CN103267861A

CN103267861A - Application of cysteine-rich secretory protein 1 in manufacturing product for diagnosing palate carcinoma

Info

Publication number: CN103267861A
Application number: CN2013101909197A
Authority: CN
Inventors: 朱进安
Original assignee: SUZHOU MAERTAI NEW MATERIAL CO Ltd
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2013-05-22
Filing date: 2013-05-22
Publication date: 2013-08-28
Anticipated expiration: 2033-05-22
Also published as: CN103267861B

Abstract

The invention relates to application of cysteine-rich secretory protein 1 in manufacturing a medical appliance for diagnosing palate 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 palate carcinoma and the self detection of palate carcinoma postoperative patients and instructs the treatment of the palate 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

Being rich in halfcystine secretory protein 1 uses in preparation diagnosis palate cancer product

Technical field

The present invention relates to a kind of halfcystine secretory protein 1 that is rich in uses in preparation diagnosis palate cancer product.

Background technology

Palate cancer (carcinoma of the palate) should only limit to the primary cancerous swelling of hard palate by the UICC classification; The soft palate cancer should be listed oropharynx cancer scope in.The hard palate carcinogenesis is in the squama cancer of hard palate, and the many highly differentiation of cell develop general the comparison slowly, often invade the palatine sclerotin, cause palatal perforation.Can be to nasal cavity and maxillary sinus, to both sides development erodable gum to vertical spread.

Summary of the invention

The objective of the invention is at deficiency of the prior art, a kind of application of halfcystine secretory protein 1 in the medicine equipment of preparation diagnosis palate cancer of being rich in is provided.

For achieving the above object, the technical scheme that the present invention takes is: a kind ofly be rich in the application of halfcystine secretory protein 1 in the medicine equipment of preparation diagnosis palate cancer, described medicine equipment is diagnose test paper or kit, and the diagnosis sample of described medicine equipment is saliva.

The diagnosis fault value of described medicine equipment 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 palate cancer, palate cancer postoperative patient detects, reliably, do not have wound, inexpensive method, instruct the treatment of palate cancer; 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 palate cancer.

Accompanying drawing 2 is testing result synoptic diagram of the CRISP1 diagnose test paper of palate cancer.

Accompanying drawing 3 is testing process synoptic diagram of the CRISP1 diagnose test paper of palate cancer.

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 palate tissue, palate cancerous 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 palate cancerous tissues, and negative in normal structure.

Embodiment 2 enzyme-linked immunosorbent assays (Elisa)

Totally totally 90 examples, wherein palate cancer (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. palate cancer 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 palate cancer

Diagnosis index	AUC	Sensitivity	Specificity	False positive rate	False negative rate	The Youden index
							Urine CRISP1 10 ng/ml	0.850	88.7%	37.2%	61.7%	12.7%	0.259
Urine CRISP1 40 ng/ml	0.809	70.4%	85.1%	12.9%	28.6%	0.555

(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 palate cancer of saliva CRISP1, its AUC reaches 0.850.Be diagnostic threshold with CRISP1 10ng/ml, its diagnostic sensitivity to the palate cancer is 88.7%, and specificity is 37.2%, and false positive rate is 61.7%, and false negative rate is 12.7%; Be diagnostic threshold with CRISP1 40 ng/ml, its sensitivity is 70.4%, 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 palate cancers

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

Please refer to accompanying drawing 2, accompanying drawing 2 is testing result synoptic diagram of the CRISP1 diagnose test paper of palate cancer.

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 palate cancer.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 the palate cancer, 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 is rich in the application of halfcystine secretory protein 1 in the medicine equipment of preparation diagnosis palate cancer, and described medicine equipment is diagnose test paper or kit, and the diagnosis sample of described medicine equipment is saliva.

2. application according to claim 1, the diagnosis fault value of described medicine equipment are that saliva is rich in halfcystine secretory protein 1 concentration 10-40ng/ml.