CN101782572A - Urinary polyamine detection kit - Google Patents
Urinary polyamine detection kit Download PDFInfo
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Abstract
The invention relates to a urinary polyamine detection kit, which comprises a detection strip, a luminescent detection instrument, a sampling device and a sample injector, wherein the detection strip comprises polyacrylamide gel, polyamine standard product, marked polyamine antigen, solid-phase antibody resin, luminescent agent, micro electrode and a plurality of plastic grooves. The invention also relates to a preparation method of the detection strip and an application of the urinary polyamine detection kit. The urinary polyamine detection kit can detect polyamine tumor markers in the urine sample, has the advantages of high sensitivity, high specificity, simple and rapid operation method, and the like, and is particularly used for ordinary families and the grass-root units to perform the tumor early prediction, the evaluation of curative effect and recrudescency monitoring.
Description
Technical field
The present invention relates to a kind of detection kit, be specifically related to a kind of urinary polyamine detection kit.
Background technology
Tumour is serious threat human life and healthy malignant disease, and diagnosing early and treating is one of the most effective means of resisting at present cancer.Tumor markers is to be present in tumor tissues itself or to be secreted to blood or other body fluid or stimulate and obviously be different from by its content of human body cell generation a class material of normal reference value because of tumor tissues by what tumor tissues produced.Tumor markers is being brought into play more and more important effect in the middle of early diagnosis of tumor.
Polyamines comprises putrescine, spermidine and spermine, is that class concentration in tumour cell significantly differs from the organic molecule tumor markers in the normal cell.The quick growth of tumour greatly depends on the higher polyamines concentration in the oncocyte, the polyamine content in tumor patient blood and the urine also be proportionate with the tumor development process (Nature Reviews, 2004,4:781-791).
Tumour quick detection kit in the market lays particular emphasis on the detection of tumor markers in the blood sample more.But there are the sampling characteristics that difficulty is big, technical requirement is high in the detection of blood sample tumor markers, is unfavorable for the use of basic unit and average family.
Russel in 1971 studies show that the cancer patient urinate in the polyamine excretion increase.Minor N 1, N12-diacetyl spermine, N1, the phenomenon of two kinds of diacetyl polyamine of N8-diacetyl spermin had been found in urine, to drain in recent years.Japan and the pure medicine of light (Wako) have been developed a kind of kit of measuring diacetyl spermine in the urine in view of the above.This kit is to use the ELISA method of distinct antibodies to the diacetyl spermine.On mini disk, scribble antigen (diacetyl spermine).Urine sample and diacetyl spermine titer and anti-diacetyl spermine distinct antibodies are reacted, and remaining antibody and the diacetyl spermine above the dish carry out combination; With the anti-rabbit IgG of HRP mark antibody response, carry out quantitatively then according to color development reaction by HRP catalysis.This kit comprises: antigen is individual changes 1 of mini disk, diacetyl spermine standard items 250 μ l * 2, antibody diluent 20mL, anti-diacetyl spermine antibody (* 100) 60 μ l, HRP mark-anti-IgG antibody (* 80) 70 μ l, 2 in OPD tablet, matrix liquid 30mL, reaction stop solution 15mL, concentrated cleaning solution (* 20) 30mL, dilution mutually with coiling 1.
Said method need be through processing procedures such as repeatedly dilution, washings, and the running time is long, and operating personnel's professional standards are had relatively high expectations, and is unfavorable for promoting the use of in grass-roots unit.Technical only based on ELISA, sensitivity and specificity are all lower, and it is single to detect target.
In addition, the traditional detection method precision is not high, complex steps, and instrument requires high.Chemoluminescence method has been applied to the detection of tumor markers with its outstanding superiority in clinical, but also only rests on the analyzing and testing of a handful of tumor markers at present, as AFP, hCG, CEA etc.Because used instrument mostly is import, cost an arm and a leg, chemoluminescence method is comparatively extensive in some big-and-middle-sized hospital application at home, and use also seldom at some basic hospitals, and reagent is based on traditional luminous agent such as Lu Lainuo and derivant thereof, and these all can't satisfy the needs of domestic development and universal oncotherapy and detection means.
Summary of the invention
The purpose of this invention is to provide a kind of urinary polyamine detector bar, its outside is a plastics colloid groove, on the colloid groove vinyl cover is arranged, built-in polyacrylamide gel colloid, comprise microelectrode and a plurality of plastic channel on the described colloid, it puts in order and is followed successively by: negative microelectrode, labelled antigen groove, standard items loading slot and sample pipetting volume groove, two antibody grooves, two luminescence-producing reaction grooves, positive microelectrode; Wherein, standard items loading slot and sample pipetting volume groove parallel arranged, two antibody groove parallel arranged, two luminescence-producing reaction groove parallel arranged; Wrap polyamines antigen, polyamines standard items, insolubilized antibody resin, the luminous agent that is labeled in described labelled antigen groove, standard items loading slot, antibody groove, the luminescence-producing reaction groove respectively.
Preferably, described luminous agent is the different Lu Lainuo of isothiocyanato.
A further object of the present invention provides a kind of urinary polyamine detection kit, and it comprises described urinary polyamine detector bar, also comprises the luminous detection instrument, preferably, also comprises sampler and sample injector.
Wherein said detector bar, sampler and sample injector are disposable product; Preferably, described sampler and sample injector are this area disposable plastic goods commonly used, and scale is all arranged on it, are beneficial to sampling; Described luminous detection instrument is portable luminous detection instrument.
Preferably, kit of the present invention comprises: 50 of disposable detector bars, 50 of disposable sampler, 50 of disposable sample injectors, 1 in portable luminous detection instrument.
Described portable luminous detection instrument is at Weak-luminescence measuring instrument (the China Patent No. ZL96241369.0 of Institute of Biophysics, Academia Sinica's development, its trade name is faint chemiluminescence and bioluminescence measuring instrument, model: improve on basis KA-08), except that display screen and operation board, also increased mechanical hook-up (comprising fishhook), luminescence detection apparatus, signal processing apparatus and the USB interface that is connected with computer of operation detection bar.
As shown in Figure 1, (China Patent No. ZL96241369.0) compares with prior art, and the present invention comprises its improved part:
1, increased the mechanical hook-up 18 of operation detection bar, it has the functions such as vinyl cover 22 that eject and regain detector bar support disc, displacement and recovery detector bar;
2, luminescence detection apparatus 19 has been moved on to the top from the below of detector bar import 2 (being the sample chamber), and linked to each other by the link 21 on the vinyl cover 22 of fishhook 20 and detector bar, described luminescence detection apparatus can detect luminous intensity;
3, signal processing apparatus 23 is linked to each other with display screen 3, operation board 4 and USB interface 5, described signal processing apparatus 23 can produce the luminous detection result, it is the information Recognition bar code, described information Recognition bar code was made up of luminous detection numerical value and time on date, in the display screen 3 of detector, show, can be by USB interface 5 input computers;
4, do not contain cooling system, saved the step that the luminous detection instrument is lowered the temperature and cooled off.
Another object of the present invention provides the preparation method of described urinary polyamine detector bar, comprises the steps: 1) the mark standard antigen; 2) preparation insolubilized antibody resin preparation polyamines antibody, 3); 4) preparation detector bar.
Wherein, step 1) comprises with polyamines standard items and luminous agent mixing and is dissolved in the damping fluid that sonicated keeps in Dark Place, and removes free luminous agent by column chromatography, obtains the polyamines antigen of mark; Preferably, described luminous agent is the different Lu Lainuo of isothiocyanato, and described damping fluid is the phosphate buffer of pH 7.3, and described column chromatography is the desalting column chromatogram;
Step 2) comprises the polyamines antigen of getting mark, add the Fu Shi adjuvant by 1: 1 volume ratio, ultrasonic emulsification in the ice bath, respectively select 3 points in rabbit back backbone both sides, intracutaneous is respectively injected the equal-volume mixed liquor of described polyamines and Fu Shi adjuvant, immunity three times, each 10 days at interval, get rabbit arterial blood after 30 days, behind the separation of serum-40 ℃ frozen, obtain the monoclonal antibody of described polyamines; Wherein, the Fu Shi Freund's complete adjuvant is used in immunity for the first time, uses the Fu Shi Freund with immunity for the third time for the second time;
Step 3) comprises that with ion exchange resin be that the toluene solution of 2%3-aminopropyl-triethoxysilane, the ethanolic solution that volume ratio is 5% glutaraldehyde are handled with 50%SDS, volume ratio respectively, be that 7.3 phosphate buffer, antibody phosphate buffer are handled with pH respectively then, obtain described insolubilized antibody resin;
Step 4) comprises that the pH that will contain little peroxidase is 7.3 phosphate buffer as reactant liquor and bag by in standard items loading slot, sample pipetting volume groove, antibody groove, labelled antigen groove; With pH is that 10.8 phosphate buffer and hydrogen peroxide mixed liquor bag are by in the luminescence-producing reaction groove; Polyamines antigen, insolubilized antibody resin, the luminous agent of described polyamines standard items, mark are wrapped respectively by in standard items loading slot, labelled antigen groove, antibody groove, luminescence-producing reaction groove, obtain described detector bar;
The check and analysis principle of kit of the present invention is: with luminous agent, and--preferred different Lu Lainuo of isothiocyanato (ILITC)--is marked on the antigen, utilize immune competitive reaction takes place between the antigen of determined antigen and ILITC mark and the antibody, add the ILITC effect on microperoxisome and the unreacted antigen then, the generation luminescence phenomenon detects the polyamine content in the urine sample.
The check and analysis process of kit of the present invention is as follows: the polyamines antigen electrophoresis of the standard items on the detector bar, testing sample and mark to the antibody groove, with insolubilized antibody immune competitive reaction is taken place; Unreacted labelled antigen is continued electrophoresis to the luminescence-producing reaction groove from the antibody groove, and the different Lu Lainuo of the isothiocyanato on this antigen reacts with microperoxisome in the luminescence-producing reaction groove, and the luminous detection instrument detects luminous intensity and provides the information Recognition bar code.
Another object of the present invention provides the application in the polyamines class tumor markers in detecting urine sample of described urinary polyamine detection kit.The user can send to the green medicine bioengineering of Beijing spring breeze Science and Technology Ltd. with the information Recognition bar code by mobile phone or internet, company is revised according to the luminous contrast reading of standard with remote computer and data analysis software (early detection is analyzed comparison system software CL0189.16), provide final reading then, and the information Recognition bar code analyzed, relatively and monitoring polyamines composition, preserve the long-term dynamics testing result, by communication tools such as phone or mobile phones analysis result is fed back to the client, and provide follow-up treatment suggestion according to analyzing data.
Five technology of utilizing urinary polyamine detection kit of the present invention detect three polyamine species compositions in the urine sample simultaneously, the large-scale remote analysis instrument that these five technology are respectively the electrophoretic techniques of antibody mediated immunity identification, auto injection and the enrichment antigen of high specific, highly sensitive chemiluminescence detection, portable luminous detection instrument and high automation carries out long-term dynamic monitoring, analysis and comparison by the combination of above-mentioned technology to the polyamines composition.
The present invention detects the concentration of urinary polyamine by the multiple technologies coupling, and 3 orders of magnitude (0.01-80nmol/mg Cr) are crossed in sensitivity, and specificity can reach 84%.The pre-treatment process of sample is simple, and is consuming time few, and the sample detection cost is low, and sensitivity and specificity are far above additive method.
Urinary polyamine detection kit of the present invention can detect polyamines class tumor markers in the urine sample, has advantages such as high sensitivity, high specific, method of operating be easy, quick; The related reagent holding time is long, pollution-free, and no safety issue, low to the technical operation requirement can be used for tumour early prediction, the assessment of treatment curative effect and the recurrence monitoring of average family and grass-roots unit.The present invention has important practical significance and economic implications to the problem that solves the present basic unit difficulty and high cost of getting medical.
Description of drawings
Fig. 1 is the composition synoptic diagram of urinary polyamine detection kit of the present invention;
Fig. 2 is the range of linearity figure of the sensitivity experiment of urinary polyamine detection kit of the present invention.
Description of reference numerals among Fig. 1:
1. portable luminous detection instrument; 2. detector bar import; 3. display screen; 4. operation board; 5.USB interface; 6. power lead; 7. standard items loading slot; 8. antibody groove; 9. luminescence-producing reaction groove; 10. labelled antigen groove; 11. luminous agent, bag is by in luminescence-producing reaction groove 9; 12. sample pipetting volume groove; 13. negative microelectrode; 14. positive microelectrode; 15. colloid detector bar; 16. disposable sample injector (2ml); 17. disposable sampler (20ml); 18. the mechanical hook-up of operation detection bar; 19. luminescence detection apparatus is positioned at 2 top, links to each other with signal processing apparatus 23; 20. fishhook; 21. link; 22. the vinyl cover of detector bar; 23. signal processing apparatus is positioned at the back of display screen 3, links to each other with display screen 3, operation board 4, USB interface 5 and luminescence detection apparatus 19.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
Urinary polyamine detection kit of the present invention comprises as the lower part: disposable detector bar (50), disposable sampler (50), disposable sample injector (50), portable luminous detection instrument (1), instructions (1 part).
The preparation of embodiment 2 detector bars
(1) mark standard antigen
(St.Louis, MO) (each components in proportions of polyamines is a putrescine to the polyamines standard items: spermidine: spermine=8: 1: 1) in purchase from Sigma company.
With 0.5 μ M polyamines and the different Lu Lainuo (isoluminolisothiocyanate of 2 μ M isothiocyanatos, ILITC) mix and be dissolved in 2mL damping fluid (10mM phosphate buffer, pH 7.3) in, sonicated 1 minute kept in Dark Place 20 minutes, by column chromatography (PD-10 desalting column, Amersham Pharmacia Biotech, Buckinghamshire, U.K.), remove free ILITC with 10mM Tris-HCl, 0.1mM EDTA wash-out, reclaim the polyamines antigen of mark;
(2) preparation polyamines antibody
The polyamines antigen 600 μ l (100 μ g) that get mark add Fu Shi adjuvant, ultrasonic emulsification in the ice bath by 1: 1 volume ratio; Respectively select 3 points in backbone both sides, Japan large rabbit back, intracutaneous is respectively injected the equal-volume mixed liquor of 200 μ l polyamines and Fu Shi adjuvant, three times (the Fu Shi Freund's complete adjuvant is used in immunity for the first time in immunity altogether, for the second time, use the Fu Shi Freund for the third time), each 10 days at interval, get rabbit arterial blood after 30 days, behind the separation of serum-40 ℃ frozen;
The ELISA method is identified antibody titer and specificity:
Polyamines (the 100mg/ μ l) bag of getting the chromatography purifying is by ELISA check-out console (100 μ l/ hole), antibody with dilution preparation in 1: 1000,1: 3000,1: 5000,1: 10000,1: 20000 resists (each concentration is established 3 multiple holes) as one respectively, goat anti-rabbit igg-HRP is that antigen-antibody reaction is carried out in two anti-(1: 20000), TMB (tetramethyl benzidine) colour developing, the Thermo microplate reader reads the absorbance in every hole under the 450nm wavelength, with the titre and the specificity of this analyte preparation antibody.Blank well replaces one with 0.01mol/LPBST (phosphate buffer that contains 0.05%Tween-20, pH 7.4) and resists, and negative control hole is anti-with preceding rabbit anteserum (1: the 1000) replacement one of immunity.Adopt the ELISA experiment to detect 147 parts of positive sample, the result is all positive; Detect 269 parts of negative sample, the result shows that 1 part is false positive, and all the other are all negative; Antibody specificity reaches 99.6%, and the result shows that antibody specificity is very strong.
(3) preparation insolubilized antibody resin
1 gram H-103 ion exchange resin was handled 6 hours with 50% lauryl sodium sulfate (SDS) at 100 ℃, clean and drying, then the base resin was handled 6 hours in the toluene solution of 15mL 2% (volume ratio) 3-aminopropyl-triethoxysilane, room temperature treatment is 24 hours in the ethanolic solution of 2.5mL 5% (volume ratio) glutaraldehyde, with 10mM phosphate buffer (pH7.3) washing 5 hours, the gained reactant resin is at the phosphate buffer (10mM of antibody, pH7.0) handled 48 hours for 4 ℃ in, the insolubilized antibody resin that obtains (mol ratio of antibody and resin is 1: 2) can be in phosphate buffer 4 ℃ of long preservation;
(4) preparation colloid detector bar
In the colloid groove, preset polyacrylamide gel and positive and negative microelectrode, phosphate buffer (the 10mM that will contain the little peroxidase of 4 μ M, pH 7.3) be preset in standard items loading slot 7, antibody groove 8, labelled antigen groove 10, the sample pipetting volume groove 12 as reactant liquor, in luminescence-producing reaction groove 9, preset phosphate buffer (10mM, pH 10.8) and the hydrogen peroxide mixed liquor, described colloid detector bar obtained.
As shown in Figure 1, a plurality of plastic channel and negative microelectrode 13 and positive microelectrode 14 are arranged on the colloid detector bar 15.The polyamines antigen of polyamines standard items, mark, insolubilized antibody resin, luminous agent 11 wrap respectively by in standard items loading slot 7, labelled antigen groove 10, antibody groove 8, luminescence-producing reaction groove 9.With sample injector 16 urine sample is joined in the sample pipetting volume groove 12.
Testing process is as follows:
Polyamines 300V voltage 30 seconds from sample pipetting volume groove 12, standard items loading slot 7, labelled antigen groove 10 by electrophoresis to antibody groove 8, immune competitive reaction takes place with unlabelled polyamines antigen and antibody in mark in antibody groove 8; Not with antibody take place immunoreactive mark polyamines antigen 500V voltage 30 seconds from antibody groove 8 by electrophoresis to luminescence-producing reaction groove 9, the ILITC on this antigen reacts with microperoxisome in luminescence-producing reaction groove 9, produces luminescence phenomenon.
Portable luminous detection instrument in the urinary polyamine detection kit of the present invention is at Weak-luminescence measuring instrument (the China Patent No. ZL96241369.0 of Institute of Biophysics, Academia Sinica's development, its trade name is faint chemiluminescence and bioluminescence measuring instrument, model: improve on basis KA-08), except that display screen and operation board, also increased mechanical hook-up (comprising fishhook), luminescence detection apparatus, signal processing apparatus and the USB interface that is connected with computer of operation detection bar.
As shown in Figure 1, (China Patent No. ZL96241369.0) compares with prior art, and the present invention comprises its improved part:
1, increased the mechanical hook-up 18 of operation detection bar, it has the functions such as vinyl cover 22 that eject and regain detector bar support disc, displacement and recovery detector bar;
2, luminescence detection apparatus 19 has been moved on to the top from the below of detector bar import 2 (being the sample chamber), and linked to each other by the link 21 on the vinyl cover 22 of fishhook 20 and detector bar, described luminescence detection apparatus can detect luminous intensity;
3, signal processing apparatus 23 is linked to each other with display screen 3, operation board 4 and USB interface 5, described signal processing apparatus 23 can produce the luminous detection result, it is the information Recognition bar code, described information Recognition bar code was made up of luminous detection numerical value and time on date, in the display screen 3 of detector, show, can be by USB interface 5 input computers;
4, do not contain cooling system, saved the step that the luminous detection instrument is lowered the temperature and cooled off, simplified trace routine.
The course of work of portable luminous detection instrument is as follows:
1) opens luminous detection instrument power supply; Initiating key on the push dish 4, the door of detector bar import 2 is opened, and ejects the detector bar support disc;
2) detector bar 15 that will add urine sample is put into support disc; Read key on the push dish 4, detector bar enters, and the fishhook 20 of the mechanical hook-up 18 of operation detection bar links to each other with link 21 on the detector bar vinyl cover 22, and the inner energising program of start detection instrument;
3) support disc block detector bar 15 motionless in, move on the inner initialize program control fishhook 20, move on the traction link 21, vinyl cover 22 (dotted line is with top) is separated with the colloid groove, simultaneously, wrap by the spacer of all ingredients (described spacer is this area plastic sheeting or aluminium foil commonly used) with vinyl cover inner face being used to of connecting together and to separate with polyacrylamide colloid, biochemical reaction and electrophoresis begin; After luminescence detection apparatus detected signal, signal was by signal processing means processes, and was presented on the display screen, also can be derived by USB interface;
4) after the EOP (end of program), programmed control fishhook 20 moves down, and restore detector bar vinyl cover 22 positions, and the door of detector bar import 2 is opened, and the detector bar support disc ejects automatically;
5) take out detector bar 15, the end key on the push dish 4, the door of detector bar import 2 is closed.
The specificity test of embodiment 4 urinary polyamine detection kits of the present invention
The colony that is made up of at random healthy people, benign tumour patient, Urinary system inflammation patient, malignant tumor patient is carried out the specificity test, the result shows: 2 all detection of dynamic malignant tumor patient True Positive Rates are that 84%, 4 all detection of dynamic True Positive Rates continuously are 91%.
The sensitivity experiment of embodiment 5 urinary polyamine detection kits of the present invention
Get the 2ml urine sample, dilute 10,100,1000,10000 times, 3 parts of each prepared at concentrations are measured.The result shows: testing result concerns good (see figure 2) 10000 times dilution range internal linear.
The storage life experiment of embodiment 6 urinary polyamine detection kits of the present invention
Kit is positioned over 40 ℃ of normal domestic use refrigerators preserves, get 0,30,60,90,120,150 and 180 day kit respectively, carry out standard model and detect to measure its detection effect.The result proves that kit can be preserved at least under 4 ℃ do not influence the detection effect in 180 days.
The concrete operations step of embodiment 7 urinary polyamine detection kits of the present invention
1, opens the detector bar packaging bag, lie against on the table;
2, get urine sample 10-20ml with sampler;
3, with sample injector the 2ml urine sample is joined the sample pipetting volume groove;
4, open luminous detection instrument power supply; Initiating key on the push dish, the door of detector bar import is opened, and ejects the detector bar support disc;
5, the detector bar that will add urine sample is put into support disc; Read key on the push dish, detector bar enters, and reads sample and begins;
6, after 2 minutes, display screen display message identification bar code;
7, the detector bar support disc ejects automatically, takes out detector bar, the end key on the push dish 4, and the door of detector bar import is closed; Detector bar, sampler and sample injector are put into dustbin in the lump;
8, the information Recognition bar code is sent to the green medicine bioengineering of Beijing spring breeze Science and Technology Ltd. by mobile phone or internet;
9, company feeds back to the client by communication tools such as phone or mobile phones with analysis result in 24 hours.
Claims (10)
1. urinary polyamine detector bar, it is characterized in that, the described detector bar outside is colloid groove and vinyl cover, built-in polyacrylamide gel colloid, comprise microelectrode and a plurality of plastic channel on the described colloid, it puts in order and is followed successively by: negative microelectrode, labelled antigen groove, standard items loading slot and sample pipetting volume groove, two antibody grooves, two luminescence-producing reaction grooves, positive microelectrode; Wherein, standard items loading slot and sample pipetting volume groove parallel arranged, two antibody groove parallel arranged, two luminescence-producing reaction groove parallel arranged; Wrap polyamines antigen, polyamines standard items, insolubilized antibody resin, the luminous agent that is labeled in described labelled antigen groove, standard items loading slot, antibody groove, the luminescence-producing reaction groove respectively.
2. urinary polyamine detector bar according to claim 1 is characterized in that, described luminous agent is the different Lu Lainuo of isothiocyanato.
3. a urinary polyamine detection kit is characterized in that, comprises claim 1 or 2 described urinary polyamine detector bars, also comprises the luminous detection instrument, and described luminous detection instrument comprises as lower device:
The mechanical hook-up of operation detection bar can eject and regain the vinyl cover of detector bar support disc, displacement and recovery detector bar;
Luminescence detection apparatus can detect luminous intensity;
Signal processing apparatus produces the luminous detection result according to described luminous intensity, i.e. information Recognition bar code, and described information Recognition bar code was made up of luminous detection numerical value and time on date.
4. the preparation method of the described urinary polyamine detector bar of claim 1 is characterized in that, comprises the steps:
1) mark standard antigen;
2) preparation polyamines antibody;
3) preparation insolubilized antibody resin;
4) preparation detector bar.
5. preparation method according to claim 4 is characterized in that, step 1) comprises with polyamines standard items and luminous agent mixing and be dissolved in the damping fluid that sonicated keeps in Dark Place, and removes free luminous agent by column chromatography, obtains the polyamines antigen of mark.
6. preparation method according to claim 5 is characterized in that, described luminous agent is the different Lu Lainuo of isothiocyanato, and described damping fluid is the phosphate buffer of pH 7.3, and described column chromatography is the desalting column chromatogram.
7. preparation method according to claim 4, it is characterized in that step 2) comprise the polyamines antigen of getting mark, by 1: 1 volume ratio adding Fu Shi adjuvant, ultrasonic emulsification in the ice bath, respectively select 3 points in rabbit back backbone both sides, intracutaneous is respectively injected the equal-volume mixed liquor of described polyamines and Fu Shi adjuvant, immunity three times, each 10 days at interval, get rabbit arterial blood after 30 days, behind the separation of serum-40 ℃ frozen, obtain the monoclonal antibody of described polyamines; Wherein, the Fu Shi Freund's complete adjuvant is used in immunity for the first time, uses the Fu Shi Freund with immunity for the third time for the second time.
8. preparation method according to claim 4, it is characterized in that, step 3) comprises that with ion exchange resin be that the toluene solution of 2%3-aminopropyl-triethoxysilane, the ethanolic solution that volume ratio is 5% glutaraldehyde are handled with 50%SDS, volume ratio respectively, be that 7.3 phosphate buffer, antibody phosphate buffer are handled with pH respectively then, obtain described insolubilized antibody resin.
9. preparation method according to claim 4 is characterized in that, step 4) comprises that the pH that will contain little peroxidase is 7.3 phosphate buffer as reactant liquor and bag by in standard items loading slot, sample pipetting volume groove, antibody groove, labelled antigen groove; With pH is that 10.8 phosphate buffer and hydrogen peroxide mixed liquor bag are by in the luminescence-producing reaction groove; Polyamines antigen, insolubilized antibody resin, the luminous agent of described polyamines standard items, mark are wrapped respectively by in standard items loading slot, labelled antigen groove, antibody groove, luminescence-producing reaction groove, obtain described detector bar.
10. the described urinary polyamine detection kit of claim 3 application in the polyamines class tumor markers in detecting urine sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010110710A CN101782572A (en) | 2010-02-10 | 2010-02-10 | Urinary polyamine detection kit |
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| CN201010110710A CN101782572A (en) | 2010-02-10 | 2010-02-10 | Urinary polyamine detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316685A (en) * | 2014-10-10 | 2015-01-28 | 辽宁迈迪生物科技有限公司 | Diacetyl spermine detection kit and preparation method and application thereof |
| CN104678035A (en) * | 2013-11-29 | 2015-06-03 | 沈阳药科大学 | Application of plasma as liver cancer diagnostic marker |
| CN111201224A (en) * | 2017-10-16 | 2020-05-26 | 新生命医药科技有限公司 | Uropolyamines as biomarkers for prostate cancer detection |
-
2010
- 2010-02-10 CN CN201010110710A patent/CN101782572A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104678035A (en) * | 2013-11-29 | 2015-06-03 | 沈阳药科大学 | Application of plasma as liver cancer diagnostic marker |
| CN104316685A (en) * | 2014-10-10 | 2015-01-28 | 辽宁迈迪生物科技有限公司 | Diacetyl spermine detection kit and preparation method and application thereof |
| CN111201224A (en) * | 2017-10-16 | 2020-05-26 | 新生命医药科技有限公司 | Uropolyamines as biomarkers for prostate cancer detection |
| CN111201224B (en) * | 2017-10-16 | 2023-04-25 | 新生命医药科技有限公司 | Urinary polyamine as biomarker for prostate cancer detection |
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Application publication date: 20100721 |