CN203414474U - Quantitative determination kit of human epididymis epithelial secretory protein 4 - Google Patents
Quantitative determination kit of human epididymis epithelial secretory protein 4 Download PDFInfo
- Publication number
- CN203414474U CN203414474U CN201320476898.0U CN201320476898U CN203414474U CN 203414474 U CN203414474 U CN 203414474U CN 201320476898 U CN201320476898 U CN 201320476898U CN 203414474 U CN203414474 U CN 203414474U
- Authority
- CN
- China
- Prior art keywords
- box body
- secretory protein
- quantitative determination
- reagent
- magnetic separation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model discloses a quantitative determination kit of human epididymis epithelial secretory protein 4. The quantitative determination kit comprises a box body (1) and a box cover (2) above the box body, and is characterized in that the inside of the box body (1) is provided with a micropore plate (6) and a support plate (3) which is arranged on the bottom of the box body (1); the micropore plate (6) consists of 48 or 96 micropores (7); a plurality of holes (4) are formed in the support plate (3); eight reagent bottles (5) are placed in the holes (4); the eight reagent bottles (5) contain a magnetic separation reagent, an enzyme reagent, a stable reinforcing agent, a calibration product, a quality control product, a cleaning concentration solution, a diluent and a substrate solution respectively; the magnetic separation reagent contains magnetic particles which are coated by a human epididymis epithelial secretory protein 4 monoclonal antibody.
Description
Technical field
The utility model relates to a kind of people's epididymal secretory protein 4 quantitative determination reagent kits, for people's epididymal secretory protein 4 content of external quantitative measurement human serum.
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 that oophoroma early symptom is very not obvious, lacks effective early diagnosis because ovary is ensconced in bone chamber deeply, and 75% patient has belonged to late period when medical.
People's epididymal secretory protein 4(Human epididymis protein4, HE4) be a kind of novel tumor mark of developing in recent years, HE4 encoding gene is to be 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, finding that HE4(has another name called WFDC2) mRNA do not express in high expressed ，Er cancer beside organism in ovarian cancer tissue.Confirm that afterwards HE4 has the value of ovarian cancer diagnosis, 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 to improve result for the treatment of, detection angles, for measuring the method for HE4, mainly contain radioimmunoassay technology, Enzyme-multiplied immune technique and chemiluminescence etc. clinically at present.
Past be take people's epididymal secretory protein 4(HE4 that radioimmunoassay technology is representative) measure kit due to methodological restriction,, there is very large drawback in its sensitivity and antijamming capability wretched insufficiency, substantially withdraws from the market; What application was more at present is Enzyme-multiplied immune technique and chemiluminescence, wherein chemiluminescence was risen eighties of last century eighties, it is the emerging technology growing up after Enzyme-multiplied immune technique and radioimmunoassay technology, due to its high sensitivity, high specific, while method is easy, quick, mark bond is stable, and the features such as "dead" isotope damage and pollution, have obtained develop rapidly in recent years.
Current people's epididymal secretory protein 4(HE4 of publicity) Patents is totally 11, comprising chemiluminescence, Enzyme-multiplied immune technique, time-resolved fluorescence, detect correlation technique, and have no magnetic particle isolation technics, combine and be applied in immunoassay with chemiluminescence.Magnetic particle separation enzyme-linked immunoassay technology is a kind ofly to take magnetic particle as solid phase carrier of separating, immune magnetic particle isolation technics is combined with enzyme linked immunosorbent detection technology and a kind of Novel immune detection method of setting up.
Summary of the invention
The purpose of this utility model is to overcome above-mentioned deficiency, and a kind of highly sensitive, high specificity, people's epididymal secretory protein 4 quantitative determination reagent kits simple to operate are provided.
The purpose of this utility model is achieved in that
A kind of people's epididymal secretory protein 4 quantitative determination reagent kits, the lid that comprises box body and box body top, in described box body, be provided with microwell plate and the back up pad that is positioned at box body bottom, wherein microwell plate is comprised of 48 or 96 micropores, in back up pad, have a plurality of holes, in described hole, be placed with altogether eight reagent bottles, in described eight reagent bottles, contain respectively magnetic separation agent, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution, wherein magnetic separation agent contains the coated magnetic particle of people's epididymal secretory protein 4 monoclonal antibodies.
Compared with prior art, the beneficial effects of the utility model are:
The utility model people epididymal secretory protein 4 quantitative determination reagent kits have higher sensitivity and specificity, the time of shorter acquisition testing result and easier mode of operation, to the significant application value that has in the case fatality rate of the early diagnosis of ovarian epithelial carcinoma, curative effect monitoring and prognosis judgement, reduction ovarian cancer patients.
Accompanying drawing explanation
Fig. 1 is the structural representation of the utility model people epididymal secretory protein 4 quantitative determination reagent kits.
Fig. 2 is that Fig. 1 removes the vertical view after 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
Referring to Fig. 1-Fig. 3, the utility model people epididymal secretory protein 4 quantitative determination reagent kits, the lid 2 that comprises box body 1 and box body top, in described box body 1, be provided with microwell plate 6 and the back up pad of being made by polyfoam 3 that is positioned at box body 1 bottom, wherein microwell plate 6 is comprised of 48 or 96 micropores 7, in back up pad 3, have a plurality of holes 4, described hole 4 is interior is placed with eight reagent bottles 5 altogether, the interior magnetic separation agent that contains respectively of described eight reagent bottles 5, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution, wherein magnetic separation agent contains the coated magnetic particle of people's epididymal secretory protein 4 monoclonal antibodies.
Preparation method
The preparation of step 1, magnetic separation agent
One, magnetic bead buffer solution preparation
1, take TRIS 4.58g and NaCl6.81g in 1L container, after then taking 0.96g TWEEN-20 and adding suitable quantity of water in 20ml container it is dissolved completely, then pour in above-mentioned 1L container;
2, with pipettor, Proclin-300 is 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 its pH value of PH instrumentation amount, control PH between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, last 1L container is settled to 1000ml, with 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage.
Two, the preparation of magnetic separation agent
1,1.0mg disuccinimidyl suberate is dissolved in 50ulDMSO, get in the PB damping fluid of the 0.1mol/L that the anti-HE4 monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, determine the input amount of disuccinimidyl suberate, with liquid-transfering gun, draw disuccinimidyl suberate and join in above-mentioned HE4 monoclonal antibody solution, put room temperature 90min;
3, then antibody-solutions is joined in Centricon-10 concentration tube, put in high speed freezing centrifuge under 3000g concentrated 30min to volume be 0.5ml;
4, get 0.5ml magnetic bead and add in 5ml reaction cup, put into test tube rack special, through magnet adsorption, after 2 minutes, draw supernatant;
5, the PB damping fluid that at every turn adds the 0.1mol/L of 1.5ml PH9.5, mixes 30 seconds, added, removes supernatant, repetitive operation 3 times; The antibody-solutions of acquisition is joined in above-mentioned magnetic bead, mix rear room temperature reaction 4 hours;
6,37 ℃ of the TRIS solution 15 minutes that add 0.3ml 1mol/L;
7, the PB buffer solution for cleaning magnetic bead of mark that at every turn adds the 0.1mol/L of 1.5ml PH7.2, mixes 30 seconds, added, removes supernatant, repetitive operation 3 times;
8, with 100ml magnetic bead, preserve liquid magnetic bead is proceeded to 125ml vial, be 0.05% HE4 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 of acquisition is mixed according to the ratio of 1:1 by magnetic bead buffer solution, obtain magnetic separation agent in the utility model kit.
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 reagent is dissolved completely;
2, adjust PH, control PH at 7.35-7.45;
3, taking BSA 4g pours in above-mentioned beaker;
4, last beaker is settled to 400ml, with 0.2um filter, filters 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, according to 1mol antigen, add the molar ratio of the disuccinimidyl suberate of 10mol to add disuccinimidyl suberate in above-mentioned 2 solution, in 37 ℃ of constant temperature ovens, react 1.5 hours;
4, according to 3mol antigen, add the mol ratio of 1mol (HRP) to add (HRP) in above-mentioned 3 solution, the PH that then adds 1ml is that 7.4 concentration are the PB damping fluid of 0.1M, is placed in 37 degree constant temperature ovens and reacts 3 hours;
5, above-mentioned 4 solution is purified with PD-10 post, collect refined solution, according to the volume of 1:3000, add the enzyme reaction thing dilution obtaining, mix and obtain enzyme reaction thing.
The preparation of step 3, increased response agent
1, take TRIS1.56g and NaCl 4.23g in 1L container; With pipettor, Proclin-300 is measured after 0.2ml dissolves completely in the beaker of 10ml purified water, pour in above-mentioned 1L container;
2, with graduated cylinder, measure 800ml purified water in above-mentioned 1L container, fully stir until then dissolving completely adjusts pH value, control PH between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container; Finally be settled to 1000ml, after dissolving completely, with 0.2um filter, filter.
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, with graduated cylinder, measure calf serum 300ml, add in above-mentioned solution, calibration object dilution is standby.
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, take Kcl 4g, NaCl40g, sucrose 10g in 1L container;
2, after taking 0.225g Tween-20 and adding 15ml water in 100ml container it is dissolved completely, pour in above-mentioned 1L container;
3, with pipettor, Proclin-300 is measured after 0.225ml dissolves completely in the beaker that fills 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, be finally settled to 1000ml, after dissolving completely, with 0.2um filter, filter and get final product.
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, with graduated cylinder, measure 400ml purified water in 1L beaker, fully stir until PH is adjusted in dissolving completely, control its scope between 7.95-8.05;
3, with 0.2um filter, filter and collect filtrate, by purified water, be settled to 500ml, after mixing and get final product.
Two, the preparation of substrate solution B
1, take borax 5.721g, boric acid 2.474g, urea peroxide 0.1g and PC300 250ul in 1L beaker;
2, with graduated cylinder, measure 400ml purified water in 1L beaker, fully stir until dissolving completely adjusts PH to control its scope between 7.95-8.05;
3, with 0.2um filter, filter and collect filtrate, by purified water, be settled to 500ml, after mixing and get final product.
The product packing of gained is semi-manufacture; extract three parts out and could assemble through specificity, accuracy, sensitivity and stability assay approvals the epididymal secretory protein 4(HE4 that is grown up) micropore plate type magnetic granule chemoluminescence immunoassay measuring kit, be assembled into and also need to inspect by random samples qualified just can dispatch from the factory afterwards after kit.
Principle of work:
The utility model is a kind of detection method that double antibody sandwich method chemiluminescence immunoassay combines with magnetic particle isolation technics.In sample, calibration object and quality-control product, add quantitative enzyme mark HE4 antigen, in conjunction with HE4 monoclonal antibody and the stabilizing reinforcer of magnetic particle.37 ℃ hatch after, in enzyme mark HE4 antigen and sample, calibration object and quality-control product HE4 competition with in conjunction with the HE4 monoclonal antibody generation specific binding of magnetic particle.Direct precipitation in externally-applied magnetic field, does not need centrifugal separable.Remove supernatant, the compound of washing and precipitating, then adds enzyme-catalyzed chemical luminescence substrate.Substrate is catalyzed cracking under enzyme effect, forms unsettled excited state intermediate, just sends photon when 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 HE4 concentration in sample are inversely proportional to.
Using method
(1) pre-treatment of sample:
Before using this kit to test, need first take out HE4 antibody, calibration object/testing sample, HE4 antibody magnetic particle, luminous substrate liquid and the cleansing solution of horseradish peroxidase-labeled, in room temperature, place 15-30min, make them equilibrate to room temperature; Afterwards, constant temperature oven or water-bath are adjusted to 37 ℃; Be ready to suitable micro sample adding appliance and corresponding suction nozzle and check that whether Chemiluminescence Apparatus is working properly.
(2) operation steps:
1, add 50 μ l HE4 calibration objects, quality-control product, sample to be measured to corresponding micropore bottom;
2, add 50 μ l enzyme reaction things to each micropore;
3, add 50 μ l increased response agent to each micropore;
4, add 50 μ l magnetic separation agents to each micropore;
5, with plastic sheeting, cover micropore, multitube vortex mixer vibrates after microwell plate 30s gently, puts 37 ℃ of water-baths 30 minutes;
6, microwell plate is put to magnetic separator, guarantees that each micropore contacts with separator surface, precipitates 2 minutes; The separation vessel that reverses is slowly poured out supernatant, and the microwell plate of reversing is placed on filter paper together with separation vessel, firmly bounces separation vessel bottom to remove all drops that are bonded on micropore;
7, cleaning concentrate is with after 20 times of purified water dilutions, adds cleaning fluid after 200 μ l dilutions to each micropore, puts on multitube vortex mixer vibration gently and mixes 30s; During application of sample, should avoid application of sample dynamics excessive and cause magnetic bead to spill, and mix and want thoroughly;
8, repeating step is 6,7,6 one times;
9, add 100 μ l substrate solutions and mix 3 seconds to micropore, with ready luminous detector, detect rapidly;
10,, as run into high value HOOK sample, for fear of there is high value HOOK effect, suggestion clinician selects suitable extension rate to dilute sample according to all the other test indexs.
The Log value of calibration object concentration of take is horizontal ordinate, the Log value of RLU is drawn out typical curve (double logarithmic curve) for ordinate, with each test serum RLU value, on typical curve, find the concentration of the HE4 of this serum, 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 are measured simultaneously, two not remarkable parallel deviates of dose-response curve.Take national calibration object as reference substance, and the actual value of kit calibration object kit should be in the scope of 0.90-1.10 with the ratio of sign value
2) linearity of dose-response curve: use double-log Model fitting, in 4-64pmol/L concentration range, the exhausted degree value of the related coefficient of dose-response curve (r) is not less than 0.9900
3) accuracy: CV≤15%
4) limit of identification: <2pmol/l
5) quality controlled serum measured value: should be within the scope of Quality Control
6) specificity: cross reacting rate should be less than 0.2%
7) stability: 37 ℃ of kits are placed 7 days, and measurement result should meet above-mentioned 1) ~ 5) requirement
The utlity model has following advantage:
1, the utility model combines chemiluminescence with immune magnetic particle, and a kind of reaction system that approaches homogeneous phase is provided, 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 utility model carries out screening experiment and quality arbitration to starting material used, the activity that comprises 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 exploring and contrast test has finally found simply, productive rate is high, cost is low, the labeling method of reliable in quality.
3, the utility model adopts a kind of new special-purpose stabilizing reinforcer and cleaning concentrate, makes course of reaction more reliable and more stable, and experimental data is sensitive effectively, when enhancing product performance, and greatly reduces cost of products.
4, the magnetic separation agent 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, and giving the use effect phase of this kit and detecting performance provides powerful guarantee.
5, the utility model adopts microwell plate magnetic particle to integrate two kinds of technology, be mainly reflected in: (1) utilizes magnetic particle in the principle of the situation following table area maximum of same volume, can be in conjunction with more antibody, thus make the sensitivity of reaction higher, and the range of linearity is wider; (2) can measure by mass simultaneous, be applicable to screening serum in enormous quantities.
Claims (1)
1. people's epididymal secretory protein 4 quantitative determination reagent kits, the lid (2) that comprises box body (1) and box body top, it is characterized in that: in described box body (1), be provided with microwell plate (6) and be positioned at the back up pad (3) of box body (1) bottom, wherein microwell plate (6) is comprised of 48 or 96 micropores (7), in back up pad (3), have a plurality of holes (4), in described hole (4), be placed with altogether eight reagent bottles (5), in described eight reagent bottles (5), contain respectively magnetic separation agent, enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning concentrate, dilution and substrate solution, wherein magnetic separation agent contains the coated magnetic particle of people's epididymal secretory protein 4 monoclonal antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201320476898.0U CN203414474U (en) | 2013-08-06 | 2013-08-06 | Quantitative determination kit of human epididymis epithelial secretory protein 4 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201320476898.0U CN203414474U (en) | 2013-08-06 | 2013-08-06 | Quantitative determination kit of human epididymis epithelial secretory protein 4 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN203414474U true CN203414474U (en) | 2014-01-29 |
Family
ID=49977356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201320476898.0U Expired - Fee Related CN203414474U (en) | 2013-08-06 | 2013-08-06 | Quantitative determination kit of human epididymis epithelial secretory protein 4 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN203414474U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103424551A (en) * | 2013-08-06 | 2013-12-04 | 江苏福隆生物技术有限公司 | Kit for performing quantitative detection on human epididymal epithelial secretory protein 4 and detection method thereof |
-
2013
- 2013-08-06 CN CN201320476898.0U patent/CN203414474U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103424551A (en) * | 2013-08-06 | 2013-12-04 | 江苏福隆生物技术有限公司 | Kit for performing quantitative detection on human epididymal epithelial secretory protein 4 and detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103499692B (en) | Golgiosome protein GP 73 quantitative determination reagent kit and detection method thereof | |
| CN103364568B (en) | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof | |
| CN103698535B (en) | Platelet-activating factor acetylhydro-lase immue quantitative detection reagent box and preparation, method of operating | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN104089949A (en) | Quantitative gastrin-releasing peptide precursor kit, as well as preparation method and detection method thereof | |
| CN103604931A (en) | Human S100 protein detection reagent and preparation method thereof | |
| WO2007121668A1 (en) | Direct chemiluminescent reagent for magnetic separation and immunoassay method using thereof | |
| CN107543932A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin | |
| CN103592445A (en) | Kit for detecting procalcitonin | |
| CN102435752B (en) | Quantitative determination kit for human myoglobin and detection method thereof | |
| CN103076458A (en) | Dissociate human chorionic gonadotrophin beta-subunit magnetic particle chemiluminescence quantitative assay kit and its preparation method | |
| CN104034892A (en) | Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof | |
| CN102323419B (en) | Kit and detection method for quantitative determination of digoxin | |
| CN103424551B (en) | People's epididymal secretory protein 4 quantitative determination reagent kit and detection method thereof | |
| CN109211868A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection MYO | |
| CN104122394A (en) | Quantitative determination kit and detection method for human epidermal growth factor receptor 2(Her-2) | |
| CN102288766B (en) | Carbohydrate antigen 15-3 (CA15-3) quantitative measurement kit and detection method thereof | |
| CN102288767B (en) | Saccharide antigen 72-4(CA72-4) quantitative determination kit and detection method | |
| CN102323417B (en) | Kit for quantitative determination of pesinogen I (PGI) and detection method thereof | |
| CN104807997A (en) | Kit for detecting CA125 and SP17 contents based on chemiluminiscence method as well as method and application thereof | |
| CN102426255A (en) | Quantitative detection kit and detection method of free tetraiodothyronine (FT4) | |
| CN102721813A (en) | Homogeneous luminous immunoassay assay kit for prostate specific antigen and detection method therefor | |
| CN102435755A (en) | Quantitative determination kit for total triiodothyronine (TT3) and detection method thereof | |
| CN102426254B (en) | Total thyroxine (TT4) quantitative determination kit and its detection method | |
| CN107490695B (en) | The board-like chemoluminescence method detection kit and preparation method of Carbohydrate Antigen 50 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140129 Termination date: 20200806 |