CN103267858B - Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma - Google Patents
Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma Download PDFInfo
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- CN103267858B CN103267858B CN201310190886.6A CN201310190886A CN103267858B CN 103267858 B CN103267858 B CN 103267858B CN 201310190886 A CN201310190886 A CN 201310190886A CN 103267858 B CN103267858 B CN 103267858B
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- gland carcinoma
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Abstract
The invention relates to application of cysteine-rich secretory protein 1 in manufacturing a medical appliance for diagnosing salivary gland carcinoma. The medical appliance is a diagnostic test paper or kit, and the diagnostic sample of the medical appliance is saliva. The application disclosed by the invention provides the new application of the cysteine-rich secretory protein 1, provides a convenient, reliable, non-invasive and low-cost method for the large-scale population screening of the salivary gland carcinoma and the self detection of salivary gland carcinoma postoperative patients and instructs the treatment of the salivary gland carcinoma. The test paper disclosed by the invention has the advantages of fast detection, high sensitivity, high specificity, good stability, easiness and convenience for operation without any instrument or equipment, visuality and reliability in result judgment, easiness for grasp of the result judgment and convenience for self illness state monitoring of patients.
Description
Technical field
The present invention relates to a kind of albumen to apply in preparation diagnosis carcinoma of salivary gland gold test strip.
Background technology
In Oral and maxillofacial tumors, the ratio that salivary tumor transformation occurs is higher, for common disease, all can there is salivary tumor transformation in any age, benign tumour occurs in comparatively young patient, malignant tumour then mostly occurs at the larger age, men and women falls ill without significant difference, carcinoma of salivary gland is the malignant tumour occurred by salivary gland epithelial tissue, glandulae salivariae majores can be derived from, the parotid gland, salivary gland and sublingual gland, also glandulae salivariae minores (being more common in palatine gland) can be derived from, namely any have the position of salivary gland all can occur, pathological classification has: mucoepidermoid carcinoma, papillary cystadenocarcinoma, acinar cell carcinoma, adenoid cystic carcinoma, gland cancer, squama cancer and undifferentiated carcinoma etc.
Summary of the invention
The object of the invention is for deficiency of the prior art, provide a kind of and be rich in the application of cysteine secretory protein 1 in the medicine equipment of preparation diagnosis carcinoma of salivary gland.
For achieving the above object, the technical scheme that the present invention takes is: a kind of cysteine secretory protein 1 that is rich in is preparing the application in the medicine equipment diagnosing carcinoma of salivary gland, described medicine equipment is diagnose test paper or kit, and the diagnosis sample of described medicine equipment is saliva.
The diagnosis threshold of described medicine equipment is that saliva is rich in cysteine secretory protein 1 concentration 10-40ng/ml.
The invention has the advantages that: provide the novelty teabag being rich in cysteine secretory protein 1, for the large-scale crowd examination of carcinoma of salivary gland, oneself's detection of carcinoma of salivary gland postoperative patient are provided convenience, reliably, without wound, inexpensive method, instructed the treatment of carcinoma of salivary gland; Detection paper of the present invention is quick, highly sensitive, high specificity, good stability, easy and simple to handle, without the need to any instrument and equipment, and result judge intuitive and reliable, be easy to grasp, simultaneously also facilitate patient to state of an illness self-monitoring.
Accompanying drawing explanation
Accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of carcinoma of salivary gland.
Accompanying drawing 2 is testing result schematic diagrames of the CRISP1 diagnose test paper of carcinoma of salivary gland.
Accompanying drawing 3 is testing process schematic diagrames of the CRISP1 diagnose test paper of carcinoma of salivary gland.
Detailed description of the invention
Below detailed description of the invention provided by the invention is elaborated.
Embodiment 1
Below in conjunction with accompanying drawing, detailed description of the invention provided by the invention is elaborated.
The Reference numeral related in accompanying drawing and part as follows:
1. base plate 2. sample pad
3. gold size pad 4. cellulose nitrate rete
5. absorbent paper layer 6. saliva
7. chromatography direction
Embodiment 1 SABC
Experiment material: normal parotid tissue, carcinoma of salivary gland tissue; Primary antibodie is goat-anti people CRISP1 antibody, two anti-donkeys anti-sheep Dylight594 antibody.
Experimental technique:
A large amount of tissue sample is combined in a small substrate surface by organization chip exactly in an orderly manner, detects by immunohistochemical method.
1, tissue PFA fixer is fixed 5 minutes, PBS washes 10 minutes
2, antigen retrieval: expose epiope with 0.01M citrate solution.The heating in 3 minutes of the high fire of micro-wave oven repairs liquid to boiling (4 minutes), then 1 minute, twice, continuous low fiery microwave.Be cooled to room temperature, PBS washes 10 minutes.Triton rupture of membranes with 0.5% 10 minutes.
3, nonspecific proteins is closed
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 30 minutes
3) tap water 10 minutes, PBS soaks 3 minutes × 3 times, and drying or paper handkerchief suck surplus liquid (not encountering tissue), draws circle (prepare 10% in the distilled water flushing time and close serum) with groupization pen around tissue
4) in circle inner tissue, instillation 5% BSA(PBS prepares rapidly), close heterogenetic antigen (do not allow tissue dry, should add immediately after paper handkerchief sops up water), room temperature 30 minutes (preparing primary antibodie), deducts confining liquid, does not wash.
4, primary antibodie is hatched
Get rid of the BSA in section, dry residual BSA around tissue with filter paper, directly add the goat-anti people CRISP1 antibody (about 60 μ l) diluted, put into 4 DEG C, wet box and spend the night.Within second day, take out from refrigerator and need 37 DEG C of rewarming 30 min.
5, two anti-to hatch
1) primary antibodie is washed off, slide is inserted plastic slide frame, then wholely put into plastic casing, add PBS and soak to be put on microoscillator and wash 10 minutes.
2) with filter paper, the water around circle is sucked, add two anti-donkeys anti-sheep Dylight594 antibody, room temperature 30 minutes.
3) add PBS to soak and be put on microoscillator and wash 10 minutes.
6, mounting
Surplus liquid absorbed by paper handkerchief, and on slide, dropper drips DAPI mounting liquid one, and then covered, extrudes gently with tweezers, and drives bubble away, and room temperature places 1 hour.Observe.
Experimental result, organization chip immunohistochemical staining display CRISP1 secretory protein is obviously positive in 23 of 30 carcinoma of salivary gland tissues, and is negative in the normal tissue.
Embodiment 2 enzyme-linked immunosorbent assay (Elisa)
Overall totally 90 examples, wherein carcinoma of salivary gland (squamous cell carcinoma) case group 30 example; Oral cavity other diseases (periodontosis, dental pulp disease, thrush) organizes 30 examples; Normal healthy controls crowd 30 example.Employing is rich in cysteine secretory protein detection kit and is detected, and detecting step is with reference to being rich in cysteine secretory protein quantitative enzyme link detection reagent kit operation instructions.
The ELISA testing result of CRISP1:
1. carcinoma of salivary gland group is higher than other two groups, and other diseases group and Healthy People group indifference (P=0.802).
Table 1 saliva CRISP1 diagnoses the diagnostic value of carcinoma of salivary gland
| Diagnosis index | AUC | Sensitivity | Specificity | False positive rate | False negative rate | Youden index |
| Urine CRISP1 10 ng/ml | 0.811 | 89.1% | 37.3% | 60.7% | 12.9% | 0.264 |
| Urine CRISP1 40 ng/ml | 0.811 | 71.4% | 87.1% | 11.9% | 28.6% | 0.585 |
(illustrate: area under AUC:ROC curve (Receiver operating curve); AUC, more close to 1, illustrates that diagnosis effect is better; AUC has lower accuracy 0.5 ~ 0.7 time, and AUC has certain accuracy 0.7 ~ 0.9 time, has high accuracy when AUC is more than 0.9.Youden index: youden index=sensitivity+specificity-1; It is excellent diagnostics dividing value that Youden index reaches maximum corresponding value.）
The diagnostic value of saliva CRISP1 to carcinoma of salivary gland is high, and its AUC reaches 0.811.With CRISP1 10ng/ml for diagnostic threshold, it is 89.1% to the diagnostic sensitivity of carcinoma of salivary gland, and specificity is 37.3%, and false positive rate is 60.7%, and false negative rate is 12.9%; With CRISP1 40 ng/ml for diagnostic threshold, its sensitivity is 71.4%, and specificity is 87.1%, and false positive rate is 11.9%, and false negative rate is 28.6%.Saliva CRISP1 concentration 40 ng/ml is excellent diagnostics dividing value; Saliva CRISP1 concentration 10 ng/ml can be used for screening object.
Embodiment 3 prepares the golden labeling antibody compound of test paper
One, collaurum preparation
The chlorauric acid solution of 100ml 0.005%-0.02% is added in round-bottomed flask and is heated to boiling, after boiling, add the trisodium citrate (Na of freshly prepared 0.4%-2% with vigorous stirring quickly and accurately
3c
6h
5o
72H
2o) aqueous solution 1-2.2ml, after boiling 10-25 minute, continues stirring and is cooled to room temperature.Can see in this process that the color change of solution is: golden yellow → black → purple → dark blue → cerise, when the color of solution becomes transparent cerise completely, i.e. obtained required collaurum.Load in bag filter after cooling and ultra-pure water (1:5000) is dialysed three times, finally the collaurum of having dialysed is transferred in the vial of clean band spiral cover, preserve under the environment of 4 DEG C of lucifuges.
Two, the connection of gold colloid and albumen
1, the absorption of collaurum to albumen depends primarily on pH value, and close under the isoelectric point of protein or the condition of meta-alkali, the two easily forms firmly bond.If during the isoelectric point of the pH value of collaurum lower than protein, then can assemble and lose binding ability.
2, the preparation of protein solution to be marked: by albumen to be marked 4 DEG C 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.Then 100000 turns of 4 DEG C of centrifugal 1h, remove polymer.
3, the preparation of marking colloidal gold solution is waited: with 0.05 mol/l-0.3 mol/l K
2cO
3the pH value adjusting collaurum liquid is 7.5-10.0.Because colloidal gold solution may damage the electroplax of pH meter, therefore, when regulating pH, accurate pH test paper is adopted to be determined as suitable.
4, the determination of collaurum and the ratio of labelled protein consumption: according to the requirement of albumen to be marked, after collaurum is mixed up pH, packing 10 is managed, often 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 above-mentioned gold size solution, mixing.Control tube only adds 1ml dilution.After 5min, in above-mentioned each pipe, add 0.1ml 10%NaCl solution, leave standstill 2h after mixing, observed result.Control tube (not adding protein) and add each pipe of quantity not sufficient with stable colloid gold of protein, all presents by the coagulation phenomenon of red stain indigo plant; And add protein content and meet or exceed the quantitative each pipe of minimum steady and still keep red constant.Stablize the red constant minimum enzyme protein dosage of 1ml colloidal gold solution, be the minimum amount of this protein of mark, in real work, suitably can increase by 10% ~ 20%.
5, the combination of collaurum and anti-CRISP1 monoclonal antibody: by colloidal gold solution with 0.05 mol/l-0.3 mol/l K
2cO
3adjust pH to 7.5-10.0, add the anti-CRISP1 monoclonal antibody solution of having dialysed, eddy mixer mixes, and after reaction 10min, adds stabilizing agent and precipitates to prevent collaurum to be polymerized.Conventional stabilizing agent is 5% hyclone (BSA) and 1% polyethylene glycol (molecular weight 20KD).The amount added: 5%BSA makes solution final concentration be 1%; 1% polyethylene glycol adds to 1/10 of total solution.
6, the purifying of colloid gold label albumen: the colloidal gold labeled monoclonal antibody compound prepared is at the centrifugal 15min of 900rpm/min, and careful sucking-off supernatant, sediment redissolves with containing the sucrose of 5% and the 0.002M borate buffer solution of 0.05% Tween-20.Centrifuge washing twice, is finally concentrated into 1/10,4 DEG C of preservations of original volume by compound.
Embodiment 4 prepares the CRISP1 test paper of quick diagnosis carcinoma of salivary gland
Please refer to accompanying drawing 1, accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of carcinoma of salivary gland.This test paper is provided with base plate 1, base plate covers successively sample pad 2, gold size pad 3, cellulose nitrate rete 4 and absorbent paper layer 5.Described base plate 1 is PVC base plate, and saliva glass fibre inhaled by the material of sample pad 2.The making of gold size pad 3: by preparation-obtained for embodiment 3 golden labeling antibody solution specking on the glass fibre membrane that 1cm is wide, point sample amount is about 2ul/cm, 37 DEG C of dryings.The making of cellulose nitrate rete 4: by two anti-speckings on nitrocellulose membrane (NC film), as nature controlling line (C line).Using anti-CRISP1 polyclonal antibody specking distance nature controlling line 1cm place as p-wire (T line), point sample amount is about 1ul/cm.The detection threshold of described p-wire (T line) is 10-40 ng/ml.37 DEG C of dryings.Above-mentioned sample pad, gold size pad, cellulose nitrate rete, absorbent paper layer are assembled on base plate successively, are cut into the test strips that 4mm is wide, load in test card.
The detection of the CRISP1 test paper of embodiment 5 quick diagnosis carcinoma of salivary gland
Please refer to accompanying drawing 2, accompanying drawing 2 is testing result schematic diagrames of the CRISP1 diagnose test paper of carcinoma of salivary gland.
One, test paper detecting method:
1, the sample to be tested (saliva) getting 50ul is added in the S place, sample application zone of test strips;
2, in 10min postscript observed and recorded testing result.The result of observing after 20min is invalid.
Two, the judgement of testing result:
1, positive findings: please refer to accompanying drawing 2A, each appearance aubergine band on the p-wire T line and nature controlling line C line position of test strips.
, only on the nature controlling line C line of test strips, there is an aubergine band in 2, negative findings: please refer to accompanying drawing 2B.
3, null result: please refer to accompanying drawing 2C, there is not aubergine band in the nature controlling line C line of test strips.
Three, Cleaning Principle
Please refer to accompanying drawing 3, accompanying drawing 3 is testing process schematic diagrames of the CRISP1 diagnose test paper of carcinoma of salivary gland.As shown in the figure, several salivas 6 drop in sample pad 2, because of chromatography effect, liquid flows along chromatography direction 7 to absorbent paper layer 5 direction, when flowing through gold size pad 3, gold labeling antibody just can be dissolved, and the CRISP1 in saliva is combined, forming gold mark compound, continuing reach, when flowing through T line (detection line) with liquid, the anti-CRISP1 polyclonal antibody of compound on T line is combined and condenses and develop the color, when flowing through C line (nature controlling line), compound and two anti-bindings and condense colour developing, as C line does not develop the color, it is invalid to show to detect.If T line develops the color, showing that in patient's saliva, CRISP1 content is more than or equal to 10-40ng/ml, is the positive, indicates the possibility suffering from carcinoma of salivary gland, if T line does not develop the color, shows that in patient's saliva, CRISP1 content is normal.
Claims (1)
1. one kind is rich in the application of cysteine secretory protein 1 in the medicine equipment of preparation diagnosis carcinoma of salivary gland, described medicine equipment is diagnose test paper or kit, the diagnosis sample of described medicine equipment is saliva, and the diagnosis threshold of described medicine equipment is that saliva is rich in cysteine secretory protein 1 concentration 40ng/ml.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310190886.6A CN103267858B (en) | 2013-05-22 | 2013-05-22 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310190886.6A CN103267858B (en) | 2013-05-22 | 2013-05-22 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma |
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| CN103267858B true CN103267858B (en) | 2015-03-25 |
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| US20030027178A1 (en) * | 2001-03-16 | 2003-02-06 | George Vasmatzis | Methods and kits for determining a cancer diagnosis and prognosis |
| 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 |
| WO2008067065A2 (en) * | 2006-10-19 | 2008-06-05 | Shiv Srivastava | Methods, kits, and systems for diagnosing and prognosing prostate cancer using secreted biomarkers |
| CA2693546A1 (en) * | 2009-11-03 | 2011-05-03 | Universite Laval | Detection of human cysteine-rich secretory protein (crisp1) in semen and medical applications related thereto |
| US8658166B2 (en) * | 2011-11-08 | 2014-02-25 | Caldera Health Limited | Methods and materials for the diagnosis of prostate cancers |
| CN102914658A (en) * | 2012-10-24 | 2013-02-06 | 高维强 | Application of CRISP3 protein on diagnosis and treatment of prostate cancer |
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