CN204228719U - Cardiac muscle troponin I-myoglobins-creatine kinase isozyme joint inspection test strips - Google Patents
Cardiac muscle troponin I-myoglobins-creatine kinase isozyme joint inspection test strips Download PDFInfo
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Abstract
The one that provides the utility model quantitatively detects cardiac muscle troponin I-myoglobins-creatine kinase isozyme joint inspection test strips, described test strips comprises base plate and on base plate, overlaps the sample pad of stickup successively, first pad, second pad, 3rd pad, coated film and thieving paper, described first pad is coated with simultaneously the cardiac troponin second antibody of the cardiac muscle troponin I first antibody epi-position different from biotin labeled identification of two kinds of different-grain diameter fluorescent microsphere marks, described second pad is coated with the myoglobins first antibody of two kinds of different-grain diameter fluorescent microsphere marks, described 3rd pad is coated with the creatine kinase isozyme first antibody of two kinds of different-grain diameter fluorescent microsphere marks.The advantages such as test strips of the present utility model can realize three joint inspections of cardiac muscle troponin I-myoglobins-creatine kinase isozyme, and has highly sensitive, easy to use.
Description
Technical field
The utility model relates to a kind of three joint inspection fluorescent chromatographic test strips for quantitatively detecting cardiac muscle troponin I-myoglobins-creatine kinase isozyme.
Background technology
Cardiac muscle troponin I (Cardiac Troponin I, be called for short cTnI) be the special and responsive mark of cardiomyocyte cell death, it can enter in blood when myocardial damage, and do not find health or express in the skeletal muscle of damaged or its hetero-organization.CTnI can be detected in 3 to 6 hours after episode in blood samples of patients, and reaches peak value in 16 to 30 hours, in blood sample the rising of cTnI concentration can continue until symptom occur after 5 to 8 days.At present, cTnI as the label widespread use in clinical diagnosis of cardiomyocyte cell death, as: the instruction to thromboembolism treatment after the diagnosis of myocardial damage, AMI, the diagnosis to peri-operative myocardial infarction, to myocarditic diagnosis and and the antidiastole etc. of Skeletal muscle injury.
Myoglobins (Myoglobin is called for short Myo) is a small protein be made up of 153 amino acid containing protoheme, is responsible for the storage of oxygen in musculature.As the label of myocardial damage, myoglobins is as being employed for three more than ten years.Myoglobins comes across in its blood after patients symptomatic occurs 1 to 3 hours, reaches peak value within 8 to 12 hours.Compared with cardiac muscle troponin I (cTnI), myoglobins lacks Cardiac-specific.Because myoglobins is present in skeletal muscle tissue in a large number, even if slight Skeletal muscle injury can cause the remarkable rising of myoglobin concentration in blood.Therefore, myoglobins is used in clinical practice to improve the specificity of Diagnosis of Acute Myocardial Infarction together with cTnI.
Creatine kinase (Creatine Kinase is called for short CK) is mainly present in skeletal muscle, cardiac muscle and brain tissue, triformed isodynamic enzyme: CK-MM, CK-MB and CK-BB.CK and isodynamic enzyme CK-MB thereof is the Enzyme target that clinical diagnosis acute myocardial infarction AMI is conventional, and the time utilizing it to increase and gradient observe the change of illness state of patient, result for the treatment of and more after.Creatine kinase isozyme CK-MB is mainly present in human body cardiac muscle cell, and in normal human serum, the overwhelming majority is the activity of CK-MM, only has a small amount of CK-MB, and CK-MB accounts for CK total activity and is less than 5%.When the reason such as inflammation or infarct causes myocardial cell damage or necrosis, make a large amount of CK-MB be discharged in blood, the CK-MB in serum can be made to increase.Generally, CK-MB increases in 4-8 hour after acute myocardial infarction, within 24 hours, reaches peak value, recovers normal after 2-3 days.CK-MB is as conventional myocardial injury markers widespread use in clinical detection.
Utility model content
technical matters
In view of the difference of cTnI, Myo and CK-MB release dynamics after myocardial damage, cTnI, Myo and C-MB tri-joint inspections have cooperative compensating effect for the judgement of acute myocardial infarction AMI, detect than independent cTnI and there is the carly fruit drop more contributing to acute myocardial infarction AMI, thus improve the diagnosis of heart stalk and make corresponding therapeutic scheme.Therefore, three the joint inspection fluorescent chromatographic test strips developing a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme are urgently needed.
technical scheme
The utility model provides a kind of cardiac muscle troponin I-myoglobins-creatine kinase isozyme joint inspection test strips, and it is characterized in that, described test strips comprises:
1) for accepting the sample pad from whole blood, blood plasma or blood serum sample;
2) be coated with the first pad of the cardiac troponin second antibody of the cardiac muscle troponin I first antibody epi-position different from biotin labeled identification of two kinds of different-grain diameter fluorescent microsphere marks simultaneously;
3) the second pad of the myoglobins first antibody of two kinds of different-grain diameter fluorescent microsphere marks is coated with;
4) the 3rd pad of the creatine kinase isozyme first antibody of two kinds of different-grain diameter fluorescent microsphere marks is coated with;
5) coated film of three detection lines separately and a nature controlling line is coated with, wherein said three detection lines are fixed with Avidin successively, identify the myoglobins second antibody of different epi-position and identify the creatine kinase isozyme second antibody of different epi-position, and described nature controlling line is fixed with the antibody of against murine IgG;
6) for providing the thieving paper of the capillary force needed for detection; With
7) base plate, wherein said base plate overlaps successively the sample pad of stickup, the first pad, the second pad, the 3rd pad, coated film and thieving paper.
In one embodiment, the particle size range of described two kinds of different-grain diameter fluorescent microspheres is all between 50nm to 500nm.
In one embodiment, described first pad, the second pad, the 3rd pad material are glass fibre element film or polyester film.
In one embodiment, described coated film is nitrocellulose filter.
In one embodiment, the width of described test strips is between 0.2cm to 0.5cm.
beneficial effect
Three joint inspection fluorescent chromatographic test strips of a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme provided by the utility model have taken into account the range of linearity of low value sensitivity and detection, its judgement for acute myocardial infarction AMI has cooperative compensating effect, detects have the carly fruit drop more contributing to acute myocardial infarction AMI than individual event.In addition, this reagent strip preparation method is simple, can realize batch production, be conducive to large-scale promotion application, possess wide market outlook.
Accompanying drawing explanation
Fig. 1 is the schematic side view of three joint inspection fluorescent chromatographic test strips of a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme described in the utility model;
Fig. 2 is the schematic top plan view of three joint inspection fluorescent chromatographic test strips of a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme described in the utility model;
Reference numeral is expressed as: 1-sample pad, 2-first pad, 3 second pads, 4-the 3rd pad, 5-coated film, 6-thieving paper, 7-base plate, 8-detection line (cTnI), 9-detection line (Myo), 10-detection line (CK-MB) and 11-nature controlling line.
Embodiment
In order to promote that the principle for cardiac muscle troponin I of the present utility model-myoglobins-creatine kinase isozyme three joint inspections is understood, hereinafter with reference to illustrating in the accompanying drawings and being described in the embodiment in the following description book.Should understand and be not intended to limit scope of the present utility model thus.
Fig. 1 is a kind of schematic side view of three joint inspection fluorescent chromatographic test strips of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme.
As shown in Figure 1, three joint inspection fluorescent chromatographic test strips of the present utility model comprise base plate 6 and order overlap joint bonds on base plate 7 sample pad 1, first pad 2, second pad 3, the 3rd pad 4, coated film 5 and water accepting layer 6.
Sample pad 1 for accepting from whole blood, blood plasma or blood serum sample, its can by being selected from glass fibre element film or polyester film, cotton fine material makes.Sample pad 1 can play the graininess insolubles in filtered sample.Therefore, compared with commercially available part test strips, adopt being designed with of independent sample pad 1 to help improve detection sensitivity.
First pad 2, second pad 3 and the 3rd pad 4 are made up of glass fibre element film or polyester film.First pad 2 is coated with simultaneously the cardiac troponin second antibody of the cardiac muscle troponin I first antibody epi-position different from biotin labeled identification of two kinds of different-grain diameter fluorescent microsphere marks.Second pad 2 is coated with the myoglobins first antibody of two kinds of different-grain diameter fluorescent microsphere marks.3rd pad 3 is coated with the creatine kinase isozyme first antibody of two kinds of different-grain diameter fluorescent microsphere marks.Antibody for different target albumen is coated in respectively on different pads, influencing each other between antibody can be effectively reduced, which thereby enhance the sensitivity of detection.
In test strips of the present utility model, adopt double-antibody sandwich to detect in conjunction with the method for biotin-avidin Cascaded amplification to the detection of cardiac troponin, adopt the method for double-antibody sandwich to detect to the detection of myoglobins and creatine kinase isozyme.In double antibody sandwich method, use simultaneously and identify that two kinds of antibody of the different epi-position of target protein carry out specific binding target protein respectively, which thereby enhance the sensitivity of detection.
Further, the antibody of small particle diameter fluorescent microsphere mark makes the range of linearity detected can cover high level region, and the antibody of Large stone fluorescent microsphere mark is conducive to the detection of low value to reach the requirement of sensitivity.In one embodiment, the particle size range of described two kinds of different-grain diameter fluorescent microspheres, all between 50nm to 500nm, between preferred 100nm to 400nm, more preferably between 100nm to 300nm, is most preferably respectively 100nm and 300nm.Therefore, reagent strip of the present utility model can meet cardiac muscle troponin I, myoglobins and creatine kinase isozyme simultaneously and measures requirement to high sensitivity and the wide detection range of linearity.
Coated film 5 is coated with three detection lines separately and a nature controlling line.Article three, detection line 8,9 and 10 (see Fig. 2) is fixed with Avidin successively, identifies the myoglobins second antibody of different epi-position and identifies the creatine kinase isozyme second antibody of different epi-position.Nature controlling line 11 is fixed with the antibody of against murine IgG.
Detection line 8, distance between 9 and 10 can between 0.1cm to 0.6cm.In one embodiment, detection line 8, distance between 9 and 10 between 0.2cm to 0.5cm, preferably between 0.2cm to 0.4cm.Distance between detection line 10 and nature controlling line 11 can between 0.05cm to 0.8cm.In one embodiment, the distance between detection line 10 and nature controlling line 11 between 0.1cm to 0.6cm, preferably between 0.3cm to 0.5cm.Nature controlling line 11 is for determining whether reaction system is set up, and the fluorescence signal value passing through to calculate detection line 8,9 and 10 and nature controlling line 11 is quantitative accurately to carry out.
Water accepting layer 6 is for providing the capillary force needed for detection, and it can be made up of the material being selected from by velveteen, glass, is preferably made up of velveteen.
Need explanation; overlapping mode between each layer of Fig. 1; such as one deck is only exemplary illustration higher than the overlapping mode of one deck or height change gradually, such as adopts other overlapping mode such as height change grade equally in the scope of the claimed test strips of the utility model.
The entire length of test strips is between 8cm to 12cm, and overall width, between 0.2cm to 0.5cm, preferably between 0.2cm to 0.3cm, most preferably is 0.3cm.Test strips of the present utility model is being provided sensitivity and while detecting the range of linearity, can reduced the consumption of antibody, greatly saved production cost by the length and/or width reducing test strips.
Fig. 2 is the schematic top plan view of three joint inspection fluorescent chromatographic test strips of a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme described in the utility model.Content identical with Fig. 1 in Fig. 2 is repeated no more.
The utility model is further illustrated below with reference to embodiment.
Embodiment
In following embodiment, described cardiac muscle troponin I first antibody, cardiac muscle troponin I second antibody, myoglobins first antibody, myoglobins second antibody, creatine kinase isozyme first antibody, creatine kinase isozyme second antibody, dynamics, fluorescent microsphere, Avidin, biotin, 2-(N-morpholine) ethyl sulfonic acid (MES), carbodiimide (EDC), N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), N, N '-dicyclohexylcarbodiimide (DCC), pyridine, bovine serum albumin(BSA) (BSA), Tween-20, trehalose, glass fibre element film, polyester film, nitrocellulose filter, high-intensity water absorbent paper is commercially available prod.
The preparation method of three joint inspection fluorescent chromatographic test strips of described a kind of quantitative detection cardiac muscle troponin I-myoglobins-creatine kinase isozyme is:
1) preparation of sample pad
Glass fibre element film or polyester film are put into the sample pad treating fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
2) preparation of the first pad
The pre-service of A, the first pad
Glass fibre element film or polyester film are put into the pad treating fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
The preparation of two kinds of different-grain diameter fluorescent microspheres of B, labelled antibody
By cardiac muscle troponin I first antibody with boric acid-borate buffer solution 4 DEG C of dialysed overnight of 0.1M, pH8.0-8.6, adjustment concentration is 2mg/ml-5mg/ml.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing small particle diameter fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the cardiac muscle troponin I first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of cardiac muscle troponin I first antibody and fluorescent microsphere is made to be 1: 8 to 1: 60, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing Large stone fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the cardiac muscle troponin I first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of cardiac muscle troponin I first antibody and fluorescent microsphere is made to be 1: 20 to 1: 80, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
By the fluorescent microsphere of two kinds of different-grain diameters of above-mentioned mark cardiac muscle troponin I first antibody in 1: 10 to 10: 1 ratio mix, namely prepare two kinds of different-grain diameter fluorescent microspheres of labelled antibody.
The preparation of C, biotinylated antibody
By cardiac muscle troponin I second antibody with phosphate buffer 4 DEG C of dialysed overnight of 0.02M, pH7.2-7.6, adjustment concentration is 5mg/ml-10mg/ml.
The pre-activate of biotin: accurately take 300mg biotin and be dissolved in the dimethyl formamide (DMF) of 8ml, the N-hydroxy-succinamide (NHS) of 183mg is added under room temperature condition, the N of 304mg, the pyridine of N '-dicyclohexylcarbodiimide (DCC) and 0.1ml, at room temperature stirs 24 hours by reaction mixture.Cross the urea filtering reaction and produce, by silica gel chromatography after filtrate is concentrated, the white solid that recrystallization obtains in isopropyl alcohol is preactivated biotin.
Preactivated biotin is dissolved with dimethyl formamide (DMF), its final concentration is made to be 0.05M, and join in the cardiac muscle troponin I second antibody of dialysing, the mol ratio of cardiac muscle troponin I second antibody and biotin is made to be 1: 10-1: 25, room temperature reaction 1 hour, with phosphate buffer 4 DEG C of dialysed overnight of 0.02M, pH7.2-7.6.
The preparation of D, the first pad
The biotinylated antibody that two kinds of different-grain diameter fluorescent microspheres of the labelled antibody prepared by above-mentioned steps B and step C prepare fully mixes, use and quantitatively spray film instrument with on the plain film of the glass fibre that 2 μ l/cm-8 μ l/cm even application are pretreated in above-mentioned steps A or polyester film, in 35-39 DEG C of air dry oven, dry 2-4 hour, add drying agent and seal up for safekeeping for subsequent use.
3) preparation of the second pad
The pre-service of A, the second pad
Glass fibre element film or polyester film are put into the pad treating fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
The preparation of two kinds of different-grain diameter fluorescent microspheres of B, labelled antibody
By myoglobins first antibody with boric acid-borate buffer solution 4 DEG C of dialysed overnight of 0.1M, pH8.0-8.6, adjustment concentration is 2mg/ml-5mg/ml.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing small particle diameter fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the myoglobins first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of myoglobins first antibody and fluorescent microsphere is made to be 1: 8 to 1: 60, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing Large stone fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the myoglobins first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of myoglobins first antibody and fluorescent microsphere is made to be 1: 20 to 1: 80, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
By the fluorescent microsphere of two kinds of different-grain diameters of above-mentioned mark myoglobins first antibody in 1: 10 to 10: 1 ratio mix, namely prepare two kinds of different-grain diameter fluorescent microspheres of labelled antibody.
The preparation of C, the second pad
Two kinds of different-grain diameter fluorescent microsphere potpourris of the labelled antibody prepared by above-mentioned steps B use and quantitatively spray film instrument with on the plain film of the glass fibre that 2 μ l/cm-8 μ l/cm even application are pretreated in above-mentioned steps A or polyester film, in 35-39 DEG C of air dry oven, dry 2-4 hour, add drying agent and seal up for safekeeping for subsequent use.
4) preparation of the 3rd pad
The pre-service of A, the 3rd pad
Glass fibre element film or polyester film are put into the pad treating fluid (0.1M containing 1%BSA and 0.1%Tween-20, boric acid-the borate buffer solution of pH8.0-8.6 or the 0.05M containing 1%BSA and 0.1%Tween-20, the Tris-HCl damping fluid of pH7.4-8.2) middle after immersion treatment 3-5 hour, in 35-39 DEG C of air dry oven, dry 3-5 hour.
The preparation of two kinds of different-grain diameter fluorescent microspheres of B, labelled antibody
By creatine kinase isozyme first antibody with boric acid-borate buffer solution 4 DEG C of dialysed overnight of 0.1M, pH8.0-8.6, adjustment concentration is 2mg/ml-5mg/ml.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing small particle diameter fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the creatine kinase isozyme first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of creatine kinase isozyme first antibody and fluorescent microsphere is made to be 1: 8 to 1: 60, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
Use 0.05M, 2-(N-morpholine) ethyl sulfonic acid (MES) the activation buffer washing Large stone fluorescent microsphere of pH4.5-5.0, adding carbodiimide (EDC) and N-hydroxy-succinamide (NHS) makes the two final concentration be 15-60mmol, room temperature reaction 20-30 minute, abundant washing fluorescent microsphere, with 0.05M, the creatine kinase isozyme first antibody of dialysing is added after boric acid-borate buffer solution redissolution of pH8.0-8.6, the mass ratio of creatine kinase isozyme first antibody and fluorescent microsphere is made to be 1: 20 to 1: 80, room temperature reaction is after 2 hours, add the 0.02M containing 10%BSA, the phosphate buffer room temperature of pH7.2-7.6 closes 30 minutes, abundant washing fluorescent microsphere, with the 0.05M containing 1%BSA and 0.1%Tween-20, the conserving liquid of the Tris-HCl damping fluid of pH7.4-8.2 redissolves to original volume.
By the fluorescent microsphere of two kinds of different-grain diameters of above-mentioned mark first antibody in 1: 10 to 10: 1 ratio mix, namely prepare two kinds of different-grain diameter fluorescent microspheres of labelled antibody.
The preparation of C, the 3rd pad
Two kinds of different-grain diameter fluorescent microsphere potpourris of the labelled antibody prepared by above-mentioned steps B use and quantitatively spray film instrument with on the plain film of the glass fibre that 2 μ l/cm-8 μ l/cm even application are pretreated in above-mentioned steps A or polyester film, in 35-39 DEG C of air dry oven, dry 2-4 hour, add drying agent and seal up for safekeeping for subsequent use.
5) preparation of coated film
The preparation of A, detection line (detecting for cTnI): use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) Avidin is diluted to the concentration of 1mg/ml-5mg/ml, use quantitatively spray film instrument with 1.2 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of B, detection line (detecting for Myo): by myoglobins second antibody with 0.01-0.02M, Tris-HCl damping fluid 4 DEG C of dialysed overnight of pH7.4-8.2, use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) the myoglobins second antibody after dialysis is diluted to the concentration of 0.2mg/ml-4mg/ml, use quantitatively spray film instrument with 1.2 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of C, detection line (detecting for CK-MB): by creatine kinase isozyme second antibody with 0.01-0.02M, Tris-HCl damping fluid 4 DEG C of dialysed overnight of pH7.4-8.2, use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) the creatine kinase isozyme second antibody after dialysis is diluted to the concentration of 0.2mg/ml-4mg/ml, use quantitatively spray film instrument with 1.2 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of D, nature controlling line: use the coating buffer (0.01-0.02M containing 0.1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) anti-mouse IgG is diluted to the concentration of 0.5mg/ml-3mg/ml, use quantitatively spray film instrument with 0.8 μ l/cm even application on nitrocellulose filter, in 35-39 DEG C of air dry oven, dry 2-4 hour.
The preparation of E, coated film: with the confining liquid (0.01-0.02M containing 1-10%BSA and 1-10% trehalose, the Tris-HCl damping fluid of pH7.4-8.2) by the cellulose nitrate membrane closure 1-2 hour containing detection line and nature controlling line, take out in rearmounted 35-39 DEG C air dry oven and dry 2-4 hour, envelope is for subsequent use.
6) assembling of test strips
Be less than 30% in humidity, under temperature 20-30 DEG C of condition, carry out the assembling of test strips.Base plate selects PVC material, connects in turn and the partly overlapping sample pad of adjacent regions, the first pad, the second pad, the 3rd pad, coated film and thieving paper in the direction of base plate the same face.When coated film covers base plate, the position of detection line is near pad, and the position of nature controlling line is near thieving paper.Thieving paper is high-intensity water absorbent paper.Test strips is cut into after assembling.
Claims (5)
1. cardiac muscle troponin I-myoglobins-creatine kinase isozyme joint inspection test strips, it is characterized in that, described test strips comprises:
1) for accepting the sample pad from whole blood, blood plasma or blood serum sample;
2) be coated with the first pad of the cardiac troponin second antibody of the cardiac muscle troponin I first antibody epi-position different from biotin labeled identification of two kinds of different-grain diameter fluorescent microsphere marks simultaneously;
3) the second pad of the myoglobins first antibody of two kinds of different-grain diameter fluorescent microsphere marks is coated with;
4) the 3rd pad of the creatine kinase isozyme first antibody of two kinds of different-grain diameter fluorescent microsphere marks is coated with;
5) coated film of three detection lines separately and a nature controlling line is coated with, wherein said three detection lines are fixed with Avidin successively, identify the myoglobins second antibody of different epi-position and identify the creatine kinase isozyme second antibody of different epi-position, and described nature controlling line is fixed with the antibody of against murine IgG;
6) for providing the thieving paper of the capillary force needed for detection; With
7) base plate, wherein said base plate overlaps successively the sample pad of stickup, the first pad, the second pad, the 3rd pad, coated film and thieving paper.
2. test strips according to claim 1, is characterized in that, the particle size range of described two kinds of different-grain diameter fluorescent microspheres is all between 50nm to 500nm.
3. test strips according to claim 1, is characterized in that, described first pad, the second pad, the 3rd pad material are glass fibre element film or polyester film.
4. test strips according to claim 1, is characterized in that, described coated film is nitrocellulose filter.
5. test strips according to claim 1, is characterized in that, the width of described test strips is between 0.2cm to 0.5cm.
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Cited By (14)
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