CN103308692A - CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis - Google Patents
CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis Download PDFInfo
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- CN103308692A CN103308692A CN2013101909089A CN201310190908A CN103308692A CN 103308692 A CN103308692 A CN 103308692A CN 2013101909089 A CN2013101909089 A CN 2013101909089A CN 201310190908 A CN201310190908 A CN 201310190908A CN 103308692 A CN103308692 A CN 103308692A
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Abstract
The invention relates to cysteine-rich secretory protein 1 (CRISP1) test paper for oral floor carcinoma diagnosis. The test paper comprises a base plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane layer and an absorbent paper layer, wherein the nitrocellulose membrane layer contains a test line with a bovine serum albumin-salivary CRISP1 conjugate and a quality control line of a mouse second antibody, and the colloidal gold pad contains a gold-labeled salivary CRISP1 antibody complex which is formed through combining colloidal gold with a salivary CRISP1 antibody. The test paper disclosed by the invention has the advantages that the detection is rapid, the sensitivity is high, the specificity is strong, the stability is good, the operation is simple and convenient, any device/equipment is not required, the result judgment is intuitive and reliable and is easy to grasp, and meanwhile, the monitoring on the state of illness by patients is facilitated.
Description
Technical field
The present invention relates to a kind of CRISP1 gold-labeled kit, specifically, is the CRISP1 gold-labeled kit about a kind of quick diagnosis carcinoma of floor of mouth.
Background technology
The squama cancer of mucous membrane at the bottom of carcinoma of floor of mouth refers to betide mouthful.The mouth end is the crescent zone between lower gum and ventral surface of tongue, forms the bottom in oral cavity.Mucous membrane of floor of mouth covers mandibular flesh and above the musculus hyoglossus, and base portion mucous membrane and margo lateralis linguae mucous membrane are connected under rear portion and the tonsillotome front pillar, and the front then is divided into the left and right sides by glossodemus.Submaxillary duct is opened on the other mucous membrane of floor of mouth of glossodemus, is sublingual gland under the mucous membrane.Clinical manifestation: the predilection site of mucous membrane of floor of mouth cancer is the other front area of glossodemus and is equivalent to the lateral region that first and second is ground one's teeth in sleep, and both are slightly different in clinical manifestation.Carcinoma of floor of mouth is respectively organized muscle, the tongue body infringement except reaching to dark face sublingual gland, also can invade to mandibular.
Summary of the invention
The objective of the invention is for deficiency of the prior art, provide a kind of diagnose carcinoma of floor of mouth be rich in halfcystine secretory protein 1 test paper.
For achieving the above object, the technical scheme that the present invention takes is: a kind of diagnose carcinoma of floor of mouth be rich in halfcystine secretory protein 1 test paper, comprise base plate, sample pad, the gold size pad, cellulose nitrate rete and absorbent paper layer, cover successively sample pad on the described base plate, 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 threshold of described test paper is that saliva is rich in halfcystine secretory protein 1 concentration 10-40ng/ml.
Described gold size pad is comprised of glass fibre and the golden labeling antibody that is fixed thereon, and described sample pad is the water absorptivity glass layer.
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 carcinoma of floor of mouth, carcinoma of floor of mouth postoperative patient detects, reliably, without wound, inexpensive method, instruct the treatment of carcinoma of floor of mouth; 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 carcinoma of floor of mouth.
Accompanying drawing 2 is testing result schematic diagram of the CRISP1 diagnose test paper of carcinoma of floor of mouth.
Accompanying drawing 3 is testing process schematic diagram of the CRISP1 diagnose test paper of carcinoma of floor of mouth.
Embodiment
The below elaborates to embodiment provided by the invention.
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 mouth end mucous membrane tissue, carcinoma of floor of mouth 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 epiope with the 0.01M citrate solution.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 suck unnecessary liquid (not running into tissue), draws circle (distilled water flushing prepares 10% sealing serum in the time) with the groupization pen around tissue
4) splashing into rapidly 5% BSA(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 (approximately 60 μ l) that direct adding has been diluted is put into 4 ℃ in wet box and is spent the night.Second day takes out from refrigerator needs 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 water around the circle is sucked, 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 then covered is pushed gently with tweezers, 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 carcinoma of floor of mouth tissues, and negative in normal structure.
Totally totally 90 examples, wherein carcinoma of floor of mouth (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 detecting step is with reference to being rich in halfcystine secretory protein quantitative enzyme link detection reagent kit operation instructions.
The ELISA testing result of CRISP1:
1. the carcinoma of floor of mouth group is higher than other two groups, and other diseases group and Healthy People group indifference (P=0.802).
The diagnostic value of table 1 saliva CRISP1 diagnosis carcinoma of floor of mouth
| Diagnosis index | AUC | Sensitivity | Specificity | False positive rate | False negative rate | The Youden index |
| Urine CRISP1 10 ng/ml | 0.843 | 87.2% | 38.5% | 59.6% | 13.2% | 0.257 |
| Urine CRISP1 40 ng/ml | 0.843 | 70.4% | 87.6% | 12.9% | 28.6% | 0.580 |
(illustrate: the lower area of AUC:ROC curve (experimenter's performance curve); AUC illustrates that more close to 1 diagnosis effect is better; 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.）
Saliva CRISP1 is high to the diagnostic value of carcinoma of floor of mouth, and its AUC reaches 0.843.Take CRISP1 10ng/ml as diagnostic threshold, its diagnostic sensitivity to carcinoma of floor of mouth is 87.2%, and specificity is 38.5%, and false positive rate is 59.6%, and false negative rate is 13.2%; Take CRISP1 40 ng/ml as diagnostic threshold, its sensitivity is 70.4%, and specificity is 87.6%, 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, collaurum preparation
Be heated to boiling in the chlorauric acid solution adding round-bottomed flask with 100ml 0.005%-0.02%, under vigorous stirring, add quickly and accurately the trisodium citrate (Na of freshly prepared 0.4%-2% after the boiling
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.Can see in this process that the color of the solution variation is: golden yellow → black → purple → dark blue → cerise when the color of the 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 clean vial with 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 under the condition near the isoelectric point of protein or meta-alkali, the two easily forms firmly bond.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.Then 100000 turn 4 ℃ of centrifugal 1h, remove polymkeric substance.
3, the preparation of colloidal gold solution to be marked: 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.Labelled protein is done serial dilution as 5 μ g/ml～50 μ g/ml take 0.005mol/l pH9.0 borate buffer solution, get respectively 1ml, add 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 colloidal gold labeled monoclonal antibody compound 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 carcinoma of floor of mouth
Please refer to accompanying drawing 1, accompanying drawing 1 is the structural representation of the CRISP1 diagnose test paper of carcinoma of floor of mouth.This test paper is provided with base plate 1, covers successively sample pad 2, gold size pad 3, cellulose nitrate rete 4 and absorbent paper layer 5 on the base plate.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 carcinoma of floor of mouth
Please refer to accompanying drawing 2, accompanying drawing 2 is testing result schematic diagram of the CRISP1 diagnose test paper of carcinoma of floor of mouth.
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 schematic diagram of the CRISP1 diagnose test paper of carcinoma of floor of mouth.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 such 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 carcinoma of floor of mouth, does not show that then CRISP1 content is normal in patient's saliva if the T line does not develop the color.
Claims (2)
- One kind diagnose carcinoma of floor of mouth be rich in halfcystine secretory protein 1 test paper, comprise base plate, sample pad, the gold size pad, cellulose nitrate rete and absorbent paper layer, cover successively sample pad on the described base plate, the gold size pad, cellulose nitrate rete and absorbent paper layer, it is characterized in that, 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 threshold of described test paper is that saliva is rich in halfcystine secretory protein 1 concentration 10-40ng/ml.
- 2. test paper according to claim 1 is characterized in that, described gold size pad is comprised of glass fibre and the golden labeling antibody that is fixed thereon, and described sample pad is the water absorptivity glass layer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101909089A CN103308692A (en) | 2013-05-22 | 2013-05-22 | CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis |
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|---|---|---|---|
| CN2013101909089A CN103308692A (en) | 2013-05-22 | 2013-05-22 | CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267857A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing tongue cancer |
| CN103293319A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Gold-labeled test paper for rapidly diagnosing carcinoma jaw |
| CN103308694A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Gold-labeled kit for salivary gland carcinoma diagnosis |
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| US20030027178A1 (en) * | 2001-03-16 | 2003-02-06 | George Vasmatzis | Methods and kits for determining a cancer diagnosis and prognosis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267857A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing tongue cancer |
| CN103293319A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Gold-labeled test paper for rapidly diagnosing carcinoma jaw |
| CN103308694A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Gold-labeled kit for salivary gland carcinoma diagnosis |
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Application publication date: 20130918 |