CN103941007A - Immunofluorescence test strip for fast and quantitatively detecting curative effect of aspirin and preparation method of immunofluorescence test strip - Google Patents
Immunofluorescence test strip for fast and quantitatively detecting curative effect of aspirin and preparation method of immunofluorescence test strip Download PDFInfo
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- CN103941007A CN103941007A CN201410121800.9A CN201410121800A CN103941007A CN 103941007 A CN103941007 A CN 103941007A CN 201410121800 A CN201410121800 A CN 201410121800A CN 103941007 A CN103941007 A CN 103941007A
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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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
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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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
Abstract
The invention discloses an immunofluorescence test strip for fast and quantitatively detecting the curative effect of aspirin and a preparation method of the immunofluorescence test strip. The immunofluorescence test strip comprises a support piece as well as a sample gasket, a detection film and a water absorption gasket, which are sequentially overlapped and pasted on the support piece, wherein a conjugate gasket is arranged between the sample gasket and the detection film; a first layer of glass fiber gasket is arranged above one end of the conjugate gasket and a second layer of glass fiber gasket is arranged below one end of the conjugate gasket or only the first layer of glass fiber gasket is arranged above one end of the conjugate gasket or no gasket is arranged; a detection line is arranged on the detection film; the detection line is coated with a 11-dehydro thromboxane B2 (11dhTxB2) monoclonal antibody or polyclonal antibody; a control line is arranged on the other side of the detection line; the control line is coated with an anti-streptavidin (SAV) antibody; the conjugate gasket is coated with fluorescently labeled 11dhTxB2 conjugate. The immunofluorescence test strip is convenient, efficient, simple to operate, accurate in result and suitable for rapid clinical diagnosis.
Description
Technical field
The invention belongs to clinical diagnose field, be specifically related to a kind of for quantitatively detecting Immunofluorescence test paper strip of aspirin and preparation method thereof.
Background technology
Aspirin (Aspirin) is the widely used auxiliary prescription that prevents angiocardiopathy, because it can suppress the generation of thromboxane A2 (TxA2), TxA2 is vasoconstriction effectively, promotes platelet aggregation.Although aspirin is consumed by people up to a million in the whole world every year, nearest studies show that be not all individualities be all consistent for the reaction of aspirin.Studies show that, when coronary heart disease and relevant patient use blood basis platelet aggregation mode and urine 11dhTxB2 to measure, its cholesterol, triglyceride and c reactive protein level can reduce for the reaction of aspirin.Compared with asymptomatic patient, after patients with coronary heart disease Aspirin, demonstrate significantly higher urine 11dhTxB2 level.
Due to the reaction of aspirin difference to some extent, need a kind of reliable mode to analyze the individual idiosyncrasy to aspirin, help to determine whether to change treatment measure.Two basic modes of existing mensuration therapeutic effect of aspirin: platelet aggregation method and urine 11dhTxB2 determination method.Platelet aggregation method has a lot of forms, generally comprises the collection of blood samples of patients sample, the mensuration of aggegation in halfbody.Although these detections are relatively very fast, they are very sensitive for variable, and these variablees are often irrelevant with therapeutic effect of aspirin again, as warm Wei Baishi factor level, and Factor IX, and hematocrit value pipe, the fact is that hematoblastic activation only needs to regulate temperature.In addition, other platelet aggregation mode set needs special equipment, has limited doctor and move in a trace routine ability of great amount of samples.Due to these reasons, the metabolin of directly measuring TxA2 is individual more suitable to the reaction of aspirin for determining.Unfortunately, the life-span of TxA2 in blood 1 is very short, analyze like this TxA2 just very difficult, but it can resolve into a lot of metabolins and comprise 11-dehydrogenation thromboxane B2 (11dhTxB2) and 11-dehydrogenation-2 under the effect of enzyme, 3-dinor thromboxane B2 (11dh2,3DTxB2), they are absorbed by kidney, and excretion is in urine.11dhTxB2 is highly stable in urine, is TxB2 the abundantest metabolin in urine, and it has 45 minutes relatively long cycling time.And compared with measuring TxA2 in blood, for thrombus in vivo element, metabolism provides instruction more accurately to urine 11dhTxB2 level.
Immunofluorescence technique (Fluorescence Immunoassay technique) is a kind of novel immunolabelling technique that is applied to antigen-antibody using fluorescence molecule as tracer label thing.Biotin-avidin system (Biotin-Avidin-System, BAS) is a kind of very effective biological respinse amplification system.Biotin-avidin system almost can be combined with the successful various labels of current research.Strong bonded and the multistage enlarge-effect of biotin-affinity element and labelled reagent high affinity, make that BAS is immune labeled and relevant tracer analysis is sensitiveer.It has become the new technology that is widely used at present trace antigen, antibody qualitative and quantitative analysis and position observation research.The huge superiority that BAS had in actual applications, is mainly manifested in the following aspects:
(1) biotin is easily combined with the biomacromolecule such as protein and nucleic acid, and the biotin derivative of formation has not only kept original biologically active of macromolecular substances, and higher than quiet degree, tool polyvalency.Therefore, BAS has multistage amplification, the sensitivity that makes it can greatly improve detection method in the time of application.
(2) combination between Avidin and biotin has high affinity, and its reaction is height selectivity.Therefore, the multi-level amplification of BAS, carrying the highly sensitive while, does not increase nonspecific interference.And BAS binding characteristic can be not influenced because of the high dilution of reaction reagent, make it can reduce to greatest extent in actual applications the non-specific effect of reaction reagent.
(3) Avidin can be 1,000,000 times of antigen-antibody reaction in conjunction with the affinity costant of biotin, and the two is very little in conjunction with the dissociation constant that forms compound, is non-reversible reaction; And acid, alkali, denaturant, protein resolvase and organic solvent all do not affect its combination.Therefore, in actual applications, the stability of product is high for BAS, improves the degree of accuracy of measuring.(4) biotin and Avidin all can be made into multiple derivant, not only can be combined with all kinds of labelling techniques such as enzyme, fluorescein and radioactive nuclides, for detection of the Ag-Ab in body fluid, tissue or cell, hormone-acceptor and nucleic acid system and other various biological reaction systems, and can be made into affinity media, for separating of the reactant of purifying in above-mentioned each reaction system.
At present, the method that detects 11-dehydrogenation thromboxane (11dhTxB2) is mainly Enzyme-multiplied immune technique (ELISA) at present, and elisa technique exists following shortcoming: checkout equipment requires high, and cost is high; Disturbing factor is more, and repeatability is bad; Detection time is long.Therefore Enzyme-multiplied immune technique detects 11dhTxB2 and is not suitable for clinical quick diagnosis.How can produce quantitative checkout equipment fast and become the problem that needs urgent solution.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of Immunofluorescence test paper strip of energy Quantitative detection therapeutic effect of aspirin, and another object of the present invention is to provide the preparation method of this Immunofluorescence test paper strip.
In order to solve the problems of the technologies described above, the invention provides a kind of Immunofluorescence test paper strip of Quantitative detection aspirin drug effect, it comprises the sample pad that on test strips support chip and test strips support chip, overlap joint is pasted in turn, detect film and water suction pad, between described sample pad and detection film, be provided with conjugates pad, top, conjugates pad one end is provided with ground floor glass fibre pad, its below, one end is provided with second layer glass fibre pad or top, conjugates pad one end is only provided with ground floor glass fibre pad or is not provided with arbitrary pad, described detection film is provided with detection line, described detection line is coated with 11-dehydrogenation thromboxane B2 (11dhTxB2) monoclonal antibody or polyclonal antibody, described detection line another side is provided with control line, control line is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad, be coated with fluorescently-labeled 11dhTxB2 conjugates.
As a further improvement on the present invention, in the described pad of thing once in a while, the preparation of conjugates comprises the steps:
(1) preparation of Biotin-X-11dhTxB2:
(1.1) preparation of Biotin-11dhTxB2
11dhTxB2 is dissolved in phosphate buffer (PBS), the biotin (Biotin) of activation is dissolved in its activation grade of guarantee in dimethyl sulfoxide (DMSO) (DMSO), then the 11dhTxB2 being dissolved in phosphate buffer (PBS) is added in biotin (Biotin) solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number;
(1.2) preparation of Biotin-X-11dhTxB2:
First respectively bovine serum albumin(BSA) (BSA) is dissolved in to phosphate buffer (PBS), and 11dhTxB2 is dissolved in dimethyl formamide (DMF), then according to a certain percentage bovine serum albumin(BSA) (BSA) and 11dhTxB2 mixing are hatched, after reaction finishes, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with bovine serum albumin(BSA) (BSA) mean number; Subsequently, by a certain percentage the biotin (Biotin) in dimethyl sulfoxide (DMSO) (DMSO) of being dissolved in of activation is joined in BSA-11dhTxB2 stoste and reacted, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number; Above-mentioned X is bovine serum albumin(BSA) (BSA), and the available ovalbumin of bovine serum albumin(BSA) (BSA) (OVA) or keyhole limpet hemocyanin (KLH) replace;
(2) preparation of fluorescence molecule-SAV:
Anti-Streptavidin (SAV) powder is dissolved in activation in phosphate buffer (PBS), then add in fluorescence molecule powder and react being dissolved in anti-Streptavidin (SAV) in phosphate buffer (PBS), after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form fluorescence molecule-SAV stoste, and calculate each fluorescence molecule may in conjunction with the mean number of anti-Streptavidin (SAV);
(3) preparation of fluorescently-labeled 11dhTxB2 conjugates:
By Biotin-X-11dhTxB2 or the dilution of Biotin-11dhTxB2 stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, and under suitable condition, stop reaction.
The present invention also provides a kind of preparation method of Immunofluorescence test paper strip of Quantitative detection aspirin drug effect, comprises the steps:
Step 1: prepare detection line; Detection film for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line is anti-11dhTxB2 antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film, then will detect film be fully dried and toast a few days after and get final product.
Step 2: prepare control line; Described control line is anti-SAV antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film, then will detect film be fully dried and toast a few days after and get final product.
Step 3: prepare conjugates pad; The starting material of described conjugates pad are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad after pre-sealing damping fluid soaks, after fully dry, fluorescently-labeled-11dhTxB2 conjugates is coated on conjugates pad, is fully drying to obtain with coating instrument.
Step 4: prepare sample pad; Described sample pad can play preliminary filtering function by liquid towards sample, after sample pad is soaked with confining liquid, is fully drying to obtain;
Step 5: the assembling of test strips;
From bottom to top adhere to successively and detect film, water suction pad and conjugates pad at test strips support chip, on conjugates pad, be provided with ground floor glass fibre pad, second layer glass fibre pad or only establish ground floor glass fibre pad or be not provided with arbitrary pad, last layer is sample pad;
By after above-mentioned material assembling, be cut into little, i.e. the Immunofluorescence test paper strip of the described quantitative detection aspirin drug effect of system.
Adopt as above after technical scheme, its beneficial effect is:
The principle of work of the Immunofluorescence test paper strip of Quantitative detection therapeutic effect of aspirin of the present invention is: adopt immune effluent reaction principle, logical competition law is prepared from.Fluorescently-labeled 11dhTxB2 conjugates on 11dhTxB2 when inspection in sample and coupling pad is detecting chromatographic flow on film simultaneously, when the 11dhTxB2 in sample and fluorescently-labeled 11dhTxB2 conjugates chromatography to detect on film detection zone (11dhTxB2 detection line) time, thereby be coated in advance the anti-11dhTxB2 antibody detecting on film and react and form immune complex and be fixed on the detection line that detects film.11dhTxB2 in sample is more, and the fluorescently-labeled 11dhTxB2 conjugates being fixed on detection line is fewer, and the optical density value on band is just lower.Simultaneously, in testing process, unconjugated fluorescently-labeled 11dhTxB2 conjugates also can detect chromatography on film with sample, when chromatography is when detecting the control line of film, thus fluorescently-labeled 11dhTxB2 can be coated in advance the anti-SAV antibody detecting on film and react and be fixed on check plot (control line).The optical density value of the fluorescence relation that is inversely proportional in 11dhTxB2 concentration and detection line in sample.
After reaction finishes, utilize detector that the optical density of control line and detection line is analyzed, and the analyze result obtaining is carried out to computing, thereby obtain relative optical density value (RI).Then detector calculates the concentration of 11dhTxB2 and shows result according to the typical curve having set in advance in detector, and taking pg/mL as unit representation, its reaction result is as shown in table 1 below:
Table 1:
In sum, the Immunofluorescence test paper strip of Quantitative detection therapeutic effect of aspirin of the present invention, this test paper has the advantages such as convenient and swift, simple to operate, result is accurate, is suitable for clinical quick diagnosis.
Brief description of the drawings
Fig. 1 is the structural representation of the Immunofluorescence test paper strip of Quantitative detection therapeutic effect of aspirin of the present invention.
Wherein: 1-sample pad, 2-detect film, 3-water suction pad, 4-support chip, 5 conjugates pads, 6-1 ground floor glass fibre pad, 6-2 second layer glass fibre pad, 7-detection line, 8-control line.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail, can not be interpreted as it is limitation of the present invention;
Embodiment 1:
Prepare the Immunofluorescence test paper strip of Quantitative detection therapeutic effect of aspirin
Immunofluorescence test paper strip structure as shown in Figure 1, a kind of Immunofluorescence test paper strip of Quantitative detection aspirin drug effect, it comprises the sample pad 1 that on test strips support chip 4 and test strips support chip, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its below, one end is provided with second layer glass fibre pad 6-2 or conjugates pad 5 tops, one end are only provided with ground floor glass fibre pad 6-1 or are not provided with arbitrary pad, described detection film 2 is provided with detection line 7, described detection line 7 is coated with 11-dehydrogenation thromboxane B2 (11dhTxB2) monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescently-labeled 11dhTxB2 conjugates.
In thing pad 5, the preparation method of conjugates is once in a while:
The preparation of small-molecular peptides (fluorescence molecule-SAV-Biotin-X-11dhTxB2) conjugates of immunofluorescence:
(1) preparation of Biotin-X-11dhTxB2:
(1.1) preparation of Biotin-11dhTxB2
11dhTxB2 is dissolved in phosphate buffer (PBS), the biotin (Biotin) of activation is dissolved in its activation grade of guarantee in dimethyl sulfoxide (DMSO) (DMSO), then the 11dhTxB2 being dissolved in phosphate buffer (PBS) is added in biotin (Biotin) solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number;
(1.2) preparation of Biotin-X-11dhTxB2:
First respectively bovine serum albumin(BSA) (BSA) is dissolved in to phosphate buffer (PBS), and 11dhTxB2 is dissolved in dimethyl formamide (DMF), then according to a certain percentage bovine serum albumin(BSA) (BSA) and 11dhTxB2 mixing are hatched, after reaction finishes, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with bovine serum albumin(BSA) (BSA) mean number; Subsequently, by a certain percentage the biotin (Biotin) in dimethyl sulfoxide (DMSO) (DMSO) of being dissolved in of activation is joined in BSA-11dhTxB2 stoste and reacted, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number; Above-mentioned X is bovine serum albumin(BSA) (BSA), and the available ovalbumin of bovine serum albumin(BSA) (BSA) (OVA) or keyhole limpet hemocyanin (KLH) replace;
(2) preparation of fluorescence molecule-SAV:
Anti-Streptavidin (SAV) powder is dissolved in activation in phosphate buffer (PBS), then add in fluorescence molecule powder and react being dissolved in anti-Streptavidin (SAV) in phosphate buffer (PBS), after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form fluorescence molecule-SAV stoste, and calculate each fluorescence molecule may in conjunction with the mean number of anti-Streptavidin (SAV);
(3) preparation of fluorescently-labeled 11dhTxB2 conjugates:
By Biotin-X-11dhTxB2 or the dilution of Biotin-11dhTxB2 stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, and under suitable condition, stop reaction;
Wherein, detection film 2 for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line 7 is anti-11dhTxB2 antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, gets 1ul/cm to line on described detection film 2, will detect film 2 and fully be dried and baking a couple of days, to obtain final product; Control line 8 is anti-SAV antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film 2, will detect film 2 and fully be dried and baking a couple of days, to obtain final product.
Wherein, the starting material of described conjugates pad 5 are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad after pre-sealing damping fluid soaks, after dry, fluorescently-labeled 11dhTxB2 conjugates is coated on conjugates pad 5 with coating instrument, dry, to obtain final product.
Wherein, described sample pad 1 can play preliminary filtering function by liquid towards sample.After sample pad is soaked with confining liquid, dry, to obtain final product.
Above-mentioned each building block is sticked in support pad 4 by structure shown in Fig. 1, obtain the Immunofluorescence test paper strip of Quantitative detection therapeutic effect of aspirin.
Embodiment 2:
With Immunofluorescence test paper strip Quantitative detection therapeutic effect of aspirin of the present invention
Get the Immunofluorescence test paper strip of the preferred a kind of Quantitative detection therapeutic effect of aspirin of the present invention, Immunofluorescence test paper strip structure as shown in Figure 1, a kind of Immunofluorescence test paper strip of Quantitative detection aspirin drug effect, it comprises the sample pad 1 that on test strips support chip 4 and test strips support chip, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its below, one end is provided with second layer glass fibre pad 6-2 or conjugates pad 5 tops, one end are only provided with ground floor glass fibre pad 6-1 or are not provided with arbitrary pad, described detection film 2 is provided with detection line 7, described detection line 7 is coated with 11-dehydrogenation thromboxane B2 (11dhTxB2) monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescently-labeled 11dhTxB2 conjugates.
Detect according to the following steps: the patient urine sample 1) extracting, preserve sample as low temperature and need return to room temperature.2) testing sample is added on sample pad 1 to reaction.3) interpretation, by described Immunofluorescence test paper strip be placed in (Rui Lai) immunofluorescence detector with and pertinent instruments result of determination.Its result is as shown in the table:
Claims (3)
1. the Immunofluorescence test paper strip of a Quantitative detection aspirin drug effect, it comprises the sample pad (1) that on test strips support chip (4) and test strips support chip, overlap joint is pasted in turn, detect film (2) and water suction pad (3), between described sample pad (1) and detection film (2), be provided with conjugates pad (5), top, conjugates pad (5) one end is provided with ground floor glass fibre pad (6-1), its below, one end is provided with second layer glass fibre pad (6-2) or top, conjugates pad (5) one end is only provided with ground floor glass fibre pad (6-1) or is not provided with arbitrary pad, it is characterized in that: described detection film (2) is provided with detection line (7), described detection line (7) is coated with 11-dehydrogenation thromboxane B2 (11dhTxB2) monoclonal antibody or polyclonal antibody, described detection line (7) another side is provided with control line (8), control line (8) is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad (5), be coated with fluorescently-labeled 11dhTxB2 conjugates.
2. the Immunofluorescence test paper strip of Quantitative detection aspirin drug effect according to claim 1, is characterized in that: in the described pad of thing once in a while (5), the preparation of fluorescently-labeled 11dhTxB2 conjugates comprises the steps:
(1) preparation of Biotin-X-11dhTxB2:
(1.1) preparation of Biotin-11dhTxB2:
11dhTxB2 is dissolved in phosphate buffer (PBS), the biotin (Biotin) of activation is dissolved in by a certain percentage by its activation grade of guarantee in dimethyl sulfoxide (DMSO) (DMSO) that is dissolved in of activation, then the 11dhTxB2 being dissolved in phosphate buffer (PBS) is added in biotin (Biotin) solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number;
(1.2) preparation of Biotin-X-11dhTxB2:
First respectively bovine serum albumin(BSA) (BSA) is dissolved in to phosphate buffer (PBS), and 11dhTxB2 is dissolved in dimethyl formamide (DMF), then according to a certain percentage bovine serum albumin(BSA) (BSA) and 11-dehydrogenation thromboxane B2 (11dhTxB2) mixing are hatched, after reaction finishes, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with bovine serum albumin(BSA) (BSA) mean number; Subsequently, by a certain percentage the biotin (Biotin) in dimethyl sulfoxide (DMSO) (DMSO) of being dissolved in of activation is joined in BSA-11dhTxB2 stoste and reacted, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-BSA-11dhTxB2 stoste, and calculate each 11dhTxB2 molecule may in conjunction with biotin (Biotin) mean number, above-mentioned X is bovine serum albumin(BSA) (BSA), and the available ovalbumin of bovine serum albumin(BSA) (BSA) (OVA) or keyhole limpet hemocyanin (KLH) replace;
(2) preparation of fluorescence molecule-SAV:
Streptavidin (SAV) powder is dissolved in activation in phosphate buffer (PBS), then add in fluorescence molecule powder and react being dissolved in Streptavidin (SAV) in phosphate buffer (PBS), after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form fluorescence molecule-SAV stoste, and calculate each fluorescence molecule may in conjunction with the mean number of SAV;
(3) preparation of fluorescently-labeled 11dhTxB2 conjugates:
By Biotin-X-11dhTxB2 or the dilution of Biotin-11dhTxB2 stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, and under suitable condition, stop reaction, form fluorescently-labeled 11dhTxB2 conjugates.
3. a preparation method for the Immunofluorescence test paper strip of Quantitative detection aspirin drug effect, is characterized in that: comprise the steps:
Step 1: prepare detection line; Detection film (2) for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line (7) is anti-11dhTxB2 antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines described detection film (2) upper, then will detect film (2) be fully dried and toast a few days after and get final product;
Step 2: prepare control line; Described control line (8) is anti-SAV antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines described detection film (2) upper, then will detect film (2) be fully dried and toast a few days after and get final product;
Step 3: prepare conjugates pad; The starting material of described conjugates pad (5) are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad (5) after pre-sealing damping fluid soaks, after fully dry, with coating instrument, fluorescently-labeled-11dhTxB2 conjugates is coated on to conjugates pad (5) upper, is fully drying to obtain;
Step 4: prepare sample pad; Described sample pad (1) can play preliminary filtering function by liquid towards sample, after sample pad is soaked with confining liquid, is fully drying to obtain;
Step 5: the assembling of test strips;
From bottom to top adhere to successively and detect film (2), water suction pad (3) and conjugates pad (5) at test strips support chip (4), on conjugates pad (5), be provided with ground floor glass fibre pad (6-1), second layer glass fibre pad (6-2) or only establish ground floor glass fibre pad (6-1) or be not provided with arbitrary pad, last layer is sample pad (1);
By after above-mentioned material assembling, be cut into little, i.e. the Immunofluorescence test paper strip of the described quantitative detection aspirin drug effect of system.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792982A (en) * | 2015-03-10 | 2015-07-22 | 山东盛百灵医药科技有限公司 | Aspirin drug resistance monitoring reagent |
| CN105974106A (en) * | 2016-05-04 | 2016-09-28 | 山东盛百灵医药科技有限公司 | 11-Dehydro-thromboxane B2 determination kit and use thereof |
| CN107941954A (en) * | 2017-11-30 | 2018-04-20 | 菏泽惠泽临床试验研究中心 | A kind of Monitoring of drug resistance method of aspirin |
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| CN110713519A (en) * | 2019-11-02 | 2020-01-21 | 深圳市安帝宝科技有限公司 | 11-dehydrothromboxane B2 antigen and preparation method thereof |
| CN110672838A (en) * | 2019-12-04 | 2020-01-10 | 湖南新开源菲思特精准医疗科技有限公司 | Kit and method for detecting aspirin metabolism and platelet high reactivity |
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