detection that sample 1-5 obtains is respectively 20.1ug/L, 49.9ug/L, 99.9ug/L, 400.05ug/L, 800.01ug/L; Golgiosome glycoprotein-73GP73 content data 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 induce albumen PIVKA-II antibody-solutions produced to spray each self-corresponding pretreated gold mark pad respectively with the AFP antibody of fluorescent microsphere mark, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II, obtainedly comprise the gold mark pad that AFP antibody, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody produced 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 AFP Test paper card, AFP antibody and sheep anti-mouse antibody is sprayed respectively; The detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card spray golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody 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 three kinds of bags by after nitrocellulose filter, the concentration of spray solution is 1mg/ml, and quantity for spray is 1ul/cm;
3) by three cover sample pad, steps 1) prepare three cover gold mark pad, step 2) prepare three cover nitrocellulose filters, three 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.
AFP, golgiosome glycoprotein-73GP73 and vitamin K deficiency or antagonist-II induce sensitivity and the detectability contrast experiment of the albumen PIVKA-II produced:
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.The AFP known sample getting 1 ~ 20ug/L gradient concentration carries out sensitivity detection, arranges a gradient at interval of 0.5ug/L, and each gradient arranges 40 samples, record testing result.The lowest detection of the kit of result display prepared by embodiment 1 is limited to 1.2ug/L; And the lowest detectable limit of kit prepared by embodiment 2 is higher than 20ug/L.
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 (8)
1. one kind is quantitatively detected AFP, golgiosome glycoprotein-73GP73, vitamin K deficiency or antagonist-II induce the detection kit of the albumen PIVKA-II produced, comprise mutual independently AFP Test paper card, the albumen PIVKA-II Test paper card of golgiosome glycoprotein-73GP73 Test paper card and vitamin K deficiency or antagonist-II induction generation, 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 AFP Test paper card comprises AFP antibody, 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, AFP antibody on described gold mark pad, albumen PIVKA-II antibody of golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induction generation adopts fluorescent microsphere mark, each gold mark is paid somebody's debt and expected repayment later through pre-service, each gold mark 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, the concentration of each component in damping fluid is: stachyose 0.8-3g/L, alum 0.1-1g/L, Fructose Diphosphate 0.8-3g/L, sodium hexametaphosphate 0.05-0.5g/L, glycocoll 1.5-2.25g/L, the solvent of described pre-treatment buffer is water.
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 AFP Test paper card is coated with AFP antibody; 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.
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, 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 three parallel test card draw-in grooves, and described test card is embedded in described test card draw-in groove, is covered with three testing windows and three wells on described, matching with the detection line of three test cards and the position of nature controlling line respectively in the position of described three testing windows, matches with the position of the sample pad of three test cards in the position of described three wells.
7. detection kit as claimed in claim 6, is characterized in that, be also provided with the translot of connection three wells between described three wells.
8. the preparation method of the detection kit according to the arbitrary claim of claim 1 ~ 7, specifically comprises the steps:
1) induce albumen PIVKA-II antibody-solutions produced to spray each self-corresponding gold mark pad respectively with the AFP antibody of fluorescent microsphere mark, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II, obtainedly comprise the gold mark pad that AFP antibody, golgiosome glycoprotein-73GP73 antibody and vitamin K deficiency or antagonist-II induce albumen PIVKA-II antibody produced respectively;
2) on the detection line and nature controlling line of the nitrocellulose filter of AFP Test paper card, AFP antibody and sheep anti-mouse antibody is sprayed respectively; The detection line and nature controlling line of the nitrocellulose filter of golgiosome glycoprotein-73GP73 Test paper card spray golgiosome glycoprotein-73GP73 antibody and sheep anti-mouse antibody 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 three cover sample pad, steps 1) prepare three cover gold mark pad, step 2) prepare three cover nitrocellulose filters, three 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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| CN109444422A (en) * | 2018-12-17 | 2019-03-08 | 福建省立医院 | A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II |
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