CN201796036U - A test strip for quantitative detection of tumor marker colloidal gold immunochromatography - Google Patents
A test strip for quantitative detection of tumor marker colloidal gold immunochromatography Download PDFInfo
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- CN201796036U CN201796036U CN2010202906770U CN201020290677U CN201796036U CN 201796036 U CN201796036 U CN 201796036U CN 2010202906770 U CN2010202906770 U CN 2010202906770U CN 201020290677 U CN201020290677 U CN 201020290677U CN 201796036 U CN201796036 U CN 201796036U
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Abstract
The utility model discloses a test strip for quantitative detection of a tumor marker by colloidal gold immunochromatography, which comprises a sample pad, a gold-labeled antibody conjugate film, a nitrate cellulose film, a water absorbent pad and a reaction substrate. The proximal end of the nitrate cellulose film to the sample pad is the initial end, and the proximal end of the nitrate cellulose film to the water absorbent pad is the tail end. The tail end of the nitrate cellulose film is coated with the following five antibody lines in sequence: test lines T1, T2, T3 and T4 which are coated with mouse anti-human tumor marker monoclonal antibody I respectively, and a quality control line C coated with goat anti-mouse IgG. The gold-labeled antibody conjugate film is a film spray-coated with colloidal gold-labeled mouse anti-human tumor marker monoclonal antibody II. The utility model achieves the effect of rapid, accurate and quantitative detection of the tumor marker and meets the requirement for rapid detection in cancer survey and screening and home cancer prognosis monitoring.
Description
Technical field
The utility model belongs to field of biological detection, is specifically related to be used for the colloidal gold immunochromatographiassay assay quantitative detection test paper of lesion detection.
Background technology
Tumor markers (tumor marker, TM) be meant in the generation and breeding of tumour, by tumour cell itself produced or by body the tumour cell reaction is produced, the class material that the reflection tumour exists and grows comprises protein, hormone, enzyme (isodynamic enzyme) and oncoprotein etc.Not being present in the normal human that these materials have only sees among the embryo, and what have surpasses normal human's intensive amount at the tumour patient in-vivo content.Tumor markers is at cancer screening, diagnosis, judging prognosis and lapse to, estimate aspects such as treatment curative effect and people at highest risk's follow-up observation all has bigger practical value.But by measuring its existence or content auxiliary diagnosis tumour, the analysis course of disease, instructing treatment, monitoring recurrence or transfer, judging prognosis.The tumor markers overwhelming majority who detects does not clinically exist only in the malignant tumour at present, is present in benign tumour, embryonic tissue even the normal structure yet.Therefore, tumor markers dynamic chek and multinomial joint inspection are more valuable, so need a kind of quick easily tumor markers detection by quantitative means of row.
Enzyme linked immunosorbent assay analysis (ELISA) is generally adopted in the detection of tumor markers clinically, because ELISA need repeatedly be hatched, detection time is longer, is not suitable for fast detecting.But the colloidal gold immunochromatographimethod technology has simply, fast, advantage such as sensitive execute-in-place, be widely used in the plague, bird flu, had a liking for the detection of lung legion etc., domestic still do not have a report of successfully developing tumor markers detection by quantitative test strips.The utility model utilizes double antibody sandwich method, has invented the fast quantification test strip of tumor markers, thereby has satisfied the needs of fast detecting such as the generaI investigation screening of reply cancer and family's cancer prognosis monitoring.
The utility model content
The purpose of this utility model provides a kind of colloidal gold immunochromatographiassay assay quantitative detection test paper of accurate detection tumor markers.
A kind of tumor marker colloidal gold immunochromatographiassay assay quantitative detection test paper of the present utility model, comprise sample pad, golden labeling antibody bond film, nitrocellulose membrane, adsorptive pads, reaction holder, the reaction holder is positioned at bottom, nitrocellulose membrane is positioned at the middle part on the reaction holder, and golden labeling antibody bond film is positioned at a side on nitrocellulose membrane top and overlaps with it; Adsorptive pads is positioned on the nitrocellulose membrane opposite side for golden labeling antibody bond film and overlaps with nitrocellulose membrane; Sample pad is positioned at a side opposite with adsorptive pads on the nitrocellulose membrane and overlapping with golden labeling antibody bond membrane portions; Described nitrocellulose membrane is terminal to be initiating terminal near sample pad one end near adsorptive pads one end, wraps successively by 5 antibody bands, is respectively bag by the T of mouse-anti human tumor marker thing monoclonal antibody I
1, T
2, T
3, T
4Test strip and bag are by the C Quality Control band of sheep anti-mouse igg monoclonal antibody, and golden labeling antibody bond film is the film that is coated with the mouse-anti human tumor marker thing monoclonal antibody II of colloid gold label.
The package amount of mouse-anti human tumor marker thing monoclonal antibody I is 0.3-1.5ug on described every antibody band, and the package amount of sheep anti-mouse igg antibody purification is 0.15-0.75ug.
The parallel interval of 3mm is arranged between described every antibody band, and the width of described nitrocellulose membrane is 0.3cm.
Every 8-15ug mouse-anti human tumor marker thing monoclonal antibody II adopts 1ml OD
520nmValue is 1.5～2.0 colloidal gold solution mark, and the colloid gold particle diameter is 40nm.
The consumption of the mouse-anti human tumor marker thing monoclonal antibody II of colloid gold label is 5-10ul on the described golden labeling antibody bond film.
Described mouse-anti human tumor marker thing monoclonal antibody I comprises the monoclonal antibody of free state prostate specific antigen fPSA, compound state prostate specific antigen cPSA, prostate acid phosphatase PAP, NMP-22 NMP-22, bladder cancer antigen UBC or tumor of bladder antigen BTA; Described mouse-anti human tumor marker thing monoclonal antibody II comprises a kind of in the monoclonal antibody different with the described various antibody epitopes of mouse-anti human tumor marker thing monoclonal antibody I.
Described sample pad is selected from polyester film or glass fibre; Described golden labeling antibody bond film is selected from polyester film or glass fibre or filter paper fibre; Adsorptive pads is selected filter paper for use; The reaction holder is selected the PVC plate for use.
Utilize prostate cancer of the present utility model and bladder cancer detecting test paper, utilize visual or outfit hand-held gold test strip analyser, can realize the detection by quantitative of kinds of tumors mark.Detecting operation is very easy, and the ordinary people also can skillfully operate.Its detection sensitivity height of while, specificity is good, and sense cycle is short, is fit to different medical unit, and the domestic consumer uses.Can be used for the examination and the monitoring of prostate cancer and carcinoma of urinary bladder.
Description of drawings
Fig. 1 is a tumor markers detection by quantitative test paper planar structure areal map;
Fig. 2 is a tumor markers detection by quantitative test paper profile structural drawing;
Wherein: 1-sample pad, 2-gold labeling antibody bond film, 3-nitrocellulose filter, 4-adsorptive pads, 51-T
1Test strip, 52-T
2Test strip, 53-T
3Test strip, 54-T
4Test strip, 55-C Quality Control band, polyester film under the 6-, the last polyester film of 7-, the 8-max line, 9-look skin, 10-reacts holder;
Fig. 3 detects the colour developing synoptic diagram for the utility model test strips.
Embodiment
Further set forth the utility model below in conjunction with embodiment, and can not limit the utility model.
Tumor marker colloidal gold immunochromatographiassay assay quantitative detection test paper of the present utility model comprises sample pad 1, golden labeling antibody bond film 2, nitrocellulose membrane 3, adsorptive pads 4, reaction holder 10.Reaction holder 10 is positioned at bottom, and nitrocellulose membrane 3 is positioned at the middle part on the reaction holder 10, and golden labeling antibody bond film 2 is positioned at a side on nitrocellulose membrane 3 tops and overlaps with it; Adsorptive pads 4 is positioned on the nitrocellulose membrane 3 opposite side for golden labeling antibody bond film 2 and overlaps with nitrocellulose membrane 3; Sample pad 1 is positioned on the nitrocellulose membrane 3 side opposite with adsorptive pads 4 and overlaps with golden labeling antibody bond film 2.Described nitrocellulose membrane 3 is terminal to be initiating terminal near sample pad 1 one ends near adsorptive pads 4 one ends, wraps successively by 5 antibody bands, is respectively bag by the T of mouse-anti human tumor marker thing monoclonal antibody I
1, T
2, T
3, T
4Test strip and bag are by the C Quality Control band of sheep anti-mouse igg, and golden labeling antibody bond film 2 is the film that is coated with the mouse-anti human tumor marker thing monoclonal antibody II of colloid gold label.
The preparation method of test strips of the present utility model may further comprise the steps:
1, Antibody Preparation
Select commercial pairing good mouse-anti people free state prostate specific antigen (fPSA) monoclonal antibody I and II (product type: A8014-1 for use, A8014-2 Beijing Bomai Century Biotechnology Co.Ltd), to 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.4 are standby.
2, the bag quilt of nitrocellulose membrane
Bag is cushioned the preparation of liquid: the phosphate buffer of 0.02M pH 7.4 (PBS) is cushioned liquid for bag, and the rearmounted 4 ℃ of preservations of 0.22um filtering with microporous membrane degerming are standby, two weeks of the term of validity.
The preparation of confining liquid: contain the 0.02M of 0.5%BSA, the phosphate buffer of pH7.4 (PBS), it is standby that the degerming of 0.22um filtering with microporous membrane is placed on 4 ℃ of preservations, one week of the term of validity.
Being cushioned liquid with bag is 1mg/ml with 1 dilution of mouse-anti people free state prostate specific antigen monoclonal antibody, sheep anti-mouse igg monoclonal antibody dilution is 1mg/ml, use quantitative spray film device BioDot with the amount of 1ul/cm with the two with the even spray printing in the interval of 3mm on 2.5cm width nitrocellulose filter.After room temperature is dried 30 minutes, in confining liquid, soak after 30 minutes,, add drying agent and seal up for safekeeping standby in 37 ℃ of oven dry 8 hours.
3, the preparation of colloid gold label mouse-anti free state prostate specific antigen monoclonal antibody II
Cut-off directly is collaurum 20ml and the mouse-anti people free state prostate specific antigen monoclonal antibody II 200ug of 40nm respectively, under the condition of pH9.0, make its combination by the magnetic agitation vibration, add bovine serum albumin(BSA) (BSA) as stabilizing agent, and make that the BSA ultimate density is 1%, adopt supercentrifugal process (10000r/min, 30min, 4 ℃) remove unconjugated monoclonal antibody and unstabilized colloid gold particle and agglutinator thereof, the peony precipitation in the centrifuge tube bottom is collaurum-antibody complex.
4, golden labeling antibody bond film preparation
With 20ml 0.2% polyglycol damping fluid washing colloids gold-antibody complex, the centrifugal supernatant of removing, obtain the peony precipitation, precipitation behind the purifying is 20mmol/L with 2ml concentration, the borate buffer dissolving of PH 8, evenly be sprayed on the 0.8cm width glass fibre membrane with the amount of BioDot spray film instrument with 0.1ml/cm, 37 ℃ of vacuum drying 3 hours promptly obtain golden labeling antibody bond film.
5, the processing of sample pad
2.5cm width sample pad is put into sample pad Treatment Solution immersion treatment take out 37 ℃ of oven dry 3 hours after 1 hour.The sample pad treating fluid is to contain the PVA of 1%Casein and 1.0% and the 0.02M of 0.2%Tween-20, the PBS solution of pH7.4.
6, the assembling of test strips and cutting
Following all operations all must carry out in temperature 20-25 ℃ the room in humidity less than 20%.The assembling of test paper: use BioDot LMS000 type assembling instrument to require the bag that 2.5cm is wide by the nitrocellulose membrane of mouse-anti people free state prostate specific antigen monoclonal antibody I and sheep anti-mouse igg monoclonal antibody according to Fig. 2,2.5cm wide adsorptive pads, 0.8cm wide golden labeling antibody bond film, 2.5cm wide sample pad is assembled on the 8cm width transparent plastic base plate, max line and Se Pi are assembled into test strips in the covering.Use BioDot CM4000 type cutting cutter that the test paper plate that assembles is cut into the wide finished product test strips of 0.3cm.Following polyester film 6, last polyester film 7 can be kept apart golden labeling antibody bond film and reaction holder and max line 8, prevents that golden labeling antibody bond film from directly contact with adhesive sticker on the max line 8 with the reaction holder, thereby avoids golden labeling antibody bond release incomplete.Max line 8 can indicate test paper can insert the degree of depth of sample, and strengthens the compactedness that combines of golden labeling antibody bond film and nitrocellulose filter.Look skin 9 can be indicated the handheld terminal of test paper, and strengthens the compactedness that combines of adsorptive pads and nitrocellulose filter.
Prepared compound state prostate specific antigen (cPSA) equally with the method for above-mentioned 1-6 step, prostate acid phosphatase (PAP), nuclear matrix protein-22 (NMP-22), bladder cancer antigen (UBC), the colloidal gold immunochromatographimethod fast quantification of tumor markerses such as tumor of bladder antigen BTA detects test paper.More than required antibody all derive from Beijing Bomai Century Biotechnology Co.Ltd.
Tumor markers fast quantification of the present utility model detects the using method of test paper:
With sample to be measured and sample diluting liquid mixing, again test paper is inserted in the sample mix liquid, the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result.Can carry out the quantitative of tumor markers according to following two kinds of methods:
1, visual inspection: the bar number and the observation shade of counting colour developing T line, comparison titer colour developing information slip reads free state prostate specific antigen concentration from table.
Prepare free state prostate specific antigen colloidal gold immuno-chromatography test paper strip by the top condition of determining, detect the free state prostate specific antigen of variable concentrations.The free state prostate specific antigen is mixed with 1ng/ml, 3ng/ml, 5ng/ml, 7ng/ml with sample preparation liquid.Three replications as Fig. 3, shown in 4, do not have detection line colour developing during＜1ng/ml, two detection lines colour developings are arranged when only detection line colour developing, 3ng/ml during 1ng/ml; Four detection line colour rendering reactions when being arranged during 5ng/ml, three detection line colour developings, 7ng/ml are arranged; Be the observable least concentration＜1ng/ml of naked eyes; The C line does not develop the color and illustrates that test paper lost efficacy.
2, detection by quantitative:
With blank sample detection paper 20 times, read signal T/C ratio with the scanning of gold test strip analyser then.The mean value (AVERAGE) of 20 sample T/C ratios is the CUT-OFF value with 3 times of standard deviations (STDEVA) sum.Colloidal-gold strip after the colour developing is put into the scanning of gold test strip analyser, read the signal T/C ratio of each bar p-wire, can obtain free state prostate specific antigen concentration.
Stability test is deposited freshly prepd colloidal gold immuno-chromatography test paper strip 8 days in 37 ℃, detect with 1ng/ml, 3ng/ml, 5ng/ml, 7ng/ml tumor markers free state prostate specific antigen respectively, result's demonstration is compared with freshly prepd test strip, sensitivity does not obviously descend, and specificity is good.
Claims (3)
1. tumor marker colloidal gold immunochromatographiassay assay quantitative detection test paper, comprise sample pad, golden labeling antibody bond film, nitrocellulose membrane, adsorptive pads, reaction holder, the reaction holder is positioned at bottom, nitrocellulose membrane is positioned at the middle part on the reaction holder, and golden labeling antibody bond film is positioned at a side on nitrocellulose membrane top and overlaps with it; Adsorptive pads is positioned on the nitrocellulose membrane opposite side for golden labeling antibody bond film and overlaps with nitrocellulose membrane; Sample pad is positioned at a side opposite with adsorptive pads on the nitrocellulose membrane and overlapping with golden labeling antibody bond membrane portions; It is characterized in that described nitrocellulose membrane is terminal to be initiating terminal near sample pad one end near adsorptive pads one end, wraps successively by 5 antibody bands, is respectively bag by the T of mouse-anti human tumor marker thing monoclonal antibody I
1, T
2, T
3, T
4Test strip and bag are by the C Quality Control band of sheep anti-mouse igg monoclonal antibody, and golden labeling antibody bond film is the film that is coated with the mouse-anti human tumor marker thing monoclonal antibody II of colloid gold label.
2. test strips according to claim 1 is characterized in that, the parallel interval of 3mm is arranged between described every antibody band, and the width of described nitrocellulose membrane is 0.3cm.
3. test strips according to claim 1 and 2 is characterized in that described sample pad is selected from polyester film or glass fibre; Described golden labeling antibody bond film is selected from polyester film or glass fibre or filter paper fibre; Adsorptive pads is selected filter paper for use; The reaction holder is selected the PVC plate for use.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010202906770U CN201796036U (en) | 2010-08-13 | 2010-08-13 | A test strip for quantitative detection of tumor marker colloidal gold immunochromatography |
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| CN2010202906770U CN201796036U (en) | 2010-08-13 | 2010-08-13 | A test strip for quantitative detection of tumor marker colloidal gold immunochromatography |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900733A (en) * | 2010-08-13 | 2010-12-01 | 中南大学 | A test strip for quantitative detection of tumor marker colloidal gold immunochromatography and its preparation method |
| CN102221617A (en) * | 2011-06-03 | 2011-10-19 | 正元盛邦(天津)生物科技有限公司 | Early stage prostate cancer semi-quantitative diagnosis method utilizing double indication line immunity chromatography |
| CN105021813A (en) * | 2014-04-15 | 2015-11-04 | 东北师范大学 | Colloidal gold immunochromatography test paper strip for tumor detection |
| CN105929168A (en) * | 2016-07-11 | 2016-09-07 | 黑龙江省科学院高技术研究院 | Double-threshold detection test paper for breast cancer tumor marker CA153 |
| CN106802350A (en) * | 2017-03-11 | 2017-06-06 | 中国海洋大学 | A kind of pathogenicity fish enteron aisle vibrios quick detection test paper |
| CN109633158A (en) * | 2018-12-20 | 2019-04-16 | 黑龙江省科学院高技术研究院 | Breast cancer tumour marker CA153 dual threshold Rapid detection test strip and preparation method thereof |
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2010
- 2010-08-13 CN CN2010202906770U patent/CN201796036U/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900733A (en) * | 2010-08-13 | 2010-12-01 | 中南大学 | A test strip for quantitative detection of tumor marker colloidal gold immunochromatography and its preparation method |
| CN102221617A (en) * | 2011-06-03 | 2011-10-19 | 正元盛邦(天津)生物科技有限公司 | Early stage prostate cancer semi-quantitative diagnosis method utilizing double indication line immunity chromatography |
| CN105021813A (en) * | 2014-04-15 | 2015-11-04 | 东北师范大学 | Colloidal gold immunochromatography test paper strip for tumor detection |
| CN105929168A (en) * | 2016-07-11 | 2016-09-07 | 黑龙江省科学院高技术研究院 | Double-threshold detection test paper for breast cancer tumor marker CA153 |
| CN106802350A (en) * | 2017-03-11 | 2017-06-06 | 中国海洋大学 | A kind of pathogenicity fish enteron aisle vibrios quick detection test paper |
| CN106802350B (en) * | 2017-03-11 | 2019-02-26 | 中国海洋大学 | A rapid detection test strip for pathogenic fish enteric Vibrio |
| CN109633158A (en) * | 2018-12-20 | 2019-04-16 | 黑龙江省科学院高技术研究院 | Breast cancer tumour marker CA153 dual threshold Rapid detection test strip and preparation method thereof |
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| 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: 20110413 Termination date: 20160813 |