CN102323419B - Kit and detection method for quantitative determination of digoxin - Google Patents
Kit and detection method for quantitative determination of digoxin Download PDFInfo
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- CN102323419B CN102323419B CN2011102575936A CN201110257593A CN102323419B CN 102323419 B CN102323419 B CN 102323419B CN 2011102575936 A CN2011102575936 A CN 2011102575936A CN 201110257593 A CN201110257593 A CN 201110257593A CN 102323419 B CN102323419 B CN 102323419B
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Abstract
The invention discloses a kit for quantitative determination of DIGOXIN. The kit is characterized by comprising a DIGOXIN magnetic separation reagent, an enzyme reactant, a reaction strengthening agent, a diluent, a DIGOXIN calibrator, a DIGOXIN quality control material, a concentrated cleaning solution, and a substrate solution. The invention also discloses a preparation method of the kit. The kit for quantitative determination of DIGOXIN combines a chemiluminescence technology and immunomagnetic particles and provides an almost homogeneous reaction system. Compared with the prior art, the kit for quantitative determination of DIGOXIN has high detection sensitivity and specificity and good performance parameters, and greatly reduces a production cost.
Description
Technical field
The present invention relates to measure kit and the method for testing thereof of serum, especially relate to kit and the method for testing thereof of measuring digoxin (DIGOXIN) content in the serum.
Background technology
Digoxin is translated into digoxin, DIGOXIN again.Chemical name 3 β-0-2,0-dideoxy-β-D-nuclear-own pyrans glycosyl-(1 → 4)-0-2,6-dideoxy-β-D-nuclear-own pyrans glycosyl-(1 → 4)-2,6-dideoxy-β-D-nuclear-own pyrans glycosyl oxo-12 β, 14 beta-dihydroxies-5 β-heart steroid-20 (22) alkene lactones.Digoxin mark comes from a kind of steroids (Steroid) material---Digoxigenin (DIG) that extracts from digitalis class plant (digitalis and a hair flower digitalis), in disease such as medically can be used for treating various acute and chronic cardiac insufficiency and supraventricular tachycardia, auricular fibrillations and flutter.On detection angles, the method that is used for mensuration DIGOXIN at present clinically mainly contains the method for exempting from of putting, ELISA method, chemoluminescence method etc.
Past, to exempt from be that the digoxin (DIGOXIN) of representative is measured kit and is subjected to methodological restriction to put, its sensitivity and antijamming capability wretched insufficiency, there is very big drawback, withdraws from the market basically, use more be enzyme linked immunosorbent detection technology and chemiluminescence at present.It is continue Enzyme-multiplied immune technique and the emerging technology that grows up after putting immune technology the eighties that chemiluminescence rises in eighties of last century, because its high sensitivity, high specific, while method is easy, quick, the mark bond is stable, characteristics such as "dead" isotope damage and pollution have obtained develop rapidly in recent years.
Magnetic particle separation enzyme-linked immunoassay technology be a kind of be the solid phase carrier of separating with the magnetic particle, immune magnetic particle isolation technics is combined with the enzyme linked immunosorbent detection technology and a kind of novel immunologic detection method set up.Traditional E LISA method, the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface, and magnetic particle separation enzyme-linked immunoassay, the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase, thereby reaction is fast, thoroughly.Compare with traditional E LISA and to have highly sensitively, detect few advantage of time spent.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of digoxin (DIGOXIN) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out DIGOXIN and detect time with higher sensitivity and specificity and shorter acquisition testing result and easier mode of operation.
The invention provides the quantitative determination reagent kit of a kind of digoxin (DIGOXIN), its reagent that comprises has DIGOXIN magnetic separation agent, enzyme reaction thing, increased response agent, dilution, calibration object, quality-control product, cleaning fluid concentrate and substrate solution.
Described magnetic separation agent contains the magnetic microsphere that is marked with anti-DIGOXIN monoclonal antibody.
Described enzyme reaction thing is the DIGOXIN antigen that contains alkali phosphatase enzyme mark.
Described increased response agent is the damping fluid that contains Tris.
Described dilution is to contain BSA solution.
Described calibration object and quality-control product are the BSA protein solutions that contains a certain amount of DIGOXIN antigen.
Described cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300.
Described substrate solution is enzyme-catalyzed chemical luminescence substrate solution.
The preparation method of the quantitative determination reagent kit of digoxin of the present invention (DIGOXIN) comprises the steps:
The first step: the preparation process of magnetic separation agent
One, magnetic bead buffer solution formulation operations step, preparation 1L:
1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the said vesse;
2, with pipettor Proclin-300 is measured 0.2ml and in the beaker of 10ml purified water, fully after the dissolving, pour in the above-mentioned 1L container, in above-mentioned 1L container, add the 800ml purified water then, fully stir;
3, regulate its pH value of PH instrumentation amount, transfer PH, PH is between 7.95-8.05 in control;
4, taking by weighing BSA 3g pours in the above-mentioned 1L container;
5, be settled to 1000ml at last, filter with the 0.2um filter; Posting label after having filtered stores in 2-8 ℃ of freezer.
Two, the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved among the 50ul DMSO, get in the 0.1mol/L PB damping fluid that the anti-DIGOXIN monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, determine the input amount of disuccinimidyl suberate, draw disuccinimidyl suberate with liquid-transfering gun and join in the antibody-solutions of step 1, put room temperature 90min;
3, step 2 antibody-solutions is joined put into then in the Centricon-10 concentration tube in the high speed freezing centrifuge under 3000g, concentrate 30min to volume be 0.5ml;
4, get the 0.5ml magnetic bead, add in the 5ml reaction cup, put into special-purpose test tube rack, draw supernatant through magnet absorption after 2 minutes;
5, add 1.5ml PH9.5 0.1mol/L PB, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times at every turn; The antibody-solutions that step 4 is obtained joins in the above-mentioned magnetic bead, and room temperature reaction is 4 hours behind the mixing;
6,37 ℃ of the TRIS solution 15 minutes that add 0.3ml 1mol/L;
7, add 1.5ml PH 7.2 0.1mol/L PR at every turn clean the magnetic bead of mark, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times;
8, preserve liquid with the 100ml magnetic bead and change magnetic bead over to the 125ml vial, be 0.05% DIGOXIN magnetic separation agent; It is 0.1% BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02% NaN3,20% ethanol, 4 ℃ of preservations.
9, the magnetic separation agent that step 9 is obtained namely gets magnetic separation agent in the kit of the present invention with the ratio mixing of magnetic bead buffer solution according to 1: 1.
Second step: enzyme reaction thing preparation process
One, enzyme reaction thing diluent preparing operation steps, preparation 1L:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g in flask, in flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;
2, transfer PH, PH is in the 7.35-7.45 scope in control;
3, taking by weighing BSA 3g pours in the above-mentioned beaker;
4, be settled to 1000ml at last, filter namely with the 0.2um filter.
Two, the coupling of alkaline phosphatase ALP and DIGOXIN antigen
1, getting DIGOXIN antigen 1 mg is positioned in the 1ml glass tube;
2, get 200ul DMSO dissolving antigen and make the final concentration of antigen arrive 5mg/ml, fully mixing;
3, the molar ratio that adds the disuccinimidyl suberate of 5mol according to 1mol antigen adds disuccinimidyl suberate in the solution of step 2, and reaction is 1.5 hours in 37 ℃ of constant temperature ovens;
4, the mol ratio that adds the ALP of 1mol according to 5mol antigen is added ALP toward step 3 solution in, and the PH that adds 1ml then is that 7.4 concentration are the PB damping fluid of 0.1M, places 37 degree constant temperature ovens to react 3 hours;
5, with the solution of step 4 with PD-10 post purifying, collect refined solution, add above-mentioned steps one according to 1: 5000 volume) the enzyme reaction thing dilution that obtains, mix, namely get the enzyme reaction thing.
The 3rd step:
Increased response agent preparation steps, preparation 1L:
1, takes by weighing TRIS1.56g and NaCl 4.23g in the 1L container; With pipettor with Proclin-300 measure 0.2ml in the beaker of 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, transfer PH until dissolving fully, PH is between 7.35-7.45 in control;
3, take by weighing Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully.
The 4th step:
The diluent preparing step, preparation 1L:
1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully, posts label and stores in 2-8 ℃ of freezer, and the term of validity is 12 months.
The 5th step:
The preparation of calibration object and quality-control product:
Calibration object concentration is respectively 0.5,1,2.5,5,10ng/ml; Quality-control product concentration is respectively 1,5ng/ml.
The 6th step:
The cleaning concentrate preparation steps, preparation 1L:
1, takes by weighing TRIS 12.54g and NaCl 325.6g in the 1L container;
2, take by weighing 5g Tween-20 adds 20ml water it is dissolved fully in the 100ml container after, pour in the above-mentioned 1L container;
3, with pipettor with Proclin-300 measure 0.2ml in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolving fully;
5, transfer PH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filter namely with the 0.2um filter dissolving back fully.
The 7th step:
The substrate solution preparation steps, preparation 1L:
1, takes by weighing TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g and Proclin-300 0.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolving fully, transfer PH, control its scope between 7.95-8.05;
3, add 250ml Lumi-Phos 480 after, filter with the 0.2um filter and to collect filtrate, be settled to 1000ml with purified water, behind the mixing namely.
Principle of work of the present invention: a kind of detection method that the present invention combines with the magnetic particle isolation technics for the competing method chemiluminescence immune analysis method.In sample, calibration object and quality-control product, add quantitative enzyme mark DIGOXIN antigen, in conjunction with DIGOXIN monoclonal antibody and the stabilizing reinforcer of magnetic particle.37 ℃ hatch after, in enzyme mark DIGOXIN antigen and sample, calibration object and the quality-control product DIGOXIN competition with combination the DIGOXIN monoclonal antibody generation specificity combination of magnetic particle.In externally-applied magnetic field, directly precipitate, do not need centrifugal namely separable.Remove supernatant, the compound of washing and precipitating adds enzyme-catalyzed chemical luminescence substrate then.Substrate by catalytic pyrolysis, forms unsettled excited state intermediate under the enzyme effect, just send photon when the excited state intermediate is got back to ground state, forms luminescence-producing reaction, can use the luminous intensity of light-emitting appearance detection reaction.In sensing range, the luminous intensity of reaction and the DIGOXIN concentration in the sample are inversely proportional to.
Main innovation part of the present invention is:
1, kit of the present invention combines chemiluminescence with immune magnetic particle, and a kind of reaction system near homogeneous phase is provided, and compared with prior art, kit of the present invention has higher detection sensitivity and specificity, and has reached preferable performance parameter.
2, the invention discloses a kind of new special-purpose stabilizing reinforcer and cleaning fluid concentrate, make course of reaction more reliable and more stable, experimental data is effectively sensitive, when enhancing product performance, and greatly reduces cost of products;
3, the magnetic separation agent in the kit, the enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning fluid concentrate, dilution and substrate solution all are the optimization formulas under this reaction system, and giving the use effect phase of this kit and detecting performance provides powerful guarantee.
Embodiment
Embodiment 1,
One, magnetic bead buffer solution formulation operations rules: prescription sees Table 1, and 1L is example with preparation:
1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the said vesse;
2, with pipettor with Proclin-300 measure 0.2ml in the beaker of a small amount of purified water fully the dissolving after, pour in the above-mentioned 1L container; In the 1L container, add the 800ml purified water then, fully stir, reagent is dissolved fully;
3, regulate pH value, measure its pH value; Transfer PH with HCl or NaOH, measure its PH and between 7.95-8.05, namely meet the requirements;
4, taking by weighing BSA (bovine serum albumin(BSA)) 3g pours in the said vesse;
5, be settled to 1000ml at last, survey pH value, scope namely meets the requirements between 7.95-8.05, filters with the 0.2um filter; Post label after having filtered and store in 28 ℃ of freezers, the magnetic bead buffer solution term of validity is 12 months;
Magnetic bead buffer solution table 1
Two, the preparation process of magnetic separation agent
1,1.0mg DSS (disuccinimidyl suberate) is dissolved among the 50ul DMSO, namely concentration is 20mg/ml; Get in the 0.1mol/L PB damping fluid that the anti-DIGOXIN monoclonal antibody of 2mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 3.84mg/ml) is dissolved in PH 9.5 to cumulative volume be 1ml;
2, calculate the input amount of DSS, calculate according to following formula: (antibody quality/16000) * 10 * 368/C
DSS), C wherein
DSSRefer to the amount of substance concentration mol/L of DSS;
3, the DSS with liquid-transfering gun absorption respective volume joins in the antibody-solutions of step 1, puts room temperature 90min;
4, step 3 antibody-solutions is joined put into then in the Centricon-10 concentration tube in the sigma2-16k high speed freezing centrifuge under the centrifugal force of 3000g, concentrate about 30min to volume be 0.5ml;
5, get the 0.5ml magnetic bead, magnetic bead concentration is 0.5g/ml, and described magnetic bead diameter adds in the 5ml reaction cup between 0.9 μ m, puts into special-purpose test tube rack, draws supernatant through magnet absorption after 2 minutes;
6, add 1.5ml PH9.5 0.1mol/L PB, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times at every turn; The antibody-solutions that step 4 is obtained joins in the magnetic bead, and room temperature reaction is 4 hours behind the mixing, keeps the mixing state;
7, the TRIS solution 37 that adds 0.3ml 1mol/L is spent 15 minutes, and wherein the application of sample amount of TRIS adds 0.15mlTRIS for 1mg antibody;
8, add 1.5ml PH 7.2 0.1mol/L PB at every turn clean the magnetic bead of mark, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times;
9, preserve liquid with the 100ml magnetic bead and change magnetic bead over to the 125ml vial, be 0.05% DIGOXIN magnetic separation agent; It is 0.1% BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02% NaN3,20% ethanol, 4 ℃ of preservations.
10, the magnetic separation agent that step 9 is obtained is with the ratio mixing of magnetic bead buffer solution according to 1: 1;
11, post label and store in 2-8 ℃ of freezer, the magnetic separation agent term of validity is 12 months;
Embodiment 2
One, enzyme reaction thing diluent preparing working specification: prescription sees Table 2, and 1L is example with preparation:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g in flask; In flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;
2, transfer PH with HCl or NaOH, measurement makes PH in the 7.35-7.45 scope;
3, taking by weighing BSA 3g pours in the said vesse;
4, be settled to 1000ml at last, survey pH value, scope namely meets the requirements between 7.357.45, filters with the 0.2um filter; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;
Enzyme reaction thing dilution table 2
Two, the coupling of alkaline phosphatase ALP and DIGOXIN antigen
1, getting DIGOXIN antigen 1 mg is positioned in the 1ml glass tube;
2, get 200ul DMSO dissolving antigen and make the final concentration of antigen arrive 5mg/ml, fully mixing;
3, the molar ratio that adds the disuccinimidyl suberate of 5mol according to 1mol antigen adds disuccinimidyl suberate in the solution of step 2, and reaction is 1.5 hours in 37 ℃ of constant temperature ovens;
4, the mol ratio that adds the ALP of 1mol according to 5mol antigen is added ALP toward step 3 solution in, and the PH that adds 1ml then is that 7.4 concentration are the PB damping fluid of 0.1M, places 37 degree constant temperature ovens to react 3 hours;
5, with the solution PD 10 post purifying of step 4, collect refined solution, add enzyme reaction thing dilution according to 1: 5000 volume, mix, namely get the enzyme reaction thing.
Embodiment 3
Increased response agent formulation operations rules: prescription is seen (table 3), and 1L is example with preparation:
1, take by weighing TRIS (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl 4.23g are in the 1L container; With pipettor with Proclin-300 measure 0.2ml in the beaker of a small amount of purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, measure the 800ml purified water in the 1L container with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;
3, take by weighing Mak330.9g in above-mentioned 1L container;
4, last constant volume 1000ml after the dissolving, surveys pH value fully, and scope namely meets the requirements between 7.35-7.45, filters with the 0.2um filter; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;
The preparation (table 3) of increased response agent
Embodiment 4
The dilution prescription is seen (table 4), and 1L is example with preparation:
1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor Proclin-300 is measured 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml after the dissolving, filters with the 0.2um filter fully, posts label and stores in 2-8 ℃ of freezer, and the term of validity is 12 months.
The preparation (table 4) of dilution
Embodiment 5
The preparation of calibration object and quality-control product:
1, peak: maximum concentration point is X, and impact point concentration is A, B, and C, D, E, F when preparing the solution of V volume, needs the volume of adding raw material for being respectively: table 5
| Concentration | Add calibration object dilution volume | Add the X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| 0F | V-F*V/X | F*V/X |
2, to be mixed with concentration point with dilution (concrete prescription see embodiment 4) be 0.5,1,2.5,5,10ng/ml to digoxin (DIGOXIN) quantitative determination reagent kit DIGOXIN calibration object raw material (purchase in AMG Co., concentration is 10mg/ml); Quality-control product is 1,5ng/ml with the concentration point of diluent preparing.
3, fully the dissolving after, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months.
Embodiment 6:
Cleaning concentrate formulation operations rules: prescription is seen (table 6), and 1L is example with preparation:
1, takes by weighing TRIS 12.54g and NaCl 325.6g in the 1L container;
2, take by weighing 5g Tween-20 adds suitable quantity of water it is dissolved fully in the 100ml container after, pour in the said vesse;
3, with pipettor with Proclin-300 measure 0.2ml in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolving fully;
5, transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;
6, last constant volume 1000ml surveys pH value, and scope namely meets the requirements between 7.35-7.45, and filter with the 0.2um filter dissolving back fully; After having filtered, post label and store in 28 ℃ of freezers, the term of validity is 12 months;
The preparation (table 6) of cleaning concentrate
Embodiment 7
Substrate solution formulation operations rules: prescription is seen (table 7), and 1L is example with preparation:
1, takes by weighing TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g and Proclin-300 0.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCl or NaOH, measure its scope between 7.95-8.05;
3, behind the adding 250ml Lumi-Phos 480, filter collection filtrate with the 0.2um filter, be settled to 1000ml with purified water, behind the mixing, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months.
The preparation (table 7) of chemical luminous substrate
Method of testing of the present invention is as follows
1, adds 45 μ l DIGOXIN calibration objects, quality-control product, sample to be measured to corresponding test tube bottom.
2, add 60 μ l enzyme reaction things to each test tube.
3, add 60 μ l increased response agent to each test tube.
4, add 30 μ l magnetic separation agents to each test tube.
5, cover test tube with plastic sheeting, the multitube vortex mixer behind the tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently.
6, the test tube frame linking is put to magnetic separator, guarantees that every test tube all contacts with separator surface, precipitates 2 minutes.The separation vessel that reverses is slowly poured out supernatant, and the test tube of reversing is placed on the filter paper together with separation vessel, firmly bounces the separation vessel bottom to remove all drops that are bonded on the tube wall.
7, the cleaning fluid concentrate with 20 times of purified water dilutions after, add cleaning fluid after the 200 μ l dilution to each test tube, put the mixing 30s that vibrates gently on the multitube vortex mixer.Should avoid the application of sample dynamics excessive and cause magnetic bead to spill during application of sample.Mixing is wanted thoroughly.
8, repeating step 6,7,6 one times.
9, add 200 μ l substrate solutions mixing 3 seconds to the test tube, detect with ready luminous detector rapidly.
10, as running into high value HOOK sample, for fear of high value HOOK effect occurring, the suggestion clinician selects suitable extension rate that sample is diluted according to all the other test indexs.
Clinical testing:
1, detects data
Sample picks up from the normal health check-up of 1200 examples, blood donor.The sample physical examination result does not all have liver, brain, kidney, disease of digestive tract, does not have blood transfusion and major operation history in half a year, and the women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draw normal serum term of reference: 0.80-2.00ng/ml.The testing result of the kit of producing with external renowned company is compared, and kit of the present invention is more accurate, is accurate to 2 significant digits.
Notebook data is only for reference, and different regions, Different Individual and employing distinct methods detect, and measured DIGOXIN level also can be different, advises the range of normal value of each laboratory foundation oneself.The DIGOXIN value that can not only draw with this method is made diagnosis, only as the intermediate data reference role, should comprise patient's concrete condition and treatment situation in conjunction with clinical other analyses result.DIGOXIN concentration value and this kit measurement result in the sample that obtains by additive method do not have direct comparability.The sample that exceeds the kit measurement scope, system can't provide definite numerical value.Measure its definite result as desire, measure again after the suggestion dilution.The testing result of this kit only supplies clinical reference, can not be separately as the foundation of making a definite diagnosis or get rid of case, and for reaching diagnostic purpose, this testing result will be used in combination with clinical examination, medical history and other inspection.This product can be used for the mensuration of serum sample, and the reliability that is used for other body fluid samples DIGOXIN concentration determination is fully confirmed.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero calibration objects, get its mean deviation of 2 times, its corresponding concentration on typical curve is sensitivity for analysis; Sensitivity for analysis: 0.10ng/ml; Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%; Linear coefficient: r 〉=0.9900; The range of linearity: 0.10-6.0ng/ml specificity: measure the cross reacting material of high concentration, the result is as follows:
Claims (1)
1. the quantitative determination reagent kit of a digoxin DIGOXIN is characterized in that, this kit comprises DIGOXIN magnetic separation agent, enzyme reaction thing, increased response agent, dilution, DIGOXIN calibration object, DIGOXIN quality-control product, cleaning fluid concentrate and substrate solution;
Each component makes according to the method for being prepared as follows in the described kit:
One, the preparation process of magnetic separation agent
One), magnetic bead buffer solution formulation operations step, the preparation 1L:
1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the above-mentioned 1L container;
2, with pipettor Proclin-300 is measured 0.2ml and in the beaker of 10ml purified water, fully after the dissolving, pour in the above-mentioned 1L container, in above-mentioned 1L container, add the 800ml purified water then, fully stir;
3, regulate PH, PH is between 7.95-8.05 in control;
4, taking by weighing BSA 3g pours in the above-mentioned 1L container;
5, be settled to 1000ml at last, filter namely with the 0.2um filter;
Two), the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved among the 50ul DMSO, get in the 0.1mol/L PB damping fluid that the anti-DIGOXIN monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, draw in the antibody-solutions that above-mentioned disuccinimidyl suberate joins step 1 with liquid-transfering gun, put room temperature 90min;
3, step 2 antibody-solutions is joined in the concentration tube, put into then in the high speed freezing centrifuge that centrifugal 30min is concentrated into 0.5ml under 3000g;
4, get the 0.5ml magnetic bead, add in the 5ml reaction cup, draw supernatant through magnet absorption after 2 minutes, add 1.5ml PH9.50.1mol/L PB then, mixing 30 seconds adds the antibody-solutions that step 3 obtains then, and room temperature reaction is 4 hours behind the mixing;
5, add 37 ℃ of the TRIS solution 15 minutes of 0.3ml 1mol/L, add 1.5ml PH 7.2 0.1mol/L PB then and clean the magnetic bead of mark, mixing 30 seconds;
6, preserve liquid with the 100ml magnetic bead and change magnetic bead over to vial;
7, the solution that step 6 is obtained and above-mentioned magnetic bead buffer solution namely get the magnetic separation agent according to 1: 1 volume ratio mixing;
Two, the preparation process of enzyme reaction thing
One), enzyme reaction thing diluent preparing operation steps, the preparation 1L:
1, gets Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g in flask, in flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;
2, transfer PH, PH is in the 7.35-7.45 scope in control;
3, taking by weighing BSA 3g pours in the above-mentioned beaker;
4, be settled to 1000ml at last, filter namely with the 0.2um filter;
Two), the coupling of alkaline phosphatase ALP and DIGOXIN antigen
1, gets DIGOXIN antigen;
2, get 200ul DMSO dissolving antigen and make the final concentration of antigen arrive 5mg/ml, fully mixing;
3, the molar ratio that adds the disuccinimidyl suberate of 5mol according to 1mol antigen adds disuccinimidyl suberate in the solution of step 2, and reaction is 1.5 hours in 37 ℃ of constant temperature ovens;
4, the mol ratio that adds the ALP of 1mol according to 5mol antigen is added ALP toward step 3 solution in, and adding 1mlPH then is that 7.4 concentration are the PB damping fluid of 0.1M, places 37 degree constant temperature ovens to react 3 hours;
5, with the solution of step 4 with PD-10 post purifying, collect refined solution, add above-mentioned steps one according to 1: 5000 volume ratio) the enzyme reaction thing dilution that obtains, mix, namely get the enzyme reaction thing;
Three, the preparation process of increased response agent, preparation 1L:
1, get TRIS1.56g and NaCl 4.23g in the 1L container, get 0.2ml Proclin-300 in the beaker of 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
2, get the 800ml purified water in above-mentioned 1L container, fully stir until dissolving fully, transfer PH between 7.35-7.45;
3, take by weighing Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml after the dissolving, filters namely with the 0.2um filter fully;
Four, the preparation process of dilution, preparation 1L:
1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;
2, get 0.5ml Proclin-300 and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml after the dissolving, filters namely with the 0.2um filter fully;
Five, the preparation of DIGOXIN calibration object and DIGOXIN quality-control product:
The concentration of DIGOXIN antigen is respectively 0.5,1,2.5,5,10ng/ml in the DIGOXIN calibration object; The concentration of DIGOXIN antigen is respectively 1,5ng/ml in the DIGOXIN quality-control product;
Six, the preparation process of cleaning fluid concentrate, preparation 1L:
1, gets TRIS 12.54g and NaCl 325.6g in the 1L container;
2, get 5g Tween-20 and in the 100ml container, add 20ml water it is dissolved fully after, pour in the above-mentioned 1L container;
3, get 0.2ml Proclin-300 in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;
4, get the 800ml purified water in above-mentioned 1L container, fully stir, until dissolving fully;
5, transfer PH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filter namely with the 0.2um filter dissolving back fully;
Seven, the preparation process of substrate solution, preparation 1L:
1, gets TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g and Proclin-300 0.2ml is in the 1L beaker;
2, get the 600ml purified water in the 1L beaker, fully stir until dissolving fully, transfer PH between 7.95-8.05;
3, add 250ml Lumi-Phos 480, filter with the 0.2um filter and collect filtrate, be settled to 1000ml with purified water, behind the mixing namely.
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| CN102323419B true CN102323419B (en) | 2013-10-09 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103383354B (en) * | 2012-05-03 | 2016-11-09 | 北京博迈世纪生物技术有限公司 | Detection method of magnetic particle chemiluminescence kit for detecting enterotoxin SEA |
| CN103439492B (en) * | 2013-08-22 | 2015-03-11 | 顺昊细胞生物技术（天津）股份有限公司 | Kit |
| CN109470867B (en) * | 2016-11-22 | 2022-07-15 | 科美博阳诊断技术(上海)有限公司 | Immunoassay method and system for hepatitis B virus surface antibody |
| CN108196055B (en) * | 2017-11-28 | 2020-07-07 | 泰州泽成生物技术有限公司 | Kit for determining digoxin content by magnetic particle chemiluminescence method and detection method thereof |
| CN109239057A (en) * | 2018-10-18 | 2019-01-18 | 郑州标源生物科技有限公司 | A kind of stable digoxin quality-control product and preparation method |
| CN110441538A (en) * | 2019-08-23 | 2019-11-12 | 北京丹大生物技术有限公司 | A kind of immuno-chromatographic test paper strip and its application for detecting digoxin |
| CN110618261A (en) * | 2019-08-27 | 2019-12-27 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay kit for quantitatively detecting digoxin and preparation method thereof |
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