CN104360085B - A kind of AFP detection kit - Google Patents

A kind of AFP detection kit Download PDF

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CN104360085B
CN104360085B CN201410737604.4A CN201410737604A CN104360085B CN 104360085 B CN104360085 B CN 104360085B CN 201410737604 A CN201410737604 A CN 201410737604A CN 104360085 B CN104360085 B CN 104360085B
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fetoprotein
detection
alpha
afp
detection kit
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CN104360085A (en
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吉权
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Zhongyuan Huiji Biotechnology Co Ltd
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CHONGQING QIANDE BIOTECHNOLOGY Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57438Specifically defined cancers of liver, pancreas or kidney

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Abstract

The present invention relates to field of biological detection, particularly relate to detection kit of a kind of quantitative detection AFP and its production and use.The invention provides a kind of detection kit of quantitative detection AFP, comprise test card, test card comprises base plate 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, described gold mark pad comprises AFP antibody, described nitrocellulose filter is coated with detection line and nature controlling line, the AFP antibody on described gold mark pad adopts fluorescent microsphere mark.AFP is detected by fluorescent micro-ball immune chromatography technology by the detection kit of quantitative detection AFP 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

A kind of AFP detection kit
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of alpha-fetoprotein 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.
Also there is more defect in alpha-fetoprotein (AFP) detection method known at present, as comparatively loaded down with trivial details, consuming time longer in operation, is not suitable for using clinically.At present, alpha-fetoprotein (AFP) the detection kit accuracy in China market, sensitivity, repeatability are still poor.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide the kit of AFP content in a kind of Fast Measurement serum or blood plasma, for solving the problems of the prior art.
For achieving the above object and other relevant objects, the invention provides the kit of AFP content in a kind of Fast Measurement serum or blood plasma, comprise test card, described test card comprises base plate 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, described gold mark pad comprises AFP monoclonal antibody, described nitrocellulose filter is coated with detection line and nature controlling line, the AFP monoclonal antibody on described gold mark pad adopts fluorescent microsphere mark.
Preferably, AFP monoclonal antibody on described gold mark pad adopts 180nm fluorescent microsphere mark, EX (nm)=650/Em (nm)=670, has 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 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, described detection line is coated with AFP monoclonal antibody.AFP monoclonal antibody on described gold mark pad can be identical antibody with the AFP monoclonal antibody on detection line, also can be different antibody.
Preferably, nature controlling line wraps by sheep anti-mouse antibody.
Preferably, described 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, 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, 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.
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 test card draw-in groove, described test card is embedded in described test card draw-in groove, be covered with testing window and well on described, matching with the position of described detection line and nature controlling line in the position of described testing window, matches with the position of described sample pad in the position of described well.
Preferred, described in get stuck for plastics get stuck.
Preferably, described detection kit is used for the content quantitatively detecting AFP in serum or blood plasma.
The detection kit of quantitative detection AFP provided by the present invention, 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 obtained when detecting sample is compared with typical curve, the AFP content detected in sample can be obtained.
Second aspect present invention provides the preparation method of the detection kit of described quantitative detection AFP, comprises the steps:
1) with the AFP antibody-solutions spraying gold mark pad of fluorescent microsphere mark, the obtained gold mark pad comprising AFP antibody;
2) on the detection line and nature controlling line of nitrocellulose filter, spray AFP antibody and sheep anti-mouse antibody respectively, obtained bag by after nitrocellulose filter;
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter, the adsorptive pads prepared be pasted onto on base plate successively, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
Preferably, 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, 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 the purposes of detection kit at AFP detection field of described quantitative detection AFP.
Beneficial effect of the present invention is:
AFP is detected by fluorescent micro-ball immune chromatography technology by the detection kit of quantitative detection AFP provided by the present invention first, has high sensitivity and high specific concurrently, can detect the content of AFP 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 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 with the AFP antibody-solutions spraying pretreated gold mark pad of fluorescent microsphere mark, obtained bag is by the gold mark pad of AFP antibody, 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 nitrocellulose filter, spray AFP antibody-solutions and the sheep anti-mouse antibody solution of 1mg/ml respectively, quantity for spray is 1ul/cm, obtained bag by after nitrocellulose filter;
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter, the adsorptive pads prepared be pasted onto on PVC base plate successively, cutting obtains the Test paper card of wide 3-5mm; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.Standard lines 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.
AFP anti-interference 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, obtain the detection data of the AFP content detected in sample, the AFP content data and true AFP content data that detect acquisition are contrasted, obtaining accuracy affects deviate again.
Sample 1:20ug/L AFP, 50mg/L haemoglobin, 50mg/dL triglyceride;
Sample 2:50ug/L AFP, 500mg/L haemoglobin, 10mg/dL triglyceride;
Sample 3:100ug/L AFP, 100mg/L haemoglobin, 20mg/dL triglyceride;
Sample 4:400ug/L AFP, 150mg/L haemoglobin, 30mg/dL triglyceride;
Sample 5:800ug/L AFP, 200mg/L haemoglobin, 40mg/dL triglyceride;
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, 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 with the AFP antibody-solutions spraying pretreated gold mark pad of fluorescent microsphere mark, obtained bag is by the gold mark pad of AFP antibody, in solution, the mass ratio of fluorescent microsphere and antibody is 5:1, the concentration of solution is 10mg/ml, and quantity for spray is 4ul/cm;
2) on the detection line and nature controlling line of nitrocellulose filter, spray AFP antibody-solutions and the sheep anti-mouse antibody solution of 1mg/ml respectively, quantity for spray is 1ul/cm, obtained bag by after nitrocellulose filter;
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter, the adsorptive pads prepared be pasted onto on PVC base plate successively, cutting obtains the Test paper card of wide 3-5mm; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
The sensitivity of AFP 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.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.
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 the detection kit of alpha-fetoprotein, comprise test card, test card comprises base plate 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, described gold mark pad comprises alpha-fetoprotein antibody, described nitrocellulose filter is coated with detection line and nature controlling line, the alpha-fetoprotein antibody on described gold mark pad adopts fluorescent microsphere mark; Described gold mark is paid somebody's debt and expected repayment later through pre-service, the pre-treatment buffer that described gold mark pad uses when pre-service 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. the detection kit quantitatively detecting alpha-fetoprotein as claimed in claim 1, it is characterized in that, on described 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. the detection kit quantitatively detecting alpha-fetoprotein as claimed in claim 1, is characterized in that, described detection line is coated with alpha-fetoprotein antibody.
4. the detection kit quantitatively detecting alpha-fetoprotein as claimed in claim 1, is characterized in that, nature controlling line wraps by sheep anti-mouse antibody.
5. the detection kit quantitatively detecting alpha-fetoprotein as claimed in claim 1, it is characterized in that, described 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. the detection kit quantitatively detecting alpha-fetoprotein 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 test card draw-in groove, and described test card is embedded in described test card draw-in groove, is covered with testing window and well on described, matching with the position of described detection line and nature controlling line in the position of described testing window, matches with the position of described sample pad in the position of described well.
7. the detection kit quantitatively detecting alpha-fetoprotein as claimed in claim 1, is characterized in that, described detection kit is used for the content quantitatively detecting alpha-fetoprotein in serum or blood plasma.
8. the preparation method of the detection kit of the quantitative detection alpha-fetoprotein according to the arbitrary claim of claim 1 ~ 7, specifically comprises the steps:
1) with the alpha-fetoprotein antibody solution spraying of fluorescent microsphere mark pretreated gold mark pad, the obtained gold mark pad comprising alpha-fetoprotein antibody;
2) on the detection line and nature controlling line of nitrocellulose filter, spray alpha-fetoprotein antibody and sheep anti-mouse antibody respectively, obtained bag by after nitrocellulose filter;
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter, the adsorptive pads prepared be pasted onto on base plate successively, cutting obtains Test paper card; Finally Test paper is snapped fits into the obtained detection kit that gets stuck.
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CN106290842A (en) * 2015-05-13 2017-01-04 上海凯创生物技术有限公司 Cholera vibrio O 139 colloidal gold method detection kit
CN106290844A (en) * 2015-05-13 2017-01-04 上海凯创生物技术有限公司 Bacillus typhi colloidal gold method detection kit
CN106290849A (en) * 2015-05-13 2017-01-04 上海凯创生物技术有限公司 A kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box
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CN106290825A (en) * 2015-05-13 2017-01-04 上海凯创生物技术有限公司 Plasmodium vivax colloidal gold method detection kit
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