CN103257241B

CN103257241B - New application of CRISP (cysteine-rich secretory protein)1 to medical instrument

Info

Publication number: CN103257241B
Application number: CN201310191251.8A
Authority: CN
Inventors: 朱进安
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2013-05-22
Filing date: 2013-05-22
Publication date: 2015-04-29
Anticipated expiration: 2033-05-22
Also published as: CN103257241A

Abstract

The invention relates to an application of a CRISP (cysteine-rich secretory protein)1 to preparation of a medical instrument for diagnosing carcinoma of buccal mucosa. The medical instrument is a diagnostic test paper or kit. The diagnosis sample of the medical instrument is saliva. The invention has the advantages that a new application of the CRISP1 is provided, and a convenient, reliable, non-invasive and cheap method is provided for large-scale population screening of carcinoma of buccal mucosa and self-checking of patients after carcinoma of buccal mucosa surgery so as to guide treatment of carcinoma of buccal mucosa; and the test paper is quick in checking, has high sensitivity, strong specificity and good stability, is simple and convenient to operate, dispenses with any instrument, is intuitive and reliable in result judgment, is easy to master and is simultaneously convenient for the patients to self-monitor the conditions.

Description

The novelty teabag of a kind of CRISP1 in medicine equipment

Technical field

The present invention relates to a kind of novelty teabag of albumen, specifically, is about the novelty teabag of a kind of CRISP1 in medicine equipment.

Background technology

Cheek cancer usually means and betides carcinoma of buccal mucosa.Person habitually belongs to carcinoma of facial skin to betide skin of cheek.Cheek cancer is general without obvious pain in early days, causes patient often to incur loss through delay and seeks medical advice, and when infiltrating the deep tissues such as muscle or concurrent infection when cancerous swelling, occurs obvious pain, companion's limitation of mouth opening in various degree, until teeth clenched.After tooth surrounding tissue is got involved, toothache or odontoseisis can be there is.Due to cancer knurl infiltrate ulcer formed, particularly occur together infect time, can cause local secondary hemorrhage, pain increases the weight of.Patient often has submandibular lymph nodes to increase.Lymphadenovaris may, due to cancer tumor metastasis, also may be caused by infection.Because cheek cancer is infiltrative growth, local relapse is high, and except infiltration is shown shallow limitation cheek mucous membrane cancer can be considered to adopt simple freezing and radiotherapy among a small circle, centering patients with terminal, at present advocates to adopt the complex treatment based on operation more.

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 cheek cancer.

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 cheek cancer, 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 cheek cancer, oneself's detection of cheek cancer postoperative patient are provided convenience, reliably, without wound, inexpensive method, instructed the treatment of cheek cancer; 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 cheek cancer.

Accompanying drawing 2 is testing result schematic diagrames of the CRISP1 diagnose test paper of cheek cancer.

Accompanying drawing 3 is testing process schematic diagrames of the CRISP1 diagnose test paper of cheek cancer.

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 BT, cheek cancerous 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 cheek cancerous tissues, and is negative in the normal tissue.

Embodiment 2 enzyme-linked immunosorbent assay (Elisa)

Overall totally 90 examples, wherein cheek cancer (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. cheek cancer 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 cheek cancer

Diagnosis index	AUC	Sensitivity	Specificity	False positive rate	False negative rate	Youden index
							Urine CRISP1 10 ng/ml	0.808	88.2%	38.3%	59.7%	11.7%	0.265
Urine CRISP1 40 ng/ml	0.808	72.3%	85.2%	11.9%	28.6%	0.575

(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 cheek cancer is high, and its AUC reaches 0.808.With CRISP1 10ng/ml for diagnostic threshold, it is 88.2% to the diagnostic sensitivity of cheek cancer, and specificity is 38.3%, and false positive rate is 59.7%, and false negative rate is 11.7%; With CRISP1 40 ng/ml for diagnostic threshold, its sensitivity is 72.3%, and specificity is 85.2%, 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 cheek cancer

Please refer to accompanying drawing 1, accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of cheek cancer.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 cheek cancer

Please refer to accompanying drawing 2, accompanying drawing 2 is testing result schematic diagrames of the CRISP1 diagnose test paper of cheek cancer.

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 cheek cancer.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 cheek cancer, if T line does not develop the color, shows that in patient's saliva, CRISP1 content is normal.

Claims

1. one kind is rich in the application of cysteine secretory protein 1 in the medicine equipment of preparation diagnosis cheek cancer, 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.