CN104897631A - Near infrared fluorescence detection kit for glycogen phosphorylase isoenzyme BB - Google Patents
Near infrared fluorescence detection kit for glycogen phosphorylase isoenzyme BB Download PDFInfo
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- CN104897631A CN104897631A CN201510292240.8A CN201510292240A CN104897631A CN 104897631 A CN104897631 A CN 104897631A CN 201510292240 A CN201510292240 A CN 201510292240A CN 104897631 A CN104897631 A CN 104897631A
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Abstract
The invention discloses a near infrared fluorescence detection kit for glycogen phosphorylase isoenzyme BB and application of the near infrared fluorescence detection kit. The near infrared fluorescence detection kit comprises a reagent strip on a backboard, wherein the reagent strip comprises a sample pad, a glass fiber film marked by an immunofluorescence probe, a nitrocellulose membrane coated by the anti-glycogen phosphorylase isoenzyme BB antibody, and absorbent paper. According to the invention, by combining with the near infrared fluorescence detection technology, the detection kit can rapidly and accurately detect the glycogen phosphorylase isoenzyme BB in human whole blood, serum and plasma.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of GPBB Near-Infrared Absorption Method fluorescence detection reagent kit and uses thereof.
Background technology
GPBB (GPBB) is the enzyme that in a kind of cardiac muscle cell, content is higher.Glycogen phosphorylase is glycogenolytic key enzyme, decomposition of glycogen compound is formed at cardiac muscle cell's sarcoplasmic reticulum, the decomposition of glycogen that this compound causes ischemic is responsive especially, make it form to change, form raised path between farm fields dissolubility GPBB (GPBB), permeate through cell membranes enters blood.
GPBB (GPBB) is responsive to myocardial ischemia-anoxemia, and can be released into blood in myocardial damage early stage (being less than 4h), be a good index of acute myocardial injury early diagnosis.Being better than cardiogram to the early diagnosis of acute myocardial ischemia, is index the most responsive in 4h after acute myocardial infarction AMI (AMI) the patient episode found at present.This feature of GPBB compensate for the deficiency of cTnI in the early diagnosis of AMI.Along with the development of biological chemistry, immunity, molecular biology and detection technique thereof, find that the research of GPBB to the aspect such as early diagnosis, obstetrics, paediatrics of ischemia myocardial damage has much value.
The detection methods such as conventional enzyme linked immunosorbent assay (ELIsA) are consuming time, need specific installation, are unsuitable for emergency ward and the on-the-spot needs to the quick indagation of AMI.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide a kind of GPBB near-infrared fluorescent method detection kit and uses thereof, for setting up efficiently feasible GPBB near-infrared fluorescent detection technique, solve the problems of the prior art.Detection kit provided by the present invention, can be quick in conjunction with near-infrared fluorescent detection technique, detects GPBB in people's whole blood/serum/plasma accurately.
For achieving the above object and other relevant objects, the present invention is by the following technical solutions:
A first aspect of the present invention provides a kind of GPBB near-infrared fluorescent method detection kit, comprise the reagent strip be positioned on backboard, reagent strip is followed successively by sample pad, is marked with the glass fibre of immune fluorescent probe element film, the nitrocellulose filter being coated with anti-GPBB monoclonal antibody and thieving paper from application of sample end.
Preferably, described immune fluorescent probe is the near-infrared fluorescent latex beads particle of anti-GPBB monoclonal antibody bag quilt.
Preferably, the anti-GPBB monoclonal antibody of nitrocellulose filter wrapping quilt can, for identical anti-GPBB monoclonal antibody, also can be different anti-GPBB monoclonal antibodies with the anti-GPBB monoclonal antibody of described near-infrared fluorescent latex beads particle wrapping quilt.
Preferred, the anti-GPBB monoclonal antibody of described nitrocellulose filter wrapping quilt is
anti-GPBB antibody [17B6], the anti-GPBB monoclonal antibody of described near-infrared fluorescent latex beads particle wrapping quilt is
anti-GPBB antibody [17B6], or the anti-GPBB monoclonal antibody of described nitrocellulose filter wrapping quilt is
anti-GPBB antibody [17B6], the anti-GPBB monoclonal antibody of described near-infrared fluorescent latex beads particle wrapping quilt is
anti-GPBB antibody [1G6].
Preferred, the diameter of described fluorescent latex microsphere particle is 140-160nm.
Near infrared light (NIR) is the electromagnetic wave between visible ray (VIS) and mid-infrared light (MIR), and it is the electromagnetic wave of wavelength within the scope of 780nm-2526nm that ASTM defines NIR.In near-infrared region, biosome self-absorption or fluorescence intensity are very little, can avoid background interference.Simultaneously because the biquadratic of scattered light intensity and wavelength is inverse ratio, the scattering interference of near-infrared region greatly reduces.
A second aspect of the present invention provides the preparation method of described GPBB near-infrared fluorescent method detection kit, comprises the steps:
1) with damping fluid by fluorescent latex particles eccentric cleaning and by latex particle disperse, in cleaned latex particle dispersion liquid, add anti-GPBB monoclonal antibody; Add EDCA and make antibody and particle coupling; Add confining liquid again and close group unnecessary on latex particle, prepare probe solution;
2) the buffer solution spray containing anti-GPBB monoclonal antibody is added on nitrocellulose filter, dry for standby;
3) by step 1) the probe solution sized glass fibres film for preparing, drying for standby;
4) by step 2) nitrocellulose filter, the step 3 of gained) glass fibre element film, sample pad, thieving paper and the backboard assembling being marked with immune fluorescent probe of gained is prepared into GPBB near-infrared fluorescent detection kit.
Preferably, described step 1) in, then add confining liquid and close group unnecessary on latex particle, and add crown ether, prepare probe solution, described crown ether is selected from one or more the combination in two cyclohexyl-18-hats-6,15-crown ether-5,18-crown ether-6.
Preferred, step 1) concentration of crown ether is 0.1-20mg/ml in the probe solution for preparing, is more preferably 4-8mg/ml.
Preferably, described step 1) in, damping fluid is phosphate (PBS) damping fluid.
Preferred, described PBS damping fluid is the PBS damping fluid of 0.009-0.011mol/L, PH7.25-7.35.
Preferably, described step 1) in, the diameter of fluorescent latex particles is 140-160nm.
Preferably, described step 1) in, the weight ratio of latex particle, anti-GPBB monoclonal antibody, EDCA is 9-11:0.9-1.1:0.9-1.1.
In a preferred embodiment, the inventory of latex particle, anti-GPBB monoclonal antibody, EDCA is respectively 1mg, 100ug, 100ug.
Preferably, described confining liquid is monoethanolamine, and addition is appropriate, and those skilled in the art can adjust the consumption of monoethanolamine according to actual conditions.
Preferably, described step 2) in, described buffer solution is PBS damping fluid.
Those skilled in the art can choose the PBS damping fluid of debita spissitudo and pH, are more preferably 0.009-0.011mol/L, the PBS damping fluid of PH7.25-7.35.
Preferably, described step 2) in, also containing crown ether in the buffer solution containing anti-GPBB monoclonal antibody, described crown ether is selected from one or more the combination in two cyclohexyl-18-hats-6,15-crown ether-5,18-crown ether-6.
Preferred, described is 0.1-20mg/ml containing the concentration of crown ether in the buffer solution of anti-GPBB monoclonal antibody, is more preferably 4-8mg/ml.
Preferably, described step 2) in, in the buffer solution containing anti-GPBB monoclonal antibody, the concentration of anti-GPBB monoclonal antibody is 1.8-2.2mg/ml.
Preferably, described step 2) in, the spray dosage of the buffer solution of anti-GPBB monoclonal antibody is 0.9-1.1ul/cm.
Preferably, described step 2) in, the actual conditions of oven dry is 36-38 DEG C of oven for drying.
Described spray adds process and utilizes trace of albumin point membranous system.
Preferably, described step 1) in anti-GPBB monoclonal antibody be
anti-GPBB antibody [17B6], described step 2) in anti-GPBB monoclonal antibody be
anti-GPBB antibody [17B6], or described step 1) in anti-GPBB monoclonal antibody be A
anti-GPBB antibody [17B6], described step 2) in anti-GPBB monoclonal antibody be
anti-GPBB antibody [1G6].
Preferably, described step 3) in, dry actual conditions is that vacuum drier is dry.
A third aspect of the present invention provides the purposes of described GPBB near-infrared fluorescent method detection kit at GPBB detection field.
Described purposes is specially and uses described GPBB near-infrared fluorescent method detection kit to detect the GPBB in sample, and described sample is selected from one or more the combination in people's whole blood, serum or blood plasma.
Detection kit provided by the present invention with people's whole blood/serum/plasma for detect sample, in conjunction with near-infrared fluorescent detection technique, can be quick, detect the GPBB in patient's sample accurately.Described kit adopts immunofluorescence chromatography, its most I is measured and can be reached 0.6pg/ml, easy to detect, cost is low, highly sensitive, specificity is good, 20-30% lower than similar quantitative Diagnosis reagent price, has saved social medical treatment cost greatly, and in testing process, sample collection and process are simply, result can be obtained fast and accurately, be applicable to very much accident scene and basic unit's use, and may be used in clinical detection, for clinical treatment provides qualitative foundation.
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 Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
In various embodiments of the present invention, test specimen is provided by Pudong New District the People's Hospital.Near-infrared fluorescent detector, is developed by Kaichuang Biotechnology Co. Ltd., Shanghai and provides.
Embodiment 1
1. immunofluorescence anti-GPBB antibody particle preparation:
With the phosphate buffer of 0.01mol/L, PH7.3 ± 0.05 diameter be the fluorescent latex particles eccentric cleaning 2 times of 150nm and latex particle is disperseed, adding in cleaned latex particle
anti-GPBB antibody [17B6]; Add EDCA and make antibody and particle coupling; Add confining liquid and close group unnecessary on latex particle, then add two cyclohexyl-18-hats-6.By the antibody to be marked of every milligram of microballoon latex 100 microgram, the EDCA consumption mark of 100 micrograms.The confining liquid that the microballoon latex of every milligram finally adds the monoethanolamine equivalent being equivalent to 20 micrograms is closed, and the final concentration of crown ether is 6mg/ml.
2. the preparation of kit:
Dilute with the PBS damping fluid of 0.01M pH7.2
anti-GPBB antibody [17B6] is to concentration 2.0mg/ml, and adding crown ether, the final concentration of crown ether is 6mg/ml, and solution spray is added on the nitrocellulose filter in suitable aperture by recycling trace of albumin point membranous system, carry out spray film by the amount of 1ul/cm, 37 DEG C of oven for drying are for subsequent use.
With the probe prepared (obtained being marked with of step 1
the near-infrared fluorescent latex beads of Anti-GPBB antibody [17B6]) solution impregnation glass fiber membrane, drying for standby in vacuum drier.
By bag by the nitrocellulose filter of anti-GPBB antibody, be marked with immune fluorescent probe glass fibre element film, sample pad, thieving paper and backboard assembling be prepared into GPBB near-infrared fluorescent method detection kit.
Embodiment 2
Kit detects:
With the GPBB standard items that the GPBB of genetic engineering synthesis and PBS damping fluid difference configuration concentration are 0.4pg/ml, 0.6pg/ml, 0.8pg/ml, 1pg/ml, 2pg/ml, 4pg/ml, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 300pg/ml, 400pg/ml, 500pg/ml, 1000pg/ml, using 0pg/ml as blank, the equal replication of standard items of blank and each concentration 6 times.Standard items and kit all balance and start to detect to room temperature again, drip 3 samples (about 120-150ul) with dropper in the well of each kit.When 15 minutes, detect fluorescence signal with near-infrared fluorescent detector, and to judge with fluorescent instrument, analyser be AD value 0-10000 to the sensing range of fluorescence signal.With GPBB concentration for horizontal ordinate, it is ordinate that near-infrared fluorescent detector detects fluorescence signal (mean value), Criterion curve.On typical curve, along with the rising of standard concentration, the fluorescence signal value recorded also increases.
Calculate known according to typical curve, this kit can partly detect the sample of 0.6pg/ml, all detects the sample of 1.0pg/ml, its minimum detectability≤1.0pg/ml, and has good linear in the scope of 1-1000pg/ml.
Crowd interior maximum CVs of each concentration is 4.2%, average CVs is 3.56% (sample of each concentration is tested with criticizing), each concentration batch between maximum CVs be 5.4%, average CVs shows that kit provided by the present invention has good repeatability 4.6% (sample of each concentration divides 5 batches to test).
Nd GPBB is selected to carry out recovery testu, add GPBB standard items, the concentration of GPBB is adjusted to 2pg/ml, 4pg/ml, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml respectively, the equal replication of sample of blank and each concentration 6 times.Crowd interior maximum CVs of each concentration is 4.3%, and average CVs is 3.6%, and the recovery of standard addition of each concentration is 97.5%-100.1%, shows that kit provided by the present invention has good accuracy.
GPBB near-infrared fluorescent method detection kit sealing provided by the present invention is preserved, and under depositing in 4 DEG C and room temperature (about 25 DEG C), observes the impact of different storage temperature on stabilization of kit.The kit being stored in 4 DEG C takes out weekly 4 boxes, detects the GPBB standard items of 0.6pg/ml, 0.8pg/ml, 1.0pg/ml, 1.5pg/ml respectively; The kit being stored in room temperature (25 DEG C) takes out 4 boxes in every 3 days, detects the GPBB standard items of 0.6pg/ml, 0.8pg/ml, 1.0pg/ml, 1.5pg/ml respectively.Empirical tests, paper box can preserve 24 months at 4 DEG C, and minimum detectability and repeatability do not have significant change, at room temperature can preserve 18 months, and minimum detectability and repeatability do not have significant change; Within the preservable time limit, kit all can reach the detection sensitivity of 1.0pg/ml.
In sum, the present invention 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 (9)
1. a GPBB near-infrared fluorescent method detection kit, comprise the reagent strip be positioned on backboard, reagent strip is followed successively by sample pad, is marked with the glass fibre of immune fluorescent probe element film, the nitrocellulose filter being coated with anti-GPBB monoclonal antibody and thieving paper from application of sample end, and described immune fluorescent probe is the near-infrared fluorescent latex beads particle of anti-GPBB monoclonal antibody bag quilt.
2. detection kit according to claim 1, it is characterized in that, described nitrocellulose filter wrapping the anti-GPBB monoclonal antibody of the anti-GPBB monoclonal antibody of quilt and described near-infrared fluorescent latex beads particle wrapping quilt is identical anti-GPBB monoclonal antibody, or is different anti-GPBB monoclonal antibodies.
3. detection kit according to claim 1, is characterized in that, the anti-GPBB monoclonal antibody of described nitrocellulose filter wrapping quilt is
anti-GPBB antibody [17B6], the anti-GPBB monoclonal antibody of described near-infrared fluorescent latex beads particle wrapping quilt is
anti-GPBB antibody [17B6], or the anti-GPBB monoclonal antibody of described nitrocellulose filter wrapping quilt is
anti-GPBB antibody [17B6], the anti-GPBB monoclonal antibody of described near-infrared fluorescent latex beads particle wrapping quilt is
anti-GPBB antibody [1G6].
4. detection kit according to claim 1, is characterized in that, the diameter of described fluorescent latex microsphere particle is 140-160nm.
5. the preparation method of the detection kit according to the arbitrary claim of claim 1-4, comprises the steps:
1) with damping fluid by fluorescent latex particles eccentric cleaning and by latex particle disperse, in cleaned latex particle dispersion liquid, add anti-GPBB monoclonal antibody; Add EDCA and make antibody and particle coupling; Add confining liquid again and close group unnecessary on latex particle, prepare probe solution;
2) the buffer solution spray containing anti-GPBB monoclonal antibody is added on nitrocellulose filter, dry for standby;
3) by step 1) the probe solution sized glass fibres film for preparing, drying for standby;
4) by step 2) nitrocellulose filter, the step 3 of gained) glass fibre element film, sample pad, thieving paper and the backboard assembling being marked with immune fluorescent probe of gained is prepared into GPBB near-infrared fluorescent detection kit.
6. preparation method according to claim 5, it is characterized in that, described step 1) in, add confining liquid again and close group unnecessary on latex particle, and add crown ether, prepare probe solution, described crown ether is selected from one or more the combination in two cyclohexyl-18-hats-6,15-crown ether-5,18-crown ether-6;
And/or, step 1) concentration of crown ether is 0.1-20mg/ml in the probe solution for preparing;
And/or, described step 1) in, damping fluid is PBS damping fluid;
And/or, described step 1) in, the weight ratio of latex particle, anti-GPBB monoclonal antibody, EDCA is 9-11:0.9-1.1:0.9-1.1;
And/or described confining liquid is monoethanolamine.
7. preparation method according to claim 5, it is characterized in that, described step 2) in, also containing crown ether in buffer solution containing anti-GPBB monoclonal antibody, described crown ether is selected from one or more the combination in two cyclohexyl-18-hats-6,15-crown ether-5,18-crown ether-6;
And/or described is 0.1-20mg/ml containing the concentration of crown ether in the buffer solution of anti-GPBB monoclonal antibody;
And/or the concentration of anti-GPBB monoclonal antibody is 1.8-2.2mg/ml in the buffer solution containing anti-GPBB monoclonal antibody;
And/or the spray dosage of the buffer solution of anti-GPBB monoclonal antibody is 0.9-1.1ul/cm;
And/or, described step 2) in, described buffer solution is PBS damping fluid.
8. the detection kit according to the arbitrary claim of claim 1-4 is in the purposes of GPBB detection field.
9. purposes according to claim 8, it is characterized in that, described purposes is specially and uses described GPBB near-infrared fluorescent method detection kit to detect the GPBB in sample, and described sample is selected from one or more the combination in people's whole blood, serum or blood plasma.
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Cited By (1)
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| CN110794135A (en) * | 2019-10-31 | 2020-02-14 | 安徽大千生物工程有限公司 | GPBB latex enhanced turbidimetry detection kit and preparation and use methods thereof |
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