CN103941016B - The use in conjunction of CST1 and carcinomebryonic antigen - Google Patents

Abstract

Do you the invention discloses CST1 (Cystatin? SN) with the use in conjunction of carcinomebryonic antigen (CEA), Cystatin is specially? SN and CEA detects the application in cancer of the stomach or gastro-intestinal stromal tumor markers in preparation; The invention also discloses the trapping agent of cancer of the stomach or gastro-intestinal stromal tumor markers and the kit containing this trapping agent, obtained kit has that specificity is good, sensitivity advantages of higher, can be used in the curative effect evaluation in the early diagnosis of cancer of the stomach or GISTs, therapeutic process and the transfer and relapse monitoring after treatment, its diagnostic result is early than clinical symptoms.

Description

The use in conjunction of CST1 and carcinomebryonic antigen

Technical field

The invention belongs to medical science, relate to the use in conjunction of CST1 and carcinomebryonic antigen.

Background technology

Cancer of the stomach is one of malignant tumour of serious threat human health, and mortality ratio occupies the second of each nauseating tumour.Cancer of the stomach 934,000 example is newly sent out in the current whole world every year, wherein has nearly 400,000 in China, and ill and mortality ratio all exceedes the twice of world average level.Along with the application of gastroscope and modern imaging technology, the diagnosis of early carcinoma of stomach brings up to about 10, early treatment cure rate reaches 95 percent, but is that middle and advanced stage is just sought medical advice more than ninety percent in current Chinese inpatient, and five year survival rate is less than 1/5th.The general curative effect of raising cancer of the stomach depends on that early discovery, early diagnosis, early treatment and the recurrence after treating detect to a great extent, and therefore the tumor markers of searching high sensitivity and high specific is of great importance to the early diagnosis of cancer of the stomach and recurrence monitoring.

GISTs (GIST) incidence of disease is (1-2)/100,000 according to the literature, accounts for the 0.1%-0.3% of gastroenteric tumor.Be 4000-6000 example in the annual new cases of the U.S., annual morbidity is about (11-14.5)/1,000,000.Onset peak is crowd between 50-70 year, and less than 40 years old rare, and teenager is rare.GIST major part betides stomach (50 ~ 70%) and small intestine (20 ~ 30%), and Colon and rectum accounts for 10 ~ 20%, and esophagus accounts for 0 ~ 6%, rare behind mesenterium, nethike embrane and abdominal cavity.Patient GIST 20-30% is pernicious, and about have 11 ~ 47% existing transfers when first time is medical, transfer is main in liver and abdominal cavity.The expression of c-kit gene function gain mutation and c-kit protein product CD117 is there is in gastrointestinal stromal tumors.Research finds that GIST occurs mainly to cause tyrosine kinase continuous activation to make mutant cell proliferation out of control relevant with C-KIT gene mutation.80-88%GIST comes from C-KIT gene function gain mutation, i.e. CD117.According to Chinese diagnosis of gastro-intestinal stromal tumors treatment National Consensus in 2008, pathological diagnosis flow process should do the inspection of histogenic immunity group to doubtful GIST, the CD117 positive then can be made a definite diagnosis, feminine gender does C-KIT abrupt climatic change again, the positive is GIST, and feminine gender carries out PDGFR genetic test again, and the positive can be made a definite diagnosis, feminine gender may be wild type GIST or other tumour, if desmin test positive may be then liomyoma; S-100 is that the positive then considers neurinoma, and should do DOG1 SABC further as the two feminine gender and detect, the positive then can make a definite diagnosis GIST.Therefore, GIST definitive pathological diagnosis program is complicated, and GIST transfer and relapse rate is high, and particularly along biopsy path, or during operation, knurl body ulceration etc. very easily brings Implantation matastasis.Therefore, clinically GIST diagnosis and treatment bottleneck is broken through in the urgent need to highly sensitive Virus monitory mark.

In recent years, along with deepening continuously of studying tumor pathogenesis, find the endogenous inhibitor of Cystatin family as cathepsin mainly cysteine proteinase, in the generation of tumour, development, invasion and m etastasis process, play very important effect.Result of study shows, the expression of several members in different tumour of Cystatin family rises to some extent, the expression of such as CystatinC in oophoroma and incidence cancer has the rising of varying level, the expression of stefinA in non-small cell lung cancer increases to some extent, the expression of CystatinF in kinds of tumors has remarkable increase, be likely owing to occurring in tumour, the participation of cathepsin is needed in evolution, its expression is first induced to increase, reactivation body itself stress mechanism, the expression of Cystatin is caused to rise to suppress the cathepsin active contained.But the expression of Cystatin not with tumour develop into positive correlation, such as in glioma, the low expression of CystatinC means the late period of disease, the life cycle of patient is shorter, and be easy to recurrence, this may be the later stage to tumour, and some other the level of mechanism to Cystatin that have again regulates and controls, to promote the further deterioration of tumour.CST1 (CystatinSN) is mankind Cystatin family member, by CST1 gene code, containing 141 amino acid, two disulfide bond are had in molecule, molecular weight is 16.4Kda, for typical secretory protein, be distributed in multiple Fluids and secretions, as tears, saliva, serum, blood plasma etc.Bibliographical information, the expression of CST1 in stomach organization is higher than normal gastric mucosa, and the expressive site in gastric carcinoma cell lines is more consistent with stomach organization, and expression rate reduces with the differentiation degree reduction of clone; Analysis of clinical shows, the expression of CSTl is by stages relevant to invasive depth, DISTANT METASTASES IN and TNM; Survival analysis shows, 5 years survival rates that CST1 expresses positive group are significantly higher than without expression group; Cox regretional analysis shows, CSTl is an independent prognostic factor; Thus prompting CST1 may play the effect of similar TIF in the generation of cancer of the stomach, evolution.

CEA is found in colon cancer and fetal gut tissue at first, therefore named carcinomebryonic antigen.CEA raises and is common in colorectal cancer, cancer of pancreas, cancer of the stomach, small-cell carcinoma of the lung, breast cancer, medullary carcinoma of thyroid gland etc.But the diseases such as smoking, the gestational period and angiocardiopathy, diabetes, nonspecific colonitis, the patients serum CEA of 15% ~ 53% also can raise, so CEA is not the specificity marker of malignant tumour, diagnosis only has auxiliary value.The clinical meaning that CEA detects in Serum Obtained From Advance Gastric Cancer is: (1) relevant to progressive stage poorly differentiated adenocarcinoma-and also infiltrate with tumor size, serosal surface and be correlated with Lymph Node Metastasis; (2) can contact with liver parenchyma and point out hepatic metastases by mediate tumor cell; (3) can with other index use in conjunction, point out as combined with cytolgical examination in peritoneal lavage fluid that the peritonaeum of cancer of the stomach recurs, abdominal cavity is planted, but accuracy comparatively CA125 is poor; (4) chemotherapeutic efficacy of cancer of the stomach can be evaluated with other index use in conjunction, if CEA level decline scope is more than 50% or be down to normal range and continue to can be used as treatment efficiency index in more than 4 weeks; (5) relation of itself and prognosis remains and is disputing on, but most result is thought, continues to increase prompting patients with gastric cancer prognosis mala after treating.But, have no the report of associating CystatinSN and CEA marker detection cancer of the stomach or GISTs at present.

Summary of the invention

In view of this, a kind of CST1 (CystatinSN) and carcinomebryonic antigen (CEA) is the object of the present invention is to provide to diagnose in preparation and indicate the application in the mark of cancer of the stomach or GISTs; Two of object of the present invention is the trapping agent providing cancer of the stomach or gastro-intestinal stromal tumor markers; Three of object of the present invention is to provide the detection kit containing above-mentioned trapping agent; Four of object of the present invention is to provide kit to set up the method calculating diagnosis and indication cancer of the stomach or GISTs threshold value.

For achieving the above object, the invention provides following technical scheme:

1. CST1 and carcinomebryonic antigen are diagnosed in preparation and are indicated the application in the mark of cancer of the stomach or GISTs, the amino acid sequence of described CST1 is as shown in SEQIDNO.1, and the amino acid sequence of described carcinomebryonic antigen is as shown in SEQIDNO.2.

Preferably, described diagnosis and indication are diagnosis, curative effect evaluation or transfer and relapse monitoring.

2. the trapping agent of cancer of the stomach or gastro-intestinal stromal tumor markers, described mark is CST1 and carcinomebryonic antigen, the amino acid sequence of described CST1 is as shown in SEQIDNO.1, and the amino acid sequence of described carcinomebryonic antigen is as shown in SEQIDNO.2.

Preferably, described trapping agent is the specific antibody identifying CST1 and carcinomebryonic antigen.

3. the kit containing described trapping agent.

Preferably, described kit is the kit detecting CST1 and carcinomebryonic antigen concentration in serum.

Preferred, described kit is enzyme-linked immunologic detecting kit.

Preferred, described kit contains the solid phase carrier being coated with monoclonal antibody, biotin labeled polyclonal antibody and chromogenic substrate.

Most preferred, described monoclonal antibody is mouse-anti people CystatinSN monoclonal antibody, and described polyclonal antibody is the anti-human CystatinSN polyclonal antibody of rabbit, and described chromogenic substrate is tetramethyl benzidine.

4. utilize described kit to calculate the method for diagnosis and indication cancer of the stomach or GISTs threshold value, CST1 and carcinomebryonic antigen concentration is detected with described kit, utilize P=exp (-6.119-0.053*a+0.509*b)/[1+exp (-6.119-0.053*a+0.509*b)] to calculate cancer of the stomach threshold value P or utilize P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)] to calculate GISTs threshold value P, when P is more than or equal to 0.75, it is the positive; When P is less than 0.75, it is feminine gender;

Wherein a represents CST1 concentration, and unit is that pg/mL, b represent carcinomebryonic antigen concentration, and unit is ng/mL.

Beneficial effect of the present invention: the invention discloses the new mark detecting cancer of the stomach or GISTs, combine for diagnosis by CystatinSN and CEA and indicate cancer of the stomach or GISTs, above-mentioned two marks are utilized jointly to detect the highly sensitive and specificity of cancer of the stomach or GISTs all higher than detection unique identification thing, the sensitivity detecting cancer of the stomach is 82.4%, and the sensitivity detecting GISTs is 78.3%; The specificity detecting cancer of the stomach is 93.9%, and the sensitivity detecting GISTs is 84.6%; The invention also discloses the trapping agent of cancer of the stomach or gastro-intestinal stromal tumor markers and the kit containing this trapping agent, generate a reagent box has easy to use, reproducible, the advantage such as be easy to carry, can be used in the early diagnosis of cancer of the stomach or GISTs, curative effect evaluation or transfer recurrence monitoring etc., and testing result is for judging that cancer of the stomach or GISTs are early than clinical symptoms, to treat in advance for doctor and intervention provides guidance.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 is the typical curve that CystatinSN enzyme-linked immunologic detecting kit detects CystatinSN albumen.

Fig. 2 is the typical curve that CEA enzyme-linked immunologic detecting kit detects CEA albumen.

Fig. 3 is that CystatinSN-CEA combined detection kit detects cancer of the stomach ROC curve.

Fig. 4 is that CystatinSN-CEA combined detection kit detects GISTs ROC curve.

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.

The reagent that the present invention uses is as follows: mouse-anti people CystatinSN monoclonal antibody purchased from American R & D company (article No. is: MAB1296); The anti-human CystatinSN polyclonal antibody of rabbit, CystatinSN protein standard substance are purchased from Sino Biological Inc..CEA monoclonal antibody, CEA polyclonal antibody, CEA protein standard substance is purchased from Sino Biological Inc. (article No. of CEA monoclonal antibody and CEA Anti-TNF-α is respectively: 11077-MM08 and 11077-MM07).

Embodiment 1 sets up CystatinSN Virus monitory reaction system and optimization thereof

With concentration be 5 μ g/mL mouse-anti people CystatinSN monoclonal antibody bags by elisa plate, wrap under 4 DEG C of conditions and spent the night, wash plate; Then be that in the BSA of 2%, room temperature closes 2 hours at massfraction, wash plate; Concentration is respectively 0pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, the CystatinSN protein standard substance (amino acid sequence of coding CystatinSN albumen is as shown in SEQIDNO.1) of 400pg/mL, 800pg/mL, 1600pg/mL and blood serum sample add in shut, in 37 DEG C of reactions 1 hour, wash plate; Then be the anti-human CystatinSN polyclonal antibody of rabbit of 0.5 μ g/mLHRP mark by concentration, react 1 hour under 37 DEG C of conditions, wash plate; React 2-3 minute with tetramethyl benzidine (TMB) again, be finally the sulfuric acid cessation reaction of 2M by concentration, and under 450nm condition, detect OD value (Fig. 1).Result shows, and the CystatinSN range of linearity is 50pg/mL-1600pg/mL, and in linear wide standards product linearly dependent coefficient r >=0.990, the recovery is in 90% ~ 110% scope.

Detection system is optimized, comparison is respectively by U.S. R & D, the CystatinSN polyclonal antibody that the CystatinSN protein standard substance that the CystatinSN monoclonal antibody that Britain Abcam and U.S. NOVUSBIOLOGICALS3 different company produces, R & D company of the U.S. and Sino Biological Inc. produce and U.S. R & D, Britain Abcam and Sino Biological Inc. produce.Result shows, the product that CystatinSN monoclonal antibody preferred R & D company provides, and best effort concentration is 5 μ g/mL; CystatinSN protein standard substance and CystatinSN polyclonal antibody preferred Sino Biological Inc. product, best effort concentration is 0.5 μ g/mL, at optimum conditions, background OD value < 0.1 can be realized, effectively can distinguish negative group and positive group, and there is statistical significance.

The determination of solid phase carrier: the ELISA Plate that U.S. Corning, German Greiner, U.S. Thermo and Denmark Nunc4 different manufacturers is produced is compared.Result show, corning company of the U.S. (article No. is: 9018) and thermo(article No. be: 468667) ELISA Plate of company meets background OD value < 0.1, and signal to noise ratio (S/N ratio) is higher.

The selection of coating buffer: be coated in the buffer system required for solid phase carrier according to antibody, it is buffer salt solution that ELISA commonly uses coating buffer, phosphate buffer (pH7.5) and carbonate buffer solution (pH9.6) is used to detect bag by the impact of environment on reaction system respectively, result display carbonate buffer solution (pH9.6) can meet blank group OD value < 0.1, blank group of effective differentiation, negative group and positive group, signal to noise ratio (S/N ratio) is higher.

The selection of thinning agent: the dilution effect that compared for 2 kinds of commercialization thinning agents (respectively purchased from Tianjin Bo Meike Bioisystech Co., Ltd (article No. BMKF017-1) and Xi Tang bio tech ltd, Shanghai (article No. C0901)) and self-control thinning agent by experiment; main from protected protein ability, the dilution effect of thinning agent is evaluated in self stability two aspect.The best results of result display self-control thinning agent, the concentration of self-control thinning agent each component is as follows: 3mMEDTA, massfraction be 0.5% BSA, 1 × PBS, massfraction be 0.05% Tween-20 and massfraction be 0.02% thimerosal (pH6.0).

The selection of stabilizing agent: use 3 kinds of stabilizing agents (3 kinds of stabilizing agents are specific as follows: stabilizing agent I: massfraction is the sucrose of 3%, volume fraction be 8% glycerine and massfraction be the NaCl of 1.3%; Stabilizing agent II: massfraction is the sucrose of 3%, volume fraction is the glycerine of 8%, massfraction be 0.1% EDTA and massfraction be the NaCl of 1.3%, stabilizing agent III: volume fraction is the PBS of 68.8%, volume fraction be 30% hyclone and massfraction be the thimerosal of 0.2%) to dilute monoclonal antibody, protein standard substance and polyclonal antibody to concentration be respectively 0.5mg/mL, 0.16ng/mL and 50 μ g/mL, is 1:100 dilution during use by volume.And detect in the 0th day, the 7th day and the 14th day, result display stabilizing agent III best results, its component is: the hyclone that the PBS that volume fraction is 68.8%, volume fraction are 30%, massfraction are the thimerosal of 0.2%.

By the key component determining detection system above, and set up CystatinSN-CEA Virus monitory kit.

Embodiment 2 sets up CEA Virus monitory system and optimization thereof

With concentration be 3 μ g/mL mouse-anti people CEA monoclonal antibody bags by elisa plate, wrapping by condition is wrap to be spent the night under 4 DEG C of conditions, washes plate; Then be that in the BSA of 2%, room temperature closes 2 hours at massfraction, wash plate; Concentration is respectively 0pg/mL, 39.0625pg/mL, 78.125pg/mL, 156.25pg/mL, 312.5pg/mL, 625pg/mL, 1250pg/mL) CEA protein standard substance (amino acid sequence of coding CEA albumen is as shown in SEQIDNO.2) and blood serum sample add in shut, in 37 DEG C of reactions 1 hour, wash plate; Then adding concentration is the anti-human CEA polyclonal antibody of rabbit that 0.5 μ g/mLHRP marks, and reacts 1 hour, wash plate under 37 DEG C of conditions; React 2-3 minute with tetramethyl benzidine (TMB) again, be finally the sulfuric acid cessation reaction of 2M by concentration, and under 450nm condition, detect OD value (Fig. 2).Result shows, and the CEA range of linearity is 39.0625pg/mL-1250pg/mL, and in range of linearity internal linear correlation coefficient r >=0.990, the recovery is in 90% ~ 110% scope.

Detection system is optimized: comparison U.S. R & D, the CEA polyclonal antibody that the CEA protein standard substance that the CEA monoclonal antibody that Britain Abcam and U.S. NOVUSBIOLOGICALS3 different company produces, U.S. R & D and Sino Biological Inc. produce and U.S. R & D, Britain Abcam and Sino Biological Inc. produce.Result shows, the product (article No. is: 11077-MM08) that the preferred Sino Biological Inc. of CEA monoclonal antibody provides, and best effort concentration is 3 μ g/mL; CEA protein standard substance and the preferred Sino Biological Inc. of CEA polyclonal antibody provide product, and the best effort concentration of CEA protein standard substance is the best effort concentration of 1.6ng/mL, CEA polyclonal antibody is 0.5 μ g/mL.At optimum conditions, background OD value < 0.1 can be realized, effectively can distinguish negative group and positive group, and there is statistical significance.

The determination of solid phase carrier: the ELISA Plate of U.S. Corning, German Greiner, U.S. Thermo and a Denmark Nunc4 different manufacturers is compared, result shows, corning company of the U.S. (article No. is: 9018) and U.S. Thermo(article No. be: 468667) ELISA Plate of company meets background OD value < 0.1, and signal to noise ratio (S/N ratio) is higher.

The selection of coating buffer: be coated in the buffer system required for solid phase carrier according to antibody, it is buffer salt solution that ELISA commonly uses coating buffer, phosphate buffer (pH7.5) and carbonate buffer solution (pH9.6) is used to detect bag by the impact of environment on reaction system respectively, result display carbonate buffer solution (pH9.6) can meet blank group OD value < 0.1, blank group of effective differentiation, negative group and positive group, signal to noise ratio (S/N ratio) is higher.

The selection of thinning agent: the dilution effect that compared for 2 kinds of commercialization thinning agents (respectively purchased from Tianjin Bo Meike Bioisystech Co., Ltd (article No. BMKF017-1) and Xi Tang bio tech ltd, Shanghai (article No. C0901)) and self-control thinning agent by experiment; main from protected protein ability, the dilution effect of thinning agent is evaluated in self stability two aspect.Result display self-control thinning agent best results, the final concentration of each component of self-control thinning agent is as follows: 3mMEDTA, massfraction be 0.5% BSA, 1 × PBS, massfraction be 0.05% Tween-20 and massfraction be 0.02% thimerosal (pH6.0).

The selection of stabilizing agent: use 3 kinds of stabilizing agents (stabilizing agent I: massfraction is the sucrose of 3%, volume fraction be 8% glycerine and massfraction be the NaCl of 1.3%; Stabilizing agent II: massfraction is the sucrose of 3%, volume fraction is the glycerine of 8%, massfraction be 0.1% EDTA and massfraction be the NaCl of 1.3%, stabilizing agent III: volume fraction is the PBS of 68.8%, volume fraction be 30% hyclone and massfraction be the thimerosal of 0.2%) to dilute monoclonal antibody, protein standard substance and polyclonal antibody to concentration be respectively 0.5mg/mL, 0.16ng/mL and 50 μ g/mL, is 1:100 dilution during use by volume.And detect in the 0th day, the 7th day and the 14th day, result display stabilizing agent III best results.

By the key component determining detection system above, and set up CEA Virus monitory kit.

Embodiment 3CystatinSN-CEA enzyme-linked immunologic detecting kit

According to setting up CystatinSN and CEA Virus monitory system construction CystatinSN-CEA enzyme-linked immunologic detecting kit in embodiment 1 and embodiment 2, concrete component is as shown in table 1:

Table 1, CystatinSN-CEA enzyme-linked immunologic detecting kit component

Evaluate CystatinSN-CEA enzyme-linked immunologic detecting kit: use CystatinSN-CEA enzyme-linked immunologic detecting kit to CystatinSN quality-control product and CEA quality-control product, respectively 160pg/mL and 80pg/mL concentration level duplicate detection 10 times, result display coefficient of variation CV≤10%; Detect same sample with 3 lot number kits, result shows interassay coefficient of variation CV≤15% of 3 lot number kits.And stabilization of kit is studied, energy stable preservation 8 months under result display sealing, 4 DEG C of conditions, stabilizability preservation 2 months under Kaifeng, 4 DEG C of conditions, transports 7 days stability under 0-4 DEG C of condition.Therefore, stabilization of kit is good, meets product Expected Results.

Embodiment 4CystatinSN-CEA combined detection kit is used for diagnosis and indication cancer of the stomach

(1) CystatinSN-CEA combined detection kit diagnosis of gastric cancer

Serum before collecting 200 routine patients with gastric cancer treatments from Shanghai tumour hospital, collects 245 routine healthy blood donation personnel serum in blood station, every routine serum 1mL simultaneously.Use CystatinSN-CEA combined detection kit to detect CystatinSN and CEA concentration in patients with gastric cancer and healthy blood donation personnel serum respectively, and draw Receiver operating curve (ROC curve) (Fig. 3) according to testing result.The area under curve of CystatinSN and CEA joint-detection, sensitivity and specificity is obtained according to ROC curve, as shown in table 2.

Table 2, CystatinSN and CEA joint-detection cancer of the stomach result

Mark	Area under curve	Sensitivity	Specificity
				Cystatin SN	0.839	60.6%	85.4%
CEA	0.756	49.8%	73.7%
				Cystatin SN+CEA	0.902	82.4%	93.9%

As shown in Table 2, the area under curve that CystatinSN and CEA detects separately is respectively 0.839 and 0.756, and sensitivity is respectively 60.6% and 49.8%, and specificity is respectively 85.4% and 73.7%; The area under curve of CystatinSN and CEA joint-detection is 0.902, and sensitivity is 82.4%, and specificity is 93.9%.Therefore, use CystatinSN-CEA combined detection kit specificity and sensitivity all higher, its testing result is more reliable.

Then according to detection gained CystatinSN and CEA marker concentration, when specificity reaches 75%, application Logistic regression and statistical method draws the judgment formula of CystatinSN and CEA joint-detection, be specially: P=exp (-6.119-0.053*a+0.509*b)/[1+exp (-6.119-0.053*a+0.509*b)] (a=CystatinSN concentration, b=CEA concentration), when P is more than or equal to 0.75, it is the positive; When P is less than 0.75, it is feminine gender.

Application: utilize CystatinSN-CEA combined detection kit to detect 100 routine doubtful patients with gastric cancer, then according to P=exp (-6.119-0.053*a+0.509*b)/[1+exp (-6.119-0.053*a+0.509*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate P value.Result shows, and in 100 routine tested samples, 53 routine P values are greater than 0.75, are patients with gastric cancer; 47 routine P values are less than 0.75, are non-patients with gastric cancer, consistent with clinical diagnoses.

(2) CystatinSN-CEA combined detection kit is used for cancer of the stomach curative effect evaluation

Serum before getting 10 routine patients with gastric cancer treatments from Shanghai tumour hospital, detect CystatinSN and CEA concentration in serum, after treatment terminates, detection is got patients serum and is detected CystatinSN and CEA concentration again.Utilize CystatinSN and CEA joint-detection judgment formula P=exp (-6.119-0.053*a+0.509*b)/[1+exp (-6.119-0.053*a+0.509*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculating P value, then and treatment rear P value assessment curative effect front according to treatment, criterion is: the decline compared with before treating of P value is less than 30%, is judged as failing to respond to any medical treatment; The decline compared with before treatment of P value is greater than 30%, is judged as that the state of an illness is improved; The decline compared with before treatment of P value is greater than 70%, and be judged as that result for the treatment of is remarkable, its assessment result is as shown in table 3.Meanwhile, doctor is according to clinical symptoms assessment cancer of the stomach curative effect, and result is as shown in table 3.

Table 3, CystatinSN-CEA combined detection kit assessment cancer of the stomach efficacy result

Patient code	Concentration change number percent before and after treatment	Clinical judgment
			1	Decline 36%	Improve
2	Raise 13%	Invalid
			3	Reduce by 67%	Improve
4	Reduce by 45%	Improve
			5	Raise 17%	Invalid
6	Reduce by 20%	Invalid
			7	Reduce by 43%	Improve
8	Reduce by 94%	Effectively
			9	Reduce by 34%	Invalid
10	Reduce by 39%	Improve

As shown in Table 3, use CystatinSN-CEA combined detection kit to be in 10 routine patients with gastric cancer to cancer of the stomach curative effect evaluation result, have 1 example treatment effectively, wherein after 5 example treatments, the state of an illness improves, and all the other 4 routine inefficacies, coincidence rate reaches 90% compared with clinical judgment.

(3) CystatinSN-CEA combined detection kit is for monitoring Metastasis of Gastric Cancer recurrence

Early gastric caacer patient after 6 examples terminate the courses for the treatment of is carried out to tracking and follows up a case by regular visits to, and within 6 weeks, gets serum first after treatment, and detect CystatinSN and CEA concentration in serum, later every three months detects once, follows the tracks of nine months, altogether detection four times.According to testing result, utilize formula P=exp (-6.119-0.053*a+0.509*b)/[1+exp (-6.119-0.053*a+0.509*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate P value, when P is more than or equal to 0.75, be transfer and relapse; When P is less than 0.75, be indicated as generation transfer and relapse, be Progression free survival, its result is as shown in table 4.9 months time, doctor evaluates Metastasis of Gastric Cancer recurrence according to clinical symptoms, and result is as shown in table 4.

Table 4, CystatinSN-CEA combined detection kit monitoring cancer of the stomach transfer recurrence result

Patient code	6 weeks	3 months	6 months	9 months	Clinical evaluation
						1	0.34	0.42	0.78	0.82	Transfer and relapse
2	0.45	0.52	0.58	0.49	Progression free survival
						3	0.65	0.61	0.63	0.69	Progression free survival
4	0.72	0.68	0.57	0.52	Progression free survival
						5	0.62	0.64	0.53	0.59	Progression free survival
6	0.47	0.62	0.79	0.81	Transfer and relapse

As shown in Table 4, kit testing result has 2 examples to occur transfer and relapse in 6 routine patients, and transfer and relapse does not occur all the other 4 examples, is Progression free survival, consistent with clinical evaluation result.And use CystatinSN-CEA combined detection kit to monitor cancer of the stomach transfer recurrence result to find early than clinical symptoms and sign, provide guidance for doctor carries out intervention in advance.

In sum, CystatinSN and CEA can as the mark of diagnosis and indication cancer of the stomach, detects the sensitivity of 2 marks and specificity higher than detection unique identification thing simultaneously, can improve the accuracy of diagnosis.

Embodiment 5CystatinSN-CEA combined detection kit is for detecting GISTs

(1) CystatinSN-CEA combined detection kit diagnosis GISTs

Serum before collecting 200 routine GISTs patient treatments from Shanghai tumour hospital, collects 245 routine blood donation personnel serum from blood station, every routine blood 1mL simultaneously.Use CystatinSN-CEA combined detection kit to detect CystatinSN and CEA concentration in GISTs and normal human serum respectively, and draw ROC curve (Fig. 4) according to testing result.Then the area under curve of CystatinSN and CEA joint-detection, sensitivity and specificity is obtained according to ROC curve, as shown in table 5.

Table 5, CystatinSN and CEA joint-detection GISTs sample results

Mark	Area under curve	Sensitivity	Specificity
				Cystatin SN	0.757	56.6%	84.6%
CEA	0.629	44.7%	63.9%
				Cystatin SN+CEA	0.827	78.3%	84.6%

As shown in Table 5, the area under curve that CystatinSN detects separately is 0.757, and sensitivity is 56.6%, and specificity is 84.6%; The area under curve of independent detection CEA is 0.629, and sensitivity is 44.7%, and specificity is 63.9%; And the area under curve of CystatinSN and CEA joint-detection is 0.827, sensitivity is 78.3%, and specificity is 84.6%.Therefore, use the sensitivity of CystatinSN and CEA combined detection kit diagnosis GISTs higher, specificity is with independent to detect CystatinSN mark consistent.

Gained CystatinSN and CEA concentration will be detected, when specificity reaches 75%, application Logistic regression and statistical method has drawn the judgment formula of CystatinSN and CEA joint-detection, be specially: P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate P value, when P is more than or equal to 0.75, be the positive; When P is less than 0.75, it is feminine gender.

Application: utilize CystatinSN-CEA combined detection kit to detect 100 routine doubtful GISTs patients, then according to P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate P value.Result shows, and in 100 routine tested samples, 17 routine P values are greater than 0.75, are GISTs patient; 83 routine P values are less than 0.75, are parenteral mesenchymoma patient, consistent with clinical diagnoses.

(2) CystatinSN-CEA combined detection kit assessment Treatment of Gastrointestinal Stromal Tumors effect

Serum before getting 10 routine GISTs patient treatments from Shanghai tumour hospital, detects CystatinSN and CEA concentration in serum, detects CystatinSN and CEA concentration again after terminating the course for the treatment of.According to CystatinSN and CEA concentration, utilize formula P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate P value, according to P value change assessment result for the treatment of, criterion is: the decline compared with before treatment of P value is less than 30%, is judged as failing to respond to any medical treatment; The decline compared with before treatment of P value is more than or equal to 30%, is judged as that the state of an illness is improved; The decline compared with before treatment of P value is more than or equal to 70%, and be judged as that result for the treatment of is remarkable, result is as shown in table 6.Meanwhile, doctor is according to the curative effect of clinical symptoms assessment GISTs, and result is as shown in table 6.

Table 6, CystatinSN-CEA combined detection kit assessment GISTs efficacy result

Patient code	Concentration change number percent before and after treatment	Clinical assessment
			1	Raise 13%	Invalid
2	Reduce by 35%	Invalid
			3	Reduce by 43%	Improve
4	Reduce by 87%	Effectively
			5	Raise 35%	Invalid
6	Reduce by 47%	Improve
			7	Raise 72%	Invalid
8	Reduce by 81%	Effectively
			9	Reduce by 24%	Invalid
10	Raise 23%	Invalid

As shown in Table 6, kit testing result is in 10 routine patients, and wherein 2 examples are effective in cure, and wherein after 2 example treatments, the state of an illness improves, and all the other 6 examples are failed to respond to any medical treatment really, and coincidence rate reaches 90% compared with clinical evaluation result.

(3) CystatinSN-CEA combined detection kit monitoring GISTs transfer recurrence

Carry out tracking to the upper gastrointestinal mesenchymoma trouble of terminating the 6 routine courses for the treatment of of Shanghai tumour hospital to follow up a case by regular visits to, within six weeks, gathers patients serum first, detect CystatinSN and CEA concentration in serum after treatment, later every three months detection once, is followed the tracks of nine months, altogether detection four times.Detection gained CystatinSN and CEA concentration are utilized formula for P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)] (a=CystatinSN concentration, unit is pg/mL; B=CEA concentration, unit is ng/mL) calculate its P value, when P is more than or equal to 0.75, be transfer and relapse; When P is less than 0.75, show transfer and relapse does not occur, be Progression free survival, result is as shown in table 7.9 months time, doctor evaluates GISTs transfer and relapse situation according to clinical symptoms, and result is as shown in table 7.

Table 7, CystatinSN-CEA combined detection kit monitoring GISTs transfer recurrence result

Patient code	6 weeks	3 months	6 months	9 months	Clinical evaluation
						1	0.56	0.49	0.52	0.58	Progression free survival
2	0.67	0.55	0.46	0.41	Progression free survival
						3	0.65	0.73	0.78	0.81	Transfer and relapse
4	0.42	0.54	0.58	0.51	Progression free survival
						5	0.31	0.43	0.71	0.82	Transfer and relapse 10-->
6	0.48	0.52	0.54	0.57	Progression free survival

As shown in Table 7, kit testing result occurs transfer recurrence, all the other 4 routine Progression free survivals after having 2 example treatments in 6 routine patients, consistent with clinical evaluation result.And use CystatinSN-CEA combined detection kit to monitor GISTs to find early than clinical symptoms and sign, provide guidance for doctor carries out intervention in advance.

In sum, CystatinSN and CEA can as the mark of diagnosis and indication GISTs, detects the sensitivity of 2 marks and specificity higher than detection unique identification thing simultaneously, can improve the accuracy of diagnosis.

What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims (10)

1. the application of reagent in preparation diagnosis and indication GISTs reagent of joint-detection CST1 and carcinomebryonic antigen, the amino acid sequence of described CST1 is as shown in SEQIDNO.1, and the amino acid sequence of described carcinomebryonic antigen is as shown in SEQIDNO.2.

2. application according to claim 1, is characterized in that: described diagnosis and indication are diagnosis, curative effect evaluation or transfer and relapse monitoring.

3. application according to claim 1, it is characterized in that: described reagent is the trapping agent of CST1 and carcinomebryonic antigen, the amino acid sequence of described CST1 is as shown in SEQIDNO.1, and the amino acid sequence of described carcinomebryonic antigen is as shown in SEQIDNO.2.

4. application according to claim 3, is characterized in that: described trapping agent is the specific antibody identifying CST1 and carcinomebryonic antigen.

5. application according to claim 3, is characterized in that: described reagent is the kit containing trapping agent described in claim 3.

6. application according to claim 5, is characterized in that: described kit is the kit detecting CST1 and carcinomebryonic antigen concentration in serum.

7. application according to claim 6, is characterized in that: described kit is enzyme-linked immunologic detecting kit.

8. application according to claim 7, is characterized in that: described kit contains the solid phase carrier being coated with monoclonal antibody, biotin labeled polyclonal antibody and chromogenic substrate.

9. application according to claim 8, it is characterized in that: described monoclonal antibody is mouse-anti human cystatin E SN monoclonal antibody, described polyclonal antibody is the anti-human CST1 polyclonal antibody of rabbit, and described chromogenic substrate is tetramethyl benzidine.

10. the application according to any one of claim 5 ~ 9, it is characterized in that: CST1 and carcinomebryonic antigen are diagnosed and indication GISTs judgment formula is, P=exp (-8.016-0.027*a+0.613*b)/[1+exp (-8.016-0.027*a+0.613*b)];

Wherein a represents CST1 concentration, and unit is that pg/mL, b represent carcinomebryonic antigen concentration, and unit is ng/mL.