CN103424551B - People's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof - Google Patents
People's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of people's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof, comprise preparation seven steps of the preparation of Magneto separate reagent, the preparation of enzyme reaction thing, the preparation of increased response agent, the preparation of calibration object dilution, calibration object and the preparation of quality-control product, the preparation of cleaning concentrate and substrate solution.The present inventor's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof have higher sensitivity and specificity, the time of shorter acquisition testing result and easier mode of operation, to having significant application value in the case fatality rate of the early diagnosis of ovarian epithelial carcinoma, curative effect monitoring and Index for diagnosis, reduction ovarian cancer patients.
Description
Technical field
The present invention relates to a kind of people's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof, measure people's epididymal secretory protein 4 content in human serum for Quantitative in vitro.
Background technology
The oophoroma in recent years incidence of disease is more and more higher, and in gynecological tumor, the incidence of disease is only second to cervix cancer and carcinoma of uterine body occupies the 3rd, but its fatal rate occupies first place.This is mainly because ovary is ensconced in bone chamber deeply, and oophoroma early symptom is very not obvious, lacks effective early diagnosis, and the patient of 75% belongs to late period when medical.
People's epididymal secretory protein 4(Human epididymis protein4, HE4) be a kind of novel tumor markers developed in recent years, HE4 encoding gene is separated in 1991 from epididymal far-end by people such as Kichhoff the earliest, the people such as Schummer in 1999 are by cDNA microarray analysis, find that HE4(has another name called WFDC2) mRNA high expressed in ovarian cancer tissue, and do not express in cancer beside organism.Confirm that HE4 has the value of ovarian cancer diagnosis afterwards, and ovarian cancer diagnosis medium sensitivity and specificity are all better than CA125 in early days.In order to improve oophoroma early diagnosis level and improve result for the treatment of, from detection angles, the method at present clinically for measuring HE4 mainly contains radioimmunoassay technology, Enzyme-multiplied immune technique and chemiluminescence etc.
People's epididymal secretory protein 4(HE4 that past is representative with radioimmunoassay technology) measure kit due to methodological restriction, its sensitivity and antijamming capability wretched insufficiency, there is very large drawback, substantially withdraw from the market; What current application was more is Enzyme-multiplied immune technique and chemiluminescence, wherein chemiluminescence is risen eighties of last century eighties, it is the emerging technology grown up after Enzyme-multiplied immune technique and radioimmunoassay technology, due to its high sensitivity, high specific, while method is easy, quick, mark bond is stablized, and the features such as "dead" isotope damage and pollution, were obtaining develop rapidly in recent years.
People's epididymal secretory protein 4(HE4 of publicity at present) Patents totally 11, detect correlation technique comprising chemiluminescence, Enzyme-multiplied immune technique, time-resolved fluorescence, and have no magnetic particle isolation technics and combine with chemiluminescence and be applied in immunoassay.Magnetic microparticle chemiluminescence immunoassay technology is a kind of is solid phase carrier of separating with magnetic particle, immune magnetic particle isolation technics is combined with chemiluminescence immunoassay detection technique and a kind of Novel immune detection method of setting up.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, a kind of highly sensitive, high specificity, people's epididymal secretory protein 4 quantitative determination reagent kit simple to operate and detection method thereof are provided.
The object of the present invention is achieved like this:
A kind of people's epididymal secretory protein 4 quantitative determination reagent kit, comprise the lid above box body and box body, be provided with microwell plate in described box body and be positioned at the back up pad bottom box body, wherein microwell plate is made up of 48 or 96 micropores, back up pad has multiple hole, eight reagent bottles are placed with altogether in described hole, respectively containing Magneto separate reagent in described eight reagent bottles, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution, wherein Magneto separate reagent contains the magnetic particle of people's epididymal secretory protein 4 monoclonal antibody bag quilt.
The present inventor's epididymal secretory protein 4 quantitative measurement detection method, described detection method comprises reagent set-up procedure and detecting step, and described reagent set-up procedure is as follows:
The preparation of step one, Magneto separate reagent
One, magnetic bead buffer solution preparation
(1), take TRIS 4.58g and NaCl6.81g in 1L container, then take 0.96g TWEEN-20 and add after suitable quantity of water makes it dissolve completely in 20ml container, then pour in above-mentioned 1L container;
(2), with pipettor Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add 800ml purified water, fully stir;
(3), regulate pH meter to measure its pH value, control pH is between 7.95-8.05;
(4), taking BSA 3g pours in above-mentioned 1L container;
(5), last 1L container is settled to 1000ml, uses 0.2um frit; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered;
Two, the preparation of Magneto separate reagent
(1), 1.0mg disuccinimidyl suberate is dissolved in 50ulDMSO, get the anti-HE4 monoclonal antibody of 2mg be dissolved in the PB damping fluid of the 0.1mol/L of 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 above-mentioned HE4 monoclonal antibody solution, put room temperature 90min;
(3), then joined in Centricon-10 concentration tube by antibody-solutions, putting into and under 3000g, concentrating 30min in high speed freezing centrifuge is 0.5ml to volume;
(4), get 0.5ml magnetic bead and add in 5ml reaction cup, put into test tube rack special, draw supernatant through magnet adsorption after 2 minutes;
(5) add the PB damping fluid of the 0.1mol/L of 1.5ml pH9.5, at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions of acquisition is joined in above-mentioned magnetic bead, room temperature reaction 4 hours after mixing;
(6) the TRIS solution 37 DEG C 15 minutes of 0.3ml 1mol/L, is added;
(7) magnetic bead that the PB buffer solution for cleaning, at every turn adding the 0.1mol/L of 1.5ml pH7.2 has marked, mixes 30 seconds, added, removes supernatant, repetitive operation 3 times;
(8), with 100ml magnetic bead conserving liquid magnetic bead is proceeded to 125ml vial, be the HE4 Magneto separate reagent of 0.05%; Magnetic bead conserving liquid formula is 0.1% BSA, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 DEG C of preservations;
(9), by the Magneto separate reagent magnetic bead buffer solution obtained mix according to the ratio of 1:1, obtain Magneto separate reagent;
The preparation of step 2, enzyme reaction thing
The preparation of step 3, increased response agent
(1) TRIS1.56g and NaCl 4.23g, is taken in 1L container; Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container;
(2), with graduated cylinder measure 800ml purified water in above-mentioned 1L container, fully stir until dissolve completely, then adjust pH, control pH is between 7.35-7.45;
(3) Mak33 0.9g, is taken in above-mentioned 1L container; Finally be settled to 1000ml, after dissolving completely, use 0.2um frit;
The preparation of step 4, calibration object dilution
The preparation of step 5, calibration object and quality-control product
The preparation of step 6, cleaning concentrate
(1) KCl 4g, NaCl40g, sucrose 10g, is taken in 1L container;
(2), take 0.225g Tween-20 and add after 15ml water makes it dissolve completely in 100ml container, pour in above-mentioned 1L container;
(3), with pipettor Proclin-300 measured after 0.225ml dissolves completely in the beaker filling 15ml purified water, pour in above-mentioned 1L container;
(4), with graduated cylinder measure 800ml purified water in above-mentioned 1L container, fully stir until dissolve completely;
(5), adjust pH, control its scope between 7.35-7.45;
(6), finally 1000ml is settled to, with 0.2um frit and get final product after dissolving completely;
The preparation of step 7, substrate solution
The detecting step of described people's epididymal secretory protein 4 is as follows:
(1), add bottom 50 μ l people epididymal secretory protein 4 calibration objects, quality-control product, sample to be measured to corresponding micropore;
(2) 50 μ l enzyme reaction things, are added in each micropore;
(3) 50 μ l increased response agent, are added in each micropore;
(4) 50 μ l Magneto separate reagent, are added in each micropore;
(5), use covered rearing with plastic film micropore, multitube vortex mixer vibrates after microwell plate 30s gently, puts 37 DEG C of water-baths 30 minutes;
(6), microwell plate puts on magnetic separator, guarantees that each micropore contacts with separator surface, precipitates 2 minutes; Supernatant poured out by the separation vessel that reverses slowly, and the microwell plate of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on micropore;
(7), after cleaning concentrate purified water dilutes 20 times, add 200 μ l dilute after cleaning fluid in each micropore, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, and mixing is wanted thoroughly;
(8), repeat step (6), (7), (6) one times;
(9), add in 100 μ l substrate solutions to micropore mix 3 seconds, detect with ready luminometer rapidly;
(10), with the Log value of calibration object concentration for horizontal ordinate, the Log value of RLU draws out typical curve (double logarithmic curve) for ordinate, on typical curve, the concentration of the HE4 of this serum is found with each test serum RLU value, wherein ordinate is luminous intensity, and horizontal ordinate is HE4 concentration (unit is pmol/L).
The present inventor's epididymal secretory protein 4 quantitative measurement detection method, the preparation of described substrate solution comprises the following steps:
One, the preparation steps of substrate solution A
(1), take borax 5.721g, boric acid 2.474g, luminol 1.0g and to iodophenol 0.1mg in 1L beaker;
(2), with graduated cylinder measure 400ml purified water in 1L beaker, fully stir until dissolve completely, adjust pH, control its scope between 7.95-8.05;
(3), with 0.2um frit collect filtrate, be settled to 500ml by purified water, after mixing and get final product;
Two, the preparation of substrate solution B
(1) borax 5.721g, boric acid 2.474g, urea peroxide 0.1g and PC300 250ul, is taken in 1L beaker;
(2), with graduated cylinder measure 400ml purified water in 1L beaker, fully stir until dissolve completely, adjust pH to control its scope between 7.95-8.05;
(3), with 0.2um frit collect filtrate, be settled to 500ml by purified water, after mixing and get final product.
Compared with prior art, the invention has the beneficial effects as follows:
The present inventor's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof have higher sensitivity and specificity, the time of shorter acquisition testing result and easier mode of operation, to having significant application value in the case fatality rate of the early diagnosis of ovarian epithelial carcinoma, curative effect monitoring and Index for diagnosis, reduction ovarian cancer patients.
Accompanying drawing explanation
Fig. 1 is the structural representation of the present inventor's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof.
Fig. 2 is the vertical view after Fig. 1 removes lid and microwell plate.
Fig. 3 is the structural representation of microwell plate in Fig. 1.
Wherein:
Box body 1
Lid 2
Back up pad 3
Hole 4
Reagent bottle 5
Microwell plate 6
Micropore 7.
Embodiment
See Fig. 1-Fig. 3, the present inventor's epididymal secretory protein 4 quantitative determination reagent kit, comprise the lid 2 above box body 1 and box body, the back up pad 3 be made up of polyfoam being provided with microwell plate 6 in described box body 1 and being positioned at bottom box body 1, wherein microwell plate 6 is made up of 48 or 96 micropores 7, back up pad 3 has multiple hole 4, eight reagent bottles 5 are placed with altogether in described hole 4, respectively containing Magneto separate reagent in described eight reagent bottles 5, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution, wherein Magneto separate reagent contains the magnetic particle of people's epididymal secretory protein 4 monoclonal antibody bag quilt.
Preparation method
The preparation of step one, Magneto separate reagent
One, magnetic bead buffer solution preparation
1, take TRIS 4.58g and NaCl6.81g in 1L container, then take 0.96g TWEEN-20 and add after suitable quantity of water makes it dissolve completely in 20ml container, then pour in above-mentioned 1L container;
2, Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container, then in above-mentioned 1L container, add 800ml purified water, fully stir;
3, regulate pH meter to measure its pH value, control pH is between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, last 1L container is settled to 1000ml, uses 0.2um frit; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered.
Two, the preparation of Magneto separate reagent
1,1.0mg disuccinimidyl suberate is dissolved in 50ulDMSO, get the anti-HE4 monoclonal antibody of 2mg be dissolved in the PB damping fluid of the 0.1mol/L of 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 above-mentioned HE4 monoclonal antibody solution, put room temperature 90min;
3, then joined in Centricon-10 concentration tube by antibody-solutions, putting into and under 3000g, concentrating 30min in high speed freezing centrifuge is 0.5ml to volume;
4, get 0.5ml magnetic bead to add in 5ml reaction cup, put into test tube rack special, draw supernatant through magnet adsorption after 2 minutes;
5, add the PB damping fluid of the 0.1mol/L of 1.5ml pH9.5 at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions of acquisition is joined in above-mentioned magnetic bead, room temperature reaction 4 hours after mixing;
6, the TRIS solution 37 DEG C 15 minutes of 0.3ml 1mol/L is added;
7, the magnetic bead that the PB buffer solution for cleaning at every turn adding the 0.1mol/L of 1.5ml pH7.2 has marked, mixes 30 seconds, added, removes supernatant, repetitive operation 3 times;
8, with 100ml magnetic bead conserving liquid, magnetic bead is proceeded to 125ml vial, be the HE4 Magneto separate reagent of 0.05%;
Magnetic bead conserving liquid formula is 0.1% BSA, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 DEG C of preservations.
9, the Magneto separate reagent magnetic bead buffer solution obtained is mixed according to the ratio of 1:1, obtain Magneto separate reagent in kit of the present invention.
The preparation of step 2, enzyme reaction thing
One, the preparation of enzyme reaction thing dilution
1, get Tris 4.846g, HCl 2847 μ l in flask, then in flask, add 800ml purified water, fully stir and reagent is dissolved completely;
2, adjust pH, control pH is at 7.35-7.45;
3, taking BSA 4g pours in above-mentioned beaker;
4, last beaker is settled to 400ml, with 0.2um frit and get final product.
Two, the coupling of horseradish peroxidase (HRP) and HE4 antigen
1, getting HE4-3-cmo-BSA antigen 1 mg is positioned in 1ml glass tube;
2, get 200ul DMSO and dissolve the final concentration arrival 5mg/ml that antigen makes antigen, then fully mix;
3, the molar ratio adding the disuccinimidyl suberate of 10mol according to 1mol antigen adds disuccinimidyl suberate in above-mentioned 2 solution, reacts 1.5 hours in 37 DEG C of constant temperature ovens;
4, the mol ratio adding (HRP) of 1mol according to 3mol antigen adds (HRP) in the solution of above-mentioned 3, and the PB damping fluid of the pH then adding 1ml to be 7.4 concentration be 0.1M, is placed in 37 degree of constant temperature ovens reactions 3 hours;
5, by the solution PD-10 post purifying of above-mentioned 4, collect refined solution, add according to the volume of 1:3000 the enzyme reaction thing dilution obtained, mix and obtain enzyme reaction thing.
The preparation of step 3, increased response agent
1, TRIS1.56g and NaCl 4.23g is taken in 1L container; Proclin-300 measured after 0.2ml dissolves completely in the beaker of 10ml purified water with pipettor, pour in above-mentioned 1L container;
2, measure 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir until dissolve completely, then adjust pH value, control pH is between 7.35-7.45;
3, Mak33 0.9g is taken in above-mentioned 1L container; Finally be settled to 1000ml, after dissolving completely, use 0.2um frit.
The preparation of step 4, calibration object dilution
1, take Tris 7.268g and Hcl 4270 μ l in the container of 1L, be settled to 600ml;
2, measure calf serum 300ml with graduated cylinder, add in above-mentioned solution, calibration object dilution is for subsequent use.
The preparation of step 5, calibration object and quality-control product
Calibration object concentration is respectively 4,8,16,32,64pmol/L; Quality-control product concentration is respectively 0.75,7.5ng/ml.
The preparation of step 6, cleaning concentrate
1, Kcl 4g, NaCl40g, sucrose 10g is taken in 1L container;
2, take 0.225g Tween-20 adds after 15ml water makes it dissolve completely in 100ml container, pours in above-mentioned 1L container;
3, Proclin-300 measured after 0.225ml dissolves completely in the beaker filling 15ml purified water with pipettor, pour in above-mentioned 1L container;
4, measure 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir until dissolve completely;
5, adjust pH, control its scope between 7.35-7.45;
6, finally 1000ml is settled to, with 0.2um frit and get final product after dissolving completely.
The preparation of step 7, substrate solution
One, the preparation steps of substrate solution A
1, take borax 5.721g, boric acid 2.474g, luminol 1.0g and to iodophenol 0.1mg in 1L beaker;
2, measure 400ml purified water in 1L beaker with graduated cylinder, fully stir until dissolve completely, adjust pH, control its scope between 7.95-8.05;
3, collect filtrate with 0.2um frit, be settled to 500ml by purified water, after mixing and get final product.
Two, the preparation of substrate solution B
1, borax 5.721g, boric acid 2.474g, urea peroxide 0.1g and PC300 250ul is taken in 1L beaker;
2, measure 400ml purified water in 1L beaker with graduated cylinder, fully stir until dissolve completely, adjust pH to control its scope between 7.95-8.05;
3, collect filtrate with 0.2um frit, be settled to 500ml by purified water, after mixing and get final product.
The product packing of gained is semi-manufacture; extract three parts out and could assemble adult's epididymal secretory protein 4(HE4 through specificity, accuracy, sensitivity and stability assay approval) micropore plate type magnetic granule chemoluminescence immunoassay measuring kit, also need after being assembled into kit that sampling observation is qualified just can dispatch from the factory afterwards.
Principle of work:
The present invention is a kind of detection method that double antibody sandwich method chemiluminescence immunoassay combines with magnetic particle isolation technics.Quantitative enzyme mark HE4 antigen, the HE4 monoclonal antibody combining magnetic particle and stabilizing reinforcer is added in sample, calibration object and quality-control product.37 DEG C hatch after, in enzyme mark HE4 antigen and sample, calibration object and quality-control product HE4 compete with the HE4 monoclonal antibody generation specific binding combining magnetic particle.Direct precipitation in externally-applied magnetic field, does not need centrifugal namely separable.Remove supernatant, the compound of washing and precipitating, then adds enzyme-catalyzed chemical luminescence substrate.Substrate is catalyzed cracking under enzyme effect, forms unstable excited state intermediate, just sends photon when excited state intermediate gets back to ground state, forms luminescence-producing reaction, can use the luminous intensity of light-emitting appearance detection reaction.In sensing range, the HE4 concentration in the luminous intensity of reaction and sample is inversely proportional to.
Using method
(1) pre-treatment of sample:
Before using this kit to test, first need take out the HE4 antibody of horseradish peroxidase-labeled, calibration object/testing sample, HE4 antibody magnetic particle, luminous substrate liquid and cleansing solution, place 15-30min in room temperature, make them equilibrate to room temperature; Afterwards, constant temperature oven or water-bath are adjusted to 37 DEG C; Get out suitable micro sample adding appliance and corresponding suction nozzle and check that whether Chemiluminescence Apparatus is working properly.
(2) operation steps:
1, add bottom 50 μ l HE4 calibration objects, quality-control product, sample to be measured to corresponding micropore;
2,50 μ l enzyme reaction things are added in each micropore;
3,50 μ l increased response agent are added in each micropore;
4,50 μ l Magneto separate reagent are added in each micropore;
5, use covered rearing with plastic film micropore, multitube vortex mixer vibrates after microwell plate 30s gently, puts 37 DEG C of water-baths 30 minutes;
6, microwell plate is put on magnetic separator, guarantees that each micropore contacts with separator surface, precipitates 2 minutes; Supernatant poured out by the separation vessel that reverses slowly, and the microwell plate of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on micropore;
7, after cleaning concentrate purified water dilutes 20 times, add 200 μ l dilute after cleaning fluid in each micropore, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, and mixing is wanted thoroughly;
8, repeat step 6,7,6 one times;
9, add in 100 μ l substrate solutions to micropore and mix 3 seconds, detect with ready luminometer rapidly;
10, as run into high level HOOK sample, in order to avoid there is high level HOOK effect, suggestion clinician selects suitable extension rate to dilute sample according to all the other test indexs.
With the Log value of calibration object concentration for horizontal ordinate, the Log value of RLU draws out typical curve (double logarithmic curve) for ordinate, on typical curve, the concentration of the HE4 of this serum is found with each test serum RLU value, wherein ordinate is luminous intensity, and horizontal ordinate is HE4 concentration (unit is pmol/L).
Performance index
1) accuracy: kit calibration object and national calibration object Simultaneously test, two not remarkable parallel deviates of dose-response curve.With national calibration object for reference substance, the actual value of kit calibration object kit should in the scope of 0.90-1.10 with the ratio of sign value
2) dose-response curve is linear: use double-log Model fitting, and in 4-64pmol/L concentration range, the exhausted angle value of the related coefficient (r) of dose-response curve is not less than 0.9900
3) accuracy: CV≤15%
4) limit of identification: <2pmol/l
5) quality controlled serum measured value: should within the scope of Quality Control
6) specificity: cross reacting rate should be less than 0.2%
7) stability: placed 7 days by kit 37 DEG C, measurement result should meet above-mentioned 1) ~ 5) item requirement
The present invention has the following advantages:
1, chemiluminescence combines with immune magnetic particle by the present invention, provides a kind of reaction system close to homogeneous phase, compared with prior art, highly sensitive, high specificity, sensing range is wide, simple to operate, no radioactivity pollute, kit cost is low, and clinical applicability is strong.
2, the present invention carries out screening experiment and quality arbitration to starting material used, comprise the activity of coated antibody, the absorption property of magnetic particle and variation size, (HRP) activity, the luminous intensity of chemical luminous substrate and lighting time interval etc., mark for (HRP) can have diverse ways, by repeatedly explore and contrast test finally have found simply, productive rate is high, cost is low, the labeling method of reliable in quality.
3, the present invention adopts a kind of new special stabilizing reinforcer and cleaning concentrate, and make course of reaction more reliable and more stable, experimental data is effectively sensitive, while enhancing product performance, and greatly reduces cost of products.
4, the Magneto separate reagent in kit, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution are all the optimization formulas under this reaction system, provide powerful guarantee to the use of this kit effect phase and detection perform.
5, the present invention adopts microwell plate magnetic particle to incorporate two kinds of technology, be mainly reflected in: (1) utilizes magnetic particle in the maximum principle of the situation following table area of same volume, can in conjunction with more antibody, thus make the sensitivity of reaction higher, the range of linearity be wider; (2) can measure by mass simultaneous, be applicable to screening serum in enormous quantities.
Claims (2)
1. people's epididymal secretory protein 4 quantitative measurement detection method of non-diagnostic object, it is characterized in that described detection method comprises reagent set-up procedure and detecting step, described reagent set-up procedure is as follows:
The preparation of step one, Magneto separate reagent
One, magnetic bead buffer solution preparation
(1), take TRIS 4.58g and NaCl6.81g in 1L container, then take 0.96g TWEEN-20 and add after suitable quantity of water makes it dissolve completely in 20mL container, then pour in above-mentioned 1L container;
(2), with pipettor Proclin-300 measured after 0.2mL dissolves completely in the beaker of 10mL purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add 800mL purified water, fully stir;
(3), regulate pH meter to measure its pH value, control pH is between 7.95-8.05;
(4), taking BSA 3g pours in above-mentioned 1L container;
(5), last 1L container is settled to 1000mL, with 0.2 μm of frit; Label is posted in 2-8 DEG C of refrigeration house storage after having filtered;
Two, the preparation of Magneto separate reagent
(1), by 1.0mg disuccinimidyl suberate be dissolved in 50 μ LDMSO, get the anti-HE4 monoclonal antibody of 2mg be dissolved in the PB damping fluid of the 0.1mol/L of pH9.5 to cumulative volume be 1mL;
(2), determine the input amount of disuccinimidyl suberate, draw disuccinimidyl suberate with liquid-transfering gun and join in above-mentioned HE4 monoclonal antibody solution, put room temperature 90min;
(3), then joined in Centricon-10 concentration tube by antibody-solutions, putting into and under 3000g, concentrating 30min in high speed freezing centrifuge is 0.5mL to volume;
(4), get 0.5mL magnetic bead and add in 5mL reaction cup, put into test tube rack special, draw supernatant through magnet adsorption after 2 minutes;
(5) add the PB damping fluid of the 0.1mol/L of 1.5mL pH9.5, at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions of acquisition is joined in above-mentioned magnetic bead, room temperature reaction 4 hours after mixing;
(6) the TRIS solution 37 DEG C 15 minutes of 0.3mL 1mol/L, is added;
(7) magnetic bead that the PB buffer solution for cleaning, at every turn adding the 0.1mol/L of 1.5mL pH7.2 has marked, mixes 30 seconds, added, removes supernatant, repetitive operation 3 times;
(8), with 100mL magnetic bead conserving liquid magnetic bead is proceeded to 125mL vial, be the HE4 Magneto separate reagent of 0.05%; Magnetic bead conserving liquid formula is 0.1% BSA, 0.05% Tween-20,0.02%NaN
3, 20% ethanol, 4 DEG C of preservations;
(9), by the Magneto separate reagent magnetic bead buffer solution obtained mix according to the ratio of 1:1, obtain Magneto separate reagent;
The preparation of step 2, enzyme reaction thing
The preparation of step 3, increased response agent
(1) TRIS1.56g and NaCl 4.23g, is taken in 1L container; Proclin-300 measured after 0.2mL dissolves completely in the beaker of 10mL purified water with pipettor, pour in above-mentioned 1L container;
(2), with graduated cylinder measure 800mL purified water in above-mentioned 1L container, fully stir until dissolve completely, then adjust pH, control pH is between 7.35-7.45;
(3) Mak33 0.9g, is taken in above-mentioned 1L container; Finally be settled to 1000mL, after dissolving completely, with 0.2 μm of frit;
The preparation of step 4, calibration object dilution
The preparation of step 5, calibration object and quality-control product
The preparation of step 6, cleaning concentrate
(1) KCl4g, NaCl40g, sucrose 10g, is taken in 1L container;
(2), take 0.225g Tween-20 and add after 15mL water makes it dissolve completely in 100mL container, pour in above-mentioned 1L container;
(3), with pipettor Proclin-300 measured after 0.225mL dissolves completely in the beaker filling 15mL purified water, pour in above-mentioned 1L container;
(4), with graduated cylinder measure 800mL purified water in above-mentioned 1L container, fully stir until dissolve completely;
(5), adjust pH, control its scope between 7.35-7.45;
(6), finally 1000mL is settled to, with 0.2 μm of frit and get final product after dissolving completely;
The preparation of step 7, substrate solution
The detecting step of described people's epididymal secretory protein 4 is as follows:
(1), add bottom 50 μ L people epididymal secretory protein 4 calibration objects, quality-control product, sample to be measured to corresponding micropore;
(2) 50 μ L enzyme reaction things, are added in each micropore;
(3) 50 μ L increased response agent, are added in each micropore;
(4) 50 μ L Magneto separate reagent, are added in each micropore;
(5), use covered rearing with plastic film micropore, multitube vortex mixer vibrates after microwell plate 30s gently, puts 37 DEG C of water-baths 30 minutes;
(6), microwell plate puts on magnetic separator, guarantees that each micropore contacts with separator surface, precipitates 2 minutes; Supernatant poured out by the separation vessel that reverses slowly, and the microwell plate of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on micropore;
(7), after cleaning concentrate purified water dilutes 20 times, add 200 μ L dilute after cleaning fluid in each micropore, to put on multitube vortex mixer vibration mixing 30s gently; Application of sample dynamics should be avoided during application of sample excessive and cause magnetic bead to spill, and mixing is wanted thoroughly;
(8), repeat step (6), (7), (6) one times;
(9), add in 100 μ L substrate solutions to micropore mix 3 seconds, detect with ready luminometer rapidly;
(10), with the Log value of calibration object concentration for horizontal ordinate, the Log value of RLU draws out typical curve for ordinate, and on typical curve, find the concentration of the HE4 of this serum with each test serum RLU value, wherein ordinate is luminous intensity, horizontal ordinate is HE4 concentration, and unit is pmol/L.
2. people's epididymal secretory protein 4 quantitative measurement detection method of a kind of non-diagnostic object according to claim 1, is characterized in that the preparation of described substrate solution comprises the following steps:
One, the preparation steps of substrate solution A
(1), take borax 5.721g, boric acid 2.474g, luminol 1.0g and to iodophenol 0.1mg in 1L beaker;
(2), with graduated cylinder measure 400mL purified water in 1L beaker, fully stir until dissolve completely, adjust pH, control its scope between 7.95-8.05;
(3), with 0.2 μm of frit collect filtrate, be settled to 500mL by purified water, after mixing and get final product;
Two, the preparation of substrate solution B
(1) borax 5.721g, boric acid 2.474g, urea peroxide 0.1g and PC300 250 μ L, is taken in 1L beaker;
(2), with graduated cylinder measure 400mL purified water in 1L beaker, fully stir until dissolve completely, adjust pH to control its scope between 7.95-8.05;
(3), with 0.2 μm of frit collect filtrate, be settled to 500mL by purified water, after mixing and get final product.
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| CN104237532A (en) * | 2014-09-10 | 2014-12-24 | 广州市达瑞抗体工程技术有限公司 | Quantitative determination kit for human epididymis secretory protein 4 |
| CN104597235B (en) * | 2015-01-31 | 2017-03-29 | 苏州大学 | A kind of people's epididymal proteins immunity detection reagent and its using method |
| CN104897901B (en) * | 2015-05-12 | 2017-07-11 | 西安金磁纳米生物技术有限公司 | A kind of method of the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles |
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| CN102095846A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for carbohydrate antigen 242 |
| EP2609429B1 (en) * | 2010-08-26 | 2017-09-20 | University Of Washington Through Its Center For Commercialization | Methods for detecting anti-he4 antibodies and methods of diagnosis and/or prognosis of conditions associated with he4-expressing cells |
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| CN203414474U (en) * | 2013-08-06 | 2014-01-29 | 江苏福隆生物技术有限公司 | Quantitative determination kit of human epididymis epithelial secretory protein 4 |
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