CN103267859A - Application of CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing gingival carcinoma - Google Patents
Application of CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing gingival carcinoma Download PDFInfo
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- CN103267859A CN103267859A CN201310190889XA CN201310190889A CN103267859A CN 103267859 A CN103267859 A CN 103267859A CN 201310190889X A CN201310190889X A CN 201310190889XA CN 201310190889 A CN201310190889 A CN 201310190889A CN 103267859 A CN103267859 A CN 103267859A
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Abstract
The invention relates to application of CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing gingival carcinoma in preparation of medical appliances for diagnosing gingival carcinoma. The test paper comprises a substrate, a sample pad, a colloidal gold pad, a nitrocellulose membranous layer and a water-absorption paper layer, wherein the nitrocellulose membranous layer contains a BSA (bovine serum albumin)-saliva CRISP1 coupling substance test line and an anti-mouse secondary antibody quality control line; and the colloidal gold pad contains a colloidal gold labeled anti-saliva CRISP1 antibody compound formed by combining colloidal gold and anti-saliva CRISP1 antibody. The test paper provided by the invention has the advantages of high detection speed, high sensitivity, high specificity, high stability, no need of any instrument or equipment, and visual and reliable judgment result, is simple to operate and easy to master, and is convenient for patients to perform self-monitoring of the state of illness.
Description
Technical field
The present invention relates to a kind of application of being rich in halfcystine secretory protein 1 test paper of diagnosing gingival carcinoma.
Background technology
Gingival carcinoma (carcinoma of the gingiva) is only second to tongue cancer in carcinoma of mouth and the cheek cancer occupies the 3rd, accounts for about 22% of carcinoma of mouth.The male sex is more than the women.Be more common in 40～60 years old.Mostly be the higher squamous cell carcinoma of differentiation degree, poor growth, early stage many non-evident symptons.See with the ulcer type more.Gingival carcinoma can betide lip buccal side gum mucous membrane, also can betide tongue, palate side gum mucous membrane.Lip buccal side gum mucous membrane and cheek mucous membrane adjoin up and down, are its separatrix with the lip buccal groove.The gum mucous membrane of lower jaw tongue side adjoined with a mouthful end, was its separatrix with the jaw lingual groove.The gum of upper jaw palate side is then mutually continuous with the palate mucous membrane, there is no clearly boundary clinically, so the scope of palate side gum does not still have final conclusion so far.
Summary of the invention
The objective of the invention is at deficiency of the prior art, provide a kind of diagnose gingival carcinoma be rich in the application of halfcystine secretory protein 1 test paper in the medicine equipment of preparation diagnosis gingival carcinoma.
For achieving the above object, the technical scheme that the present invention takes is: a kind of diagnose gingival carcinoma be rich in the application of halfcystine secretory protein 1 test paper in the medicine equipment of preparation diagnosis gingival carcinoma, described test paper, comprise base plate, sample pad, the gold size pad, cellulose nitrate rete and absorbent paper layer, cover sample pad on the described base plate successively, the gold size pad, cellulose nitrate rete and absorbent paper layer, contain bovine serum albumin(BSA)-saliva on the described cellulose nitrate rete and be rich in the p-wire of halfcystine secretory protein 1 conjugate and the nature controlling line that anti-mouse two resists, containing collaurum and anti-saliva on the described gold size pad is rich in halfcystine secretory protein 1 antibody and is combined the gold that forms and marks anti-saliva and be rich in halfcystine secretory protein 1 antibody complex, the diagnosis sample of described test paper is saliva, and the detection fault value of described test paper is that saliva is rich in halfcystine secretory protein 1 concentration 10-40ng/ml.
The invention has the advantages that: the new purposes that is rich in halfcystine secretory protein 1 is provided, provide convenience for the oneself of the large-scale crowd examination of gingival carcinoma, gingival carcinoma postoperative patient detects, reliably, do not have wound, inexpensive method, instruct the treatment of gingival carcinoma; Detection paper of the present invention is quick, highly sensitive, high specificity, good stability, easy and simple to handle, need not any instrument and equipment, and the result judge intuitive and reliable, be easy to grasp, also make things convenient for patient to state of an illness self-monitoring simultaneously.
Description of drawings
Accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of gingival carcinoma.
Accompanying drawing 2 is testing result synoptic diagram of the CRISP1 diagnose test paper of gingival carcinoma.
Accompanying drawing 3 is testing process synoptic diagram of the CRISP1 diagnose test paper of gingival carcinoma.
Embodiment
Below embodiment provided by the invention is elaborated.
Embodiment 1
Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.
The Reference numeral and the ingredient that relate in the accompanying drawing are as follows:
1. base plate 2. sample pad
3. gold size pad 4. cellulose nitrate retes
5. absorbent paper layer 6. salivas
7. chromatography direction
Embodiment 1 SABC
Experiment material: normal gingiva tissue, gingival carcinoma tissue; Primary antibodie is goat-anti people CRISP1 antibody, the anti-sheep Dylight594 of two anti-donkeys antibody.
Experimental technique:
Organization chip is combined in a large amount of tissue samples a small substrate surface exactly in an orderly manner, detects by immunohistochemical method.
1, will organize with the PFA immobile liquid and fix 5 minutes, PBS washed 10 minutes
2, antigen retrieval: expose antigen with the 0.01M citrate solution and determine family.The heating in 3 minutes of the high fire of micro-wave oven is repaired liquid to boiling (4 minutes), and continuously low fiery microwave is 1 minute, twice again.Be cooled to room temperature, PBS washed 10 minutes.Triton rupture of membranes with 0.5% 10 minutes.
3, sealing nonspecific proteins
1) PBS soaks 3 minutes * 3 times
2) 3% H
2O
2-methyl alcohol (30% H
2O
210ml+ methyl alcohol 90ml) soaking at room temperature is 30 minutes
3) the tap water flushing is 10 minutes, and PBS soaks 3 minutes * 3 times, and drying or paper handkerchief are inhaled and removed unnecessary liquid (not running into tissue), draws circle (distilled water flushing prepares 10% and seals serum in the time) with the groupization pen around tissue
4) splashing into 5% BSA(rapidly in circle inner tissue prepares with PBS), sealing heterogenetic antigen (do not allow tissue dry, should after paper handkerchief sops up water, add immediately), room temperature 30 minutes (preparing primary antibodie) is deducted confining liquid, does not wash.
4, primary antibodie is hatched
Get rid of the BSA on cutting into slices, dry tissue residual BSA on every side with filter paper, the goat-anti people CRISP1 antibody (about 60 μ l) that direct adding has been diluted is put into wet box and is spent the night for 4 ℃.From refrigerator, took out in second day and need 37 ℃ of rewarming 30 min.
5, two anti-hatching
1) primary antibodie is washed off, slide is inserted the plastic slide frame, the whole plastic casing of putting into then adds PBS and soaks to be put in and wash 10 minutes on the microoscillator.
2) with filter paper the suction of the water around the circle is gone, add the anti-sheep Dylight594 of two anti-donkeys antibody, room temperature 30 minutes.
3) adding PBS soaks to be put in and washes 10 minutes on the microoscillator.
6, mounting
Paper handkerchief is absorbed unnecessary liquid, and dropper drips one of DAPI mounting liquid on slide, and covered is pushed gently with tweezers then, and drives bubble away, and room temperature was placed 1 hour.Observe.
Experimental result, organization chip immunohistochemical staining show that the CRISP1 secretory protein is the obvious positive in 23 of 30 gingival carcinoma tissues, and negative in normal structure.
Totally totally 90 examples, wherein gingival carcinoma (squamous cell carcinoma) case group 30 examples; Oral cavity other diseases (periodontosis, dental pulp disease, thrush) group 30 examples; Normal healthy controls crowd's 30 examples.Employing is rich in halfcystine secretory protein detection kit and is detected, and detects step with reference to being rich in the quantitative elisa kit for detecting operation instructions of halfcystine secretory protein.
The ELISA testing result of CRISP1:
1. the gingival carcinoma group is higher than other two groups, and other diseases group and healthy population group indifference (P=0.802).
The diagnostic value of table 1 saliva CRISP1 diagnosis gingival carcinoma
| Diagnosis index | AUC | Sensitivity | Specificity | False positive rate | False negative rate | The Youden index |
| Urine CRISP1 10 ng/ml | 0.829 | 86.5% | 41.3% | 60.7% | 12.9% | 0.278 |
| Urine CRISP1 40 ng/ml | 0.829 | 75.4% | 85.1% | 12.9% | 28.6% | 0.605 |
(illustrate: AUC:ROC curve (experimenter's performance curve) is area down; AUC illustrates that more close to 1 diagnosis effect is more good; AUC had low accuracy at 0.5～0.7 o'clock, AUC had certain accuracy at 0.7～0.9 o'clock, and AUC has high accuracy when above 0.9.Youden index: youden index=sensitivity+specificity-1; It is the excellent diagnostics dividing value that the Youden index reaches maximum corresponding value.）
The diagnostic value height of the gingival carcinoma of saliva CRISP1, its AUC reaches 0.829.Be diagnostic threshold with CRISP1 10ng/ml, its diagnostic sensitivity to gingival carcinoma is 86.5%, and specificity is 41.3%, and false positive rate is 60.7%, and false negative rate is 12.9%; Be diagnostic threshold with CRISP1 40 ng/ml, its sensitivity is 75.4%, and specificity is 85.1%, and false positive rate is 12.9%, and false negative rate is 28.6%.Saliva CRISP1 concentration 40 ng/ml are the excellent diagnostics dividing value; Saliva CRISP1 concentration 10 ng/ml can be used for the screening purpose.
The golden labeling antibody compound of embodiment 3 preparation test paper
One, Preparation of Colloidal Gold
Be heated to boiling in the chlorauric acid solution adding round-bottomed flask with 100ml 0.005%-0.02%, the boiling back adds the trisodium citrate (Na of freshly prepared 0.4%-2% quickly and accurately under vigorous stirring
3C
6H
5O
72H
2O) aqueous solution 1-2.2ml, boil 10-25 minute after, continue to stir and to be cooled to room temperature.The change color that can see solution in this process is: golden yellow → black → purple → dark blue → cerise when the color of solution becomes transparent cerise fully, namely makes required collaurum.Pack into after the cooling in the bag filter to ultrapure water (1:5000) dialysis three times, the collaurum of getting well of will dialyse is at last transferred in the vial of clean band spiral cover, preserves under the environment of 4 ℃ of lucifuges.
Two, gold colloid and albumen is connected
1, collaurum depends primarily on the pH value to the absorption of albumen, and near the isoelectric point of protein or partially under the condition of alkali, the two forms firm bond easily.When if the pH value of collaurum is lower than the isoelectric point of protein, then can assembles and lose binding ability.
2, the preparation of protein solution to be marked: with albumen to be marked 4 ℃ of dialysed overnight in 0.003 mol/l-0.01 mol/l pH5.5-7.8 NaCl solution in advance, to remove unnecessary salt ion.100000 change 4 ℃ of centrifugal 1h then, remove polymkeric substance.
3, wait to mark the preparation of colloidal gold solution: with 0.05 mol/l-0.3 mol/l K
2CO
3Transferring the pH value of collaurum liquid is 7.5-10.0.Because colloidal gold solution may damage the electroplax of pH meter, therefore, when regulating pH, adopt accurate pH test paper to be determined as suitable.
4, determining of the ratio of collaurum and labelled protein consumption: according to the requirement of albumen to be marked, collaurum is mixed up after the pH packing 10 pipes, every pipe 1ml.It is 5 μ g/ml～50 μ g/ml that labelled protein is done serial dilution with 0.005mol/l pH9.0 borate buffer solution, gets 1ml respectively, adds in the above-mentioned gold size solution mixing.Control tube only adds the 1ml dilution.Behind the 5min, in above-mentioned each pipe, add 0.1ml 10%NaCl solution, leave standstill 2h behind the mixing, observations.The quantity not sufficient of control tube (not adding protein) and adding protein all presents the coagulation phenomenon by red stain indigo plant with each pipe of stable colloid gold; Still maintenance is red constant and the adding protein content meets or exceeds each quantitative pipe of minimum steady.Stablize the red constant minimum protein consumption of 1ml colloidal gold solution, be the minimum amount of this protein of mark, in real work, can suitably increase by 10%～20%.
5, the combination of collaurum and anti-CRISP1 monoclonal antibody: with colloidal gold solution with 0.05 mol/l-0.3 mol/l K
2CO
3Transfer pH to 7.5-10.0, add the good anti-CRISP1 monoclonal antibody solution of dialysis, the eddy mixer mixing behind the reaction 10min, adds stabilizing agent and precipitates to prevent the collaurum polymerization.Stabilizing agent commonly used is 5% hyclone (BSA) and 1% polyglycol (molecular weight 20KD).The amount that adds: it is 1% that 5%BSA makes the solution final concentration; 1% polyglycol adds to 1/10 of total solution.
6, the purifying of colloid gold label albumen: the colloid gold label antibody complex for preparing is at the centrifugal 15min of 900rpm/min, careful sucking-off supernatant, and sediment redissolves with containing 5% sucrose and the 0.002M borate buffer solution of 0.05% Tween-20.Centrifuge washing twice is concentrated into compound 1/10,4 ℃ of preservation of original volume at last.
The CRISP1 test paper of embodiment 4 preparation quick diagnosis gingival carcinomas
Please refer to accompanying drawing 1, accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of gingival carcinoma.This test paper is provided with base plate 1, covers sample pad 2, gold size pad 3, cellulose nitrate rete 4 and absorbent paper layer 5 on the base plate successively.Described base plate 1 is the PVC base plate, and the material of sample pad 2 is to inhale the saliva glass fibre.The making of gold size pad 3: on the wide glass fibre membrane of 1cm, the point sample amount is about 2ul/cm, 37 ℃ of dryings with embodiment 3 preparation-obtained golden labeling antibody solution speckings.The making of cellulose nitrate rete 4: with two anti-speckings on nitrocellulose membrane (NC film), as nature controlling line (C line).To resist CRISP1 polyclonal antibody specking at distance nature controlling line 1cm place as p-wire (T line), the point sample amount is about 1ul/cm.The detection threshold of described p-wire (T line) is 10-40 ng/ml.37 ℃ of dryings.Above-mentioned sample pad, gold size pad, cellulose nitrate rete, absorbent paper layer are assembled on the base plate successively, are cut into the wide test strips of 4mm, in the test card of packing into.
The detection of the CRISP1 test paper of embodiment 5 quick diagnosis gingival carcinomas
Please refer to accompanying drawing 2, accompanying drawing 2 is testing result synoptic diagram of the CRISP1 diagnose test paper of gingival carcinoma.
One, test paper detecting method:
1, the sample to be tested (saliva) of getting 50ul is added in the sample application zone S place of test strips;
2, in 10min postscript observed and recorded testing result.The result who observes behind the 20min is invalid.
Two, the judgement of testing result:
1, positive findings: please refer to accompanying drawing 2A, on the p-wire T line of test strips and nature controlling line C line position, an aubergine band respectively occurs.
2, negative findings: please refer to accompanying drawing 2B, only an aubergine band occurs at the nature controlling line C of test strips line.
3, null result: please refer to accompanying drawing 2C, on the nature controlling line C of test strips line, the aubergine band do not occur.
Three, detect principle
Please refer to accompanying drawing 3, accompanying drawing 3 is testing process synoptic diagram of the CRISP1 diagnose test paper of gingival carcinoma.As shown in the figure, several salivas 6 drop on the sample pad 2, because of the chromatography effect, 5 directions flow liquid along chromatography direction 7 to absorbent paper layer, when flowing through gold size pad 3, the gold labeling antibody just can be dissolved, and the CRISP1 in saliva is combined, form gold mark compound, continue reach with liquid, when flowing through T line (detection line), the anti-CRISP1 polyclonal antibody of compound on the T line is combined and condensed colour developing, when flowing through C line (nature controlling line), compound and two resistive connections close and condense colour developing, and not developing the color as the C line shows that then detection is invalid.If the colour developing of T line shows that then CRISP1 content is more than or equal to 10-40ng/ml in patient's saliva, positive, expression has the possibility of suffering from gingival carcinoma, if the T line does not develop the color and shows that then CRISP1 content is normal in patient's saliva.
Claims (1)
- One kind diagnose gingival carcinoma be rich in the application of halfcystine secretory protein 1 test paper in the medicine equipment of preparation diagnosis gingival carcinoma, described test paper, comprise base plate, sample pad, the gold size pad, cellulose nitrate rete and absorbent paper layer, cover sample pad on the described base plate successively, the gold size pad, cellulose nitrate rete and absorbent paper layer, contain bovine serum albumin(BSA)-saliva on the described cellulose nitrate rete and be rich in the p-wire of halfcystine secretory protein 1 conjugate and the nature controlling line that anti-mouse two resists, containing collaurum and anti-saliva on the described gold size pad is rich in halfcystine secretory protein 1 antibody and is combined the gold that forms and marks anti-saliva and be rich in halfcystine secretory protein 1 antibody complex, the diagnosis sample of described test paper is saliva, and the detection fault value of described test paper is that saliva is rich in halfcystine secretory protein 1 concentration 10-40ng/ml.
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2013
- 2013-05-22 CN CN201310190889.XA patent/CN103267859B/en not_active Expired - Fee Related
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| US20030027178A1 (en) * | 2001-03-16 | 2003-02-06 | George Vasmatzis | Methods and kits for determining a cancer diagnosis and prognosis |
| US20050282729A1 (en) * | 2002-05-28 | 2005-12-22 | Regents Of The Univeristy Of Minnesota | Crisp polypeptides as contraceptives and inhibitors of sperm capacitation |
| WO2007092713A2 (en) * | 2006-02-02 | 2007-08-16 | Trustees Of The University Of Pennsylvania | Microfluidic system and method for analysis of gene expression in cell-containing samples and detection of disease |
| US20100183562A1 (en) * | 2007-02-02 | 2010-07-22 | Istituto Nazionale Di Genetica Molecolare-Ingm | Sub -population of hematopoietic stem cells that express the crisp-1 protein |
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