CN102175850A - ELISA (enzyme linked immunosorbent assay) kit for detecting endothelial cell specific molecule-1 (ESM-1) of tumour marker - Google Patents

ELISA (enzyme linked immunosorbent assay) kit for detecting endothelial cell specific molecule-1 (ESM-1) of tumour marker Download PDF

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CN102175850A
CN102175850A CN2010106167166A CN201010616716A CN102175850A CN 102175850 A CN102175850 A CN 102175850A CN 2010106167166 A CN2010106167166 A CN 2010106167166A CN 201010616716 A CN201010616716 A CN 201010616716A CN 102175850 A CN102175850 A CN 102175850A
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antibody
esm
cancer
kit
stomach
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季加孚
朱昱冰
刘妮
张连海
王晓红
邢晓芳
杜红
胡颖
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BEIJING TUMOUR HOSPITAL
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Abstract

The invention provides an ELISA (enzyme linked immunosorbent assay) kit for detecting endothelial cell specific molecule-1 (ESM-1) of a tumour marker, wherein the kit comprises (1) a coated antibody, wherein the coated antibody is a mouse anti-human ESM-1 antibody; (2) a detection antibody, wherein the detection antibody is a biotinylated goat anti-human Endocan antibody; (3) an enzyme linked antibody, wherein the enzyme liked antibody is a rabbit anti-goat IgG (immunoglobulin G) marked by a horseradish peroxidase; (4) a human Endocan standard substance; and (5) a colour development substrate of the enzyme linked antibody. By the kit provided by the invention, the early diagnosis rate of gastric cancer can be improved under a relatively small wound; the biomarker is an independent prognostic factor and is relevant to vessel invasion, distant metastasis and general classification of gastric cancer, so that prognosis can be predicted, states of the illness can be monitored, and treatments can be guided.

Description

Detect the ELISA kit of tumor markers endothelial cell specific molecule-1
Technical field
The present invention relates to be used to detect the kit of tumor markers, particularly relate to the kit that detects endothelial cell specific molecule-1 (ESM-1).
Background technology
Cancer of the stomach is one of malignant tumour of modal threat human health, is that fatal rate occupies second tumour, and excision is the present unique means that may effect a radical cure, but Most patients has been late period when finding, treatment can't undergo surgery.Therefore, diagnosis promptly and accurately is a key of saving patient's life.
The existing cancer of the stomach detection method immunohistochemistry technique of cutting into slices in a organized way.But immunohistochemistry technology, step is various, and to experimenter and requirement for experiment condition height, and the dyeing of false positive and false negative is many.Therefore, the result of SABC stablizes (document 6,7,8,9,10 sees reference) inadequately.Worst is that the used sample of immunohistochemistry technique must undergo surgery and just can obtain, and has delayed the time of diagnosis and treatment, the situation in the time of can not accurately reflecting patient's first visit.
Therefore, need relevant molecular marker to be applied to the early diagnosis and the prognosis prediction of cancer of the stomach.The value performance of tumor markers is on its susceptibility and specificity.Desirable tumor markers should be the susceptibility height, high specificity, and diffusion of level and tumor tissues or lump size are proportionate in expression or the serum.
The cancer of the stomach blood serum tumor markers that uses at present has the clinical detection of CEA, CA19-9, CA72-4 (the electrochemiluminescence method detects) and CA242 (the ELISA method detects) clinically.
For tumor markers CEA,, cause false positive results to increase because the polyclonal antibody of CEA combines with irrelevant antigen in the sample easily.Therefore, CEA is not suitable as the diagnosis of gastric cancer specific parameters.Tumor markers CA19-9, CA72-4 and CA242 also exist same problem, and specificity is not high.Therefore, also there is not a kind of tumor markers special fully now to cancer of the stomach.Seek a kind of more suitable blood serum tumor markers, be applied to clinical detection, assist the cancer of the stomach early diagnosis, prediction patient prognosis situation, the development of monitoring conditions of patients, further seek the treatment novel targets, accomplish that individualized treatment (document 11,12,13, the 14 sees reference) aspect of cancer of the stomach is significant.
Summary of the invention
Through further investigation, the inventor finds the expression of endothelial cell specific molecule-1 (ESM-1, its encoding proteins is also referred to as endocan) between cancer of the stomach and the other morphology normal structure of cancer, and there were significant differences.
Based on above-mentioned discovery, an object of the present invention is to provide a kind of kit that detects tumor markers endothelial cell specific molecule-1, described kit comprises: (1) coated antibody, described coated antibody are mouse-anti people ESM-1 antibody; (2) detect antibody, described detection antibody is biotinylation goat-anti people Endocan antibody; (3) enzyme len antibody, the anti-sheep IgG of the rabbit that described enzyme len antibody is a horseradish peroxidase-labeled; (4) people Endocan standard items; (5) chromogenic substrate of the enzyme of described enzyme len antibody.
Kit of the present invention is used to detect the tumour of expressing ESM-1.The tumour of described expression ESM-1 is cancer of the stomach, kidney, breast cancer, glioma and non-small cell carcinoma, and described tumour is preferably cancer of the stomach.
Kit of the present invention is used for early diagnosis, monitoring therapeuticing effect or the prognosis evaluation of cancer of the stomach.
In kit of the present invention, the working concentration of described coated antibody is 4 μ g/mL to 1 μ g/mL, and the working concentration of described detection antibody is 10 μ g/mL to 0.1 μ g/mL, and the working concentration of described enzyme len antibody is 1 μ g/mL.Preferably, in the kit of the present invention, the working concentration of described coated antibody is 4 μ g/mL, and the working concentration of described detection antibody is 1 μ g/mL, and the working concentration of described enzyme len antibody is 1 μ g/mL.
The present invention also provides the application of endothelial cell specific molecule-1 (ESM-1) in the preparation of lesion detection kit, and described kit comprises: (1) coated antibody, described coated antibody are mouse-anti people ESM-1 antibody; (2) detect antibody, described detection antibody is biotinylation goat-anti people Endocan antibody; (3) enzyme len antibody, the anti-sheep IgG of the rabbit that described enzyme len antibody is a horseradish peroxidase-labeled; (4) people Endocan standard items; (5) chromogenic substrate of the enzyme of described enzyme len antibody.
In described application, the tumour that is detected is cancer of the stomach, kidney, breast cancer, glioma and non-small cell carcinoma.Described tumour is preferably cancer of the stomach.
In described application, the working concentration of described coated antibody is 4 μ g/mL to 1 μ g/mL, and the working concentration of described detection antibody is 10 μ g/mL to 0.1 μ g/mL, and the working concentration of described enzyme len antibody is 1 μ g/mL.Preferably, in the kit of the present invention, the working concentration of described coated antibody is 4 μ g/mL, and the working concentration of described detection antibody is 1 μ g/mL, and the working concentration of described enzyme len antibody is 1 μ g/mL.
Use kit of the present invention, can improve the early diagnostic rate of cancer of the stomach, and measurable prognosis result and result of treatment, for better, more specific treatment provides effective guidance.
Description of drawings
Typical curve, the ELISA testing result should illustrate with diagrammatic form with experimental result.
Fig. 1 has shown the differential gene dendrogram that high-throughout chip technology is analyzed.
Fig. 2 has shown the cancer of the stomach forecast model of 43 genes and the checking in second lot sample basis.
Fig. 3 has shown that ESM-1 is at the expression of mRNA level and protein level in each sample.
Fig. 4 has shown that ESM-1 expresses and single factor survival analysis (K-M curve) of patients with gastric cancer.
Fig. 5 has shown that ESM-1 is in patients with gastric cancer and normal population expression of serum difference.
Fig. 6 shown calculate the tumor patient of (a) and adjusted (b) and normal healthy controls according to formula the ESM-1 concentration ratio.
Embodiment
Endothelial cell specific molecule-1 (ESM-1), at first by Lassalle and work together and obtained from human endothelial cell cDNA library clone in 1996, be positioned human No. 5 chromosome 5q11.2, comprise three extrons and two intrones, its encoding proteins is also referred to as endocan (in some cases, also representing endothelial cell specific molecule-1 albumen with ESM-1 herein).Endocan is by vascular endothelial cell, and kidney distal tubule endothelial layer and bronchus and the secretion of lung submucosal gland body can be by TNF-α at endothelium and epithelial expression, IL-1 β, and LPS and VEGF raise, by IL-4 and IFN-γ downward modulation.The blood vessel endothelium of kidney, breast cancer, glioma and non-small cell carcinoma is crossed expression endocan, local expression level relevant with vascularization and tumor invasion (document 2,3,4,15-19 see reference).At present, except that the applicant, still there is not the report of ESM-1 expression study in cancer of the stomach.
The inventor is in earlier stage by high-throughout chip technology and interpretation of result, found the change situation of ESM-1 gene expression pattern between the other morphology normal structure of cancer of the stomach and cancer, ESM-1 is that the other normal structure differences of cancer of the stomach and cancer is expressed one of the most significant gene, and the ESM-1 nucleic acid level obviously raises in the cancer of the stomach.Subsequently, raise by ESM-1mRNA and protein expression in real-time RT-PCR and the protein imprinted further confirmation stomach organization.Further analyze the relation of ESM-1 expression and clinicopathologic features, discovery tumour epithelium is crossed expression ESM-1 and is invaded relevant with BorrmannIV type cancer of the stomach with DISTANT METASTASES IN, vascular; Single factor survival analysis shows that ESM-1 positive patient shorter survival is short; Multiplicity confirms that it is an independent prognostic factor that ESM-1 expresses.
It is that the infrastest result uses the means that stride forward to clinical practice that the serology of molecular marker detects, in view of above experimental result and ESM-1 are a kind of secretory proteins, the inventor further attempts making up the human serum detection kit---double antibodies sandwich ELISA kit.It is a kind of immunoenzyme technics that grows up after immunofluorescence and radioimmunoassay technique that enzyme connects immunosorbent mensuration (Enzyme-Linked ImmunosorbentAssay is called for short ELISA).ELISA is based on immunological response, the very high experimental technique of a kind of susceptibility that the specific reaction of antigen, antibody and enzyme are combined to the efficient catalytic effect of substrate.The basis of ELISA is the enzyme labeling of the immobilization and the antibody of antibody.The antibody that is combined in surface of solid phase carriers still keeps its immunologic competence, and the antibody of enzyme labeling had both kept its immunologic competence, keeps the activity of enzyme again.When measuring, examined the sample (measuring antigen wherein) and the antibody of surface of solid phase carriers and reacted.With the method for washing the antigen antibody complex that forms on the solid phase carrier and other materials in the liquid are separated.Add the antibody of enzyme labeling again, also be combined on the solid phase carrier by reaction.The amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment.After adding the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative test according to the depth of colour generation.Because the catalytic efficiency of enzyme is very high, has amplified immunoreactive result indirectly, makes assay method reach very high susceptibility.In vicissitudinous ELISA method, the second antibody un-marked that in antigen antibody complex, adds, and show the enzyme of reaction result and the 3rd antibody coupling of anti-second antibody.
Experimental result confirms that kit successfully constructs.This kit is 13.3% to the credible recall rate of patients with gastric cancer, and by this kit, the inventor finds that normal individual and patients with gastric cancer serum ESM-1 content are variant.Concrete outcome is seen Fig. 6 (a and b).
The advantage of kit of the present invention is: 1. introduce a kind of new relevant cancer of the stomach molecular marker ESM-1 of prognosis, by detecting this mark, can assist the clinical diagnosis of cancer of the stomach, further judge patient's prognosis situation, instruct treatment; Simultaneously, as an independent prognostic factor of cancer of the stomach, this mark also might become the treatment novel targets.2. the detection of this mark only need be drawn blood and just can be carried out, and patient institute suffer is less, and can detect at any time, helps monitoring the state of an illness.3.ELISA detection technique is at clinical practice comparative maturity, and simple and convenient, cost is moderate, is fit to clinical detection.
The present invention will be described in more detail below in conjunction with embodiment.
The ESM-1 nucleic acid level obviously raises in embodiment 1 tumor tissues, and ESM-1mRNA and protein expression raise in the stomach organization:
Early stage, we utilized the high density oligonucleotide chip technology that Associated Genes in Gastric Carcinoma has been carried out research comprehensive, larger samples, with containing 42, article 292, long oligonucleotide (24, article 000, be the reference sequences (RefSeqdatabase release 17) in the gene database, other is 24 years old, article 000, for EST and Unigene sequence (Unigene build188)) probe chip studies first batch of 46 routine cancer of the stomach, 20 routine non-tumprigenicity gastric mucosas, filters out cancer of the stomach and other normal structure differences expression of cancer and the relevant gene of cancer of the stomach biological characteristics.These genes can be used for carrying out the classification of tumour, explore the possible cause of disease of tumour, the prognosis of prediction tumor patient and the relevant target spot of definite tumour molecular therapy.Previously studying on the basis, utilizing the second batch 100 routine sample of collecting, 47 qualified examples of screening Quality Control, comprise 33 routine tumours and the other gastric mucosas of 14 routine non-tumprigenicity cancers to early stage chip results carried out platform validation and further integrated data analysis.
With FDR<0.01% is threshold value, utilization SAM screens 3768 differential genes in first sample, in second lot sample basis, screen 1499 differential genes, utilize these differential genes, the cluster that respectively first sample and second lot sample is originally had supervision, first 66 routine sample and second batch 47 routine sample successfully are distinguished into two groups of the other normal structures of cancer and cancer respectively as a result.Have more overlappingly between the differential gene that first sample and second lot sample find in this, 1211 genes are arranged, and all differential expression is remarkable in this at two lot samples, account for respectively first sample and second lot sample this 31.3% and 80.7%.(k-nearest neighbors KNN), screens 43 genes as the tomour specific molecular marker from first batch of sample to utilize the K-nearest neighbor method, in these 43 genes, COL1A1, COL4A1, SULF1, TMPRSS2, TTL etc. have reported relevant with cancer of the stomach, and other gene does not appear in the newspapers as yet, with this marker to second this cluster of lot sample, have only two routine tumor samples by cluster in normal structure, 96% (45/47) sample cluster is correct, accuracy and the consistance of marker are good.Utilize the combined method of principal component analysis (PCA) and linear regression, comprehensive two batches of tumor samples, filter out 10 genes and be used to predict the patients with gastric cancer prognosis, this cover marker checking result in first batch of sample can be distinguished into patient good prognosis and difference two groups, survival rate was respectively 63% and 10.5% (P=4.69e-5) in 5 years, checking in second lot sample basis, second lot sample originally can be divided into 5 years survival rates and be respectively 83% and 4.76% two groups (P=0.001), confirm this marker effectively, reliable.Utilize the gene enrichment to analyze GSEA, find in cancer of the stomach and cancer beside organism and the patient of good prognosis and poor prognosis group in signal path, transcription factor and the miRNA etc. that there are differences, wherein 88% tumour shows the unusual of MYC signal path.Utilize semi-supervised cluster, clinical pathology mathematic(al) parameter in conjunction with the sample case, found to express related gene with the cancer of the stomach tumor invading degree of depth, lymphatic metastasis, vascular cancer embolus and cardia cancer and cancer of the stomach differences, these genes participate in generation, development and the prediction tumour final result of cancer of the stomach closely.
The differential gene dendrogram is seen Fig. 1.(a). first 66 example tissue has the supervision dendrogram.3768 genes have differential expression (the FDR value is 0.01%) in stomach organization and normal gastric mucosa.2088 of cance high-expression genes, what hang down expression is 1680.(b). second batch 47 example tissue has the supervision dendrogram.1499 genes have differential expression (the FDR value is 0.01%) in stomach organization and normal gastric mucosa.243 of cance high-expression genes, what hang down expression is 1157.Row representative sample among the figure, on behalf of gene, different colours, row represent the variable condition of gene, redness: gene expression dose raises; Green: the gene expression dose downward modulation; Black: changes in gene expression is not obvious.
Fig. 2 has shown the cancer of the stomach forecast model of 43 genes and the checking in second lot sample basis.(a). the forecast model by 43 genomic constitutions can separate first 66 routine cancers and cancer beside organism; (b). originally carry out cluster according to these 43 gene pairs second lot samples, only 2 routine tumor tissues mistakes are assigned in the normal structure, and the accuracy of prediction is 96% (45/47).
On the basis of chip results, the inventor has carried out further research to the significant gene ESM-1 of differential expression.And verified the change of ESM-1 expression in stomach organization by real-time quantitative RT-PCR, SABC, change in the expression of protein level by the protein imprinted ESM-1 that verified.
Concrete experimentation is as follows:
1.RT-PCR with real-time RT-PCR
(1) cancer of the stomach and the total RNA of pairing normal structure extract
Cancer of the stomach and pairing normal mucosa are organized under the liquid nitrogen and grind, and leave and take part tissue and are used to extract DNA, and part tissue is used for the extraction of RNA and protein.The 50-100mg tissue adds 1ml TRIZOL (TrizolTMReagent, QIAGEN company).Room temperature was placed 5 minutes, make its abundant cracking, use chloroform extracting (every 1mlTRIZOL adds 0.2ml), behind the isopropanol precipitating (every 1mlTRIZOL adds 0.5ml), 75% ethanol (every 1mlTRIZOL adds 0.5ml) washing precipitation (, improving RNA purity) in order to remove impurity, separate so that an amount of DEPC is water-soluble the dry back of precipitation, 1% Ago-Gel identifies that ultraviolet spectrophotometer is surveyed RNA concentration ,-80 ℃ of preservations.
(2)RT-PCR
Getting the total RNA reverse transcription of 1 μ g is cDNA, and process is as follows: the total RNA of 1 μ g inserts in the ice afterwards immediately 70 ℃ of sex change 10 minutes.Add 2.0 μ l, 10 * Reverse Transcription damping fluid, 25mM MgCl then 2, 0.5 μ g Oligo (dT), 2.0 μ l 10mM dNTPs mix, 0.5 μ l RNasin (40U/l) and 15u AMV ReverseTranscriptase (using Promega reverse transcription kit (A3500---Reverse TranscriptionSystem, Promega company) herein).Hatched 15 minutes for 42 ℃ behind reaction system (the seeing Table 1) mixing, hatched 5 minutes for 95 ℃, last 4 ℃ of 5 minutes cessation reactions.
Table 1 reverse transcription system
* PCR primer sequence, product size, Tm value see Table 2
Table 2PCR primer sequence, product size, Tm value
Figure BDA0000041939320000071
The PCR reaction system sees Table 3
Table 3PCR reaction system
Figure BDA0000041939320000072
(3) fluorescent quantitation PCR in real time
1) RNA reverse transcription: the same RT-PCR of step method.
Fluorescent quantitation PCR in real time step:
The typical curve specimen preparation
With 90 μ l H 20 adds 10 μ l plasmids carries out 10 times of gradient dilution plasmids, does 5 standard points.
1. it is centrifugal slightly with the palm hydro-extractor to test required sample and reagent, mixing.
2. the volume of the required mixed liquor of calculation sample according to adding water earlier, adds primer, probe then, the arranged in order mixed liquor of last 2 * TaqManUniversal PCR Master Mix (ABI, TaqMan Universal PCR Master Mix).See Table 4.
3. the mixed liquor that configures is shaken mixing on the whirlpool oscillator, put into micro centrifuge, centrifugal slightly.
4. get PCR reaction black substrate and import 384 reaction plates, 384 reaction plates are placed on the black substrate, get 8 μ l master mixed liquors and add respectively in each hole, the intact back of packing adds 2 μ l templates in each hole, cover reaction film, puts into hydro-extractor afterwards, 4000rpm, 2 minutes.
5. the PCR cycling condition sees Table 5.
Table 4 quantitative PCR system (/ hole)
Table 5PCR cycling condition
Figure BDA0000041939320000082
6. after finishing above-mentioned steps, 384 orifice plates that added sample are placed in the ABI 7900HT type quantitative real time PCR Instrument react, the quality of examination criteria curve and sample and concentration range, the related coefficient of typical curve should be near 1.The probe that table 6 is used for this experiment and the sequence of primer.
The probe of table 6 real-time quantitative RT-PCR and primer sequence
Primer Sequence
The ESM-1 forward AACTTGCTACCGCACAGTCTCA
The ESM-1-MGB-probe FTATCTGCAAAGACTGTCCCTAP
ESM-1 is reverse CTGGCAGTTGCAGGTCTCTCT
2. organize the extraction and the protein imprinted analysis of total protein
Adopt the Trizol single stage method to extract and organize total protein.The piece of tissue of about 100mg is taken out from-70 ℃ of refrigerators, put into the mortar of liquid nitrogen precooling then, with special-purpose mortar and pestle piece of tissue is pounded powderedly, put into the Trizol of 2ml, firmly concussion, make it thorough suspension, place 5 minutes under the room temperature, add the 0.2ml chloroform according to 1ml Trizol to the nucleoprotein separation, shaking up the back room temperature placed 3 minutes, 12,000g, 4 ℃ are centrifugal 15 minutes.Supernatant is changed in the new centrifuge tube, keep lower floor's organic phase in order to extracting protein.To add the 1.5ml isopropyl alcohol in the above-mentioned organic phase precipitation, room temperature was placed 10 minutes, and 4 ℃ centrifugal, 12000g, 10 minutes.Abandon supernatant, add 2ml 0.3M guanidine hydrochloride according to 1ml Trizol in the precipitation, room temperature 20 minutes, 4 ℃ are centrifugal, 7500g, 5 minutes.Repeat twice.Abandon supernatant, vacuum drying protein 5-10 minute adds 1%SDS, smash fully down ultrasonic, and 4 ℃ then, centrifugal 10000g, 10 minutes, supernatant went to new pipe ,-20 ℃ of preservations.The purifying employing albumen precipitation kit method of albumen (Calbiochem, USA).(Pierce, Rockford USA) measure protein concentration with BCAProteinAssay Kit then.Every sample is measured 3 times, gets average.
With the 100 ℃ of sex change after 3 minutes in 2 * SDS sample sample-loading buffer of 50 μ g samples, the order application of sample, 12% SDS-PAGE separates, adopt Shi Shi electroporation (Bio-Rad) constant voltage 100V ice bath to change the pvdf membrane that film 2h forwards 0.2mm to, 37 ℃ of sealings of 5% skim milk 1 hour add an anti-monoclonal antibody anti-ESM-1 (1: 500), or mouse-anti people's monoclonal antibody tublin (1: 5000, Santa Cruz, USA), 4 ℃ of overnight incubation; Add two anti-(Goatanti-mouse IgG-HRP, 1: 1000, Santa Cruz USA), was hatched under the room temperature 1 hour, chemiluminescence (Chemilu minute scent Substrae of SuperSignal West Pico, 34080, Pierce), develop, photographic fixing.Carry out Flame Image Process through ImageQuant Totallab 2003.2 after the image scanning of immuning hybridization, with the intensity of quantitative band.
Above-mentioned experimental result is seen Fig. 3: (a), RT-PCR detects ESM-1mRNA expression in the cancer of the stomach.(b), ESM-1mRNA expresses and beta-actin density analysis relatively between the other normal structure of cancer of the stomach and cancer, and ESM-1 gene expression obviously increases (P=0.005) in the cancer of the stomach.(c), real-time quantitative RT-PCR amplification ESM-1 and beta-actin curve map between 34 routine cancer of the stomach and the other normal structure of 14 routine cancers, (P<0.000; Student).(d), the comparative result of ESM-1 expression significant difference (beta-actin correction) between cancer of the stomach and normal mucosa.
Embodiment 2 analyzes the relation of ESM-1 expression and clinicopathologic features
ESM-1 in stomach organization expression and the relation of clinical pathologic characteristic see Table 7.Chi-square Test the analysis showed that easier vascular infringement, DISTANT METASTASES IN and the Borrmann somatotype of betiding of ESM-1 positive expression is the patient of IV type.It is 67% (61/91) apparently higher than 45.3% (29/64) of no expression group that ESM-1 positive expression group patient's vascular is invaded incidence.In addition, between two groups of the incidences of DISTANT METASTASES IN difference (17.4%vs.7.5%) (P<0.05) is arranged more also.The distributive law of Borrmann somatotype between two groups also has difference, is respectively to express positive group 54.3% (50/92) and express 38.8% (26/67) of negative group.The expression of ESM-1 is invaded with patient age, sex, tumour differentiation and invasive depth and lymph node does not have obviously relevant (P>0.05).
Relation (159 routine patients with gastric cancer) between table 7ESM-1 expression and clinicopathologic features
Figure BDA0000041939320000091
Figure BDA0000041939320000101
Use the Kaplan-Meier survival analysis, estimate ESM-1 and express and 159 routine patient's CORRELATED WITH CLINICAL PROGNOSIS.Total survival rate was respectively 30.4% and 50.7% when two groups of positive and negative patients' statistics of ESM-1 expression were finished.The OS (total life span (Overall Survival)) that expresses positive group is starkly lower than and does not express group (logarithm rank test (log-ranktest): P=0.0339) (see figure 4).
Fig. 4 ESM-1 expresses the single factor survival analysis (K-M curve) with patients with gastric cancer.The ESM-1 positive patient is short than negative patient's postoperative life span, and difference has remarkable statistical significance (log-rank test:P=0.0339).
Use multifactor Cox and return survival analysis, vascular cancer embolus, lymphatic metastasis, tumour size, Borrmann somatotype, TNM reach by stages that ESM-1 expresses and whether radical operation be included into progressively in the regression model as potential high risk factor, the result shows that ESM-1, TNM reach the Borrmann somatotype by stages all can be as the independent prognostic factor.The results are shown in Table 8.
The multifactor survival analysis of table 8
Figure BDA0000041939320000102
Figure BDA0000041939320000111
Embodiment 3: be used to detect the antibody of ESM-1 and determining of testing conditions
Adopt ESM-1 human recombination protein (1810EC; R﹠amp; D Systems USA) is standard items, the production standard curve, and choose responsive special pairing coated antibody and utilize the square formation titrimetry to determine testing conditions with detection antibody.
Determine the working concentration of antibody: is 4 μ g/mL with coated antibody with the coating buffer dilution, 2 μ g/mL, 1.3 μ g/mL, four concentration of 1 μ g/mL are wrapped by 96 orifice plates (96 hole ELISA Plate by row, Corning Incorporated, the U.S.), each concentration bag is by triplex row (every row 3 holes), respectively at first of each concentration bag quilt, two, add strong positive antigen (ESM-1 human recombination protein concentration is 10000pg/ml) in the triplex row, weak positive antigen (ESM-1 human recombination protein concentration is 2000pg/ml) and negative control (normal human serum), to detect the antibody diluted is 10 μ g/mL, 1 μ g/mL, 0.1 three concentration of μ g/mL add first of each concentration bag quilt respectively, two, in three row.Used enzyme len antibody working concentration is 1 μ g/ml.Add substrate (TMB) colour developing, (0.05ml) cessation reaction reads absorbance A value (serial 21660 for 450nm, BIO-RAD Model 680) respectively for sulfuric acid, 2M to add acid.About 0.8, negative condition with reference to A value<0.1 is an optimum condition with strong positive antigen liquid A value.
Wherein coated antibody be mouse-anti people ESM-1 antibody (1: 100, MABH00011082-M02, Abnova), detecting antibody is the anti-people Endocan of biotinylation antibody (BAF1810, R﹠amp; D Systems USA), determines that through above-mentioned experiment the best effort concentration of coated antibody is 4 μ g/ml, and the best effort concentration that detects antibody is 1 μ g/ml, and the best effort concentration of enzyme len antibody is 1 μ g/ml.Term used herein " working concentration " is meant when detecting for example concentration of antibody of employed reagent.
Embodiment 4:ELISA detects step:
The Elisa method detects the used reagent of ESM-1 and comprises: 1. coated antibody: title: Anti h ESM1 (85-184) manufacturer: Abnova catalog number (Cat.No.): H00011082-M02 lot number: 09229-6D4WEPc specification: 0.1mg/Vial (0.41mg/ml); 2. detection antibody: title: Biotin-hEndocan Goat IgG manufacturer: R﹠amp; D catalog number (Cat.No.): BAF1810 lot number: KIS014051 specification: 50ug/Vial; 3. standard items: title: recombinanthEndocan manufacturer: R﹠amp; D catalog number (Cat.No.): 1810-EC lot number: Hrs0809041 specification: 50ug/Vial.
The every hole of 96 hole ELISA Plate add 1 μ g/ml antibody diluent dilution the ESM-1 monoclonal antibody (mouse-anti people ESM-1 antibody, 1: 100, MABH00011082-M02, Abnova) 50 μ l are hatched 24h for 4 ℃.Supernatant discarded is washed 3 times with PBS, and filter paper blots, and 1%BSA seals 2h (room temperature sealing), supernatant discarded, and PBS washes 6 times, and filter paper blots, and every hole adds examination criteria product and serum (standard items: Lot.Hrs0809041 packing product EZI12ZI; The serum source: 30 routine cancer of the stomach serum are from Beijing Tumour Hospital sample storehouse, medical history information is collected in the court's Data Analyzing Room, 10 routine non-cancer control serums are from clinical laboratory of Beijing Tumour Hospital) 50 μ l (dilution in 1: 2), reserve negative control hole and add PBS and do blank, hatch supernatant discarded behind the 2h for 37 ℃, PBS washes 3 times, filter paper blots, every hole adds polyclonal antibody (the anti-people Endocan of biotinylation antibody, BAF1810, R﹠amp; D Systems, USA) 1 μ g/ml, 50 μ l are hatched supernatant discarded after 1.5 hours for 37 ℃, PBS washes 3 times, filter paper blots, and adds the anti-sheep IgG of rabbit (dilution in 1: 1000) of horseradish peroxidase-labeled, and reaction is 40 minutes under the room temperature, PBS washes 3 times, add the TMB colour developing, room temperature was used the stop buffer cessation reaction after 20 minutes, microplate reader detects 450nm place absorbance, reads the OD value.
Embodiment 5:ESM-1 serum ELISA detects
According to the detection method described in the embodiment 4, (n=30) detects to the patients with gastric cancer serum sample, and with standard items and normal individual blood serum sample (n=10) in contrast.This testing result (Shanghai Yi Kesai ELISA report, 15: 13: 43 on the 13rd November in 2009) is carried out following statistical analysis:
Determining of positive rate .Cutoff value: one group of negative sample O.D mean value+2 to 3 standard deviation, this experiment Cutoff value=0.06590+2*0.005527=0.077, therefore in the 30 routine patients with gastric cancer serum, positive routine number is 4 examples, positive rate is 13.3%.
ESM-1 is in patients with gastric cancer and normal population expression of serum difference: the OD value is relatively seen Fig. 5 (a and b), OD average (normal individual)=0.0659 among the figure; OD average (tumor patient)=0.0782; Carry out independent sample T check, P=0.558.
The concentration ratio that calculates according to formula is shown in Fig. 6 (a and b).Concentration average (normal individual)=-822.5294pg/ml, concentration average (tumor patient)=-291.3754pg/ml, according to independent sample T check, P=0.597.
Adjust later concentration ratio, concentration average (normal individual)=2177.471pg/ml, concentration average (tumor patient)=2708.625pg/ml is according to independent sample T check, P=0.597.
R-Square (being the regression coefficient of typical curve) is 0.9992, represents that this kit is qualified technically.
Industrial applicibility
Kit of the present invention can be used for detection of clinical, understands patients serum ESM-1 situation, thereby assists the diagnosis of cancer of the stomach, prediction prognosis, guiding treatment. Utilize kit of the present invention, can in the littler situation of wound, improve the early diagnostic rate of cancer of the stomach, and because this biomarker is an independent prognostic factor, and with vascular infringement, DISTANT METASTASES IN and cancer of the stomach somatotype is relevant substantially, therefore can predict prognosis, the monitoring state of an illness, guiding treatment. This mark might become the new target spot of curing gastric cancer, for Individualized Treatment for Stomach Cancer brings new thinking and scheme.
List of references:
1.Liu, N., L.H.Zhang etc. " Overexpression of endothelial cell specific molecule-1 (ESM-1) in gastric cancer. " Ann Surg Oncol17 (10): 2628-39.
2.Scherpereel, A., F.Depontieu etc. (2006). and " Endocan, a new endothelial marker in humansepsis. " Crit Care Med34 (2): 532-7.
3.Grigoriu, B.D., F.Depontieu etc. (2006). and " Endocan expression and relationship withsurvival in human non-small cell lung cancer. " Clin Cancer Res12 (15): 4575-82.
4.Aitkenhead, M., S.J.Wang etc. (2002). " Identification of endothelial cell genesexpressed in an in vitro model of angiogenesis:induction of ESM-1, (beta) ig-h3, andNrCAM. " Microvasc Res63 (2): 159-71.
5.Ji, N.Y., Y.H.Kim etc. " Identification of endothelial cell-specific molecule-1as apotential serum marker for colorectal cancer. " Cancer Sci101 (10): 2248-53.
6. Xu Ping. the medical .2008 in the application J. of immunohistochemistry technique China and foreign countries in the pathological diagnosis; 21:40-41.
Li Jing if, Niu Yunyun, Zhang Yun's Chinese, kingdom mountain range etc. problem and not enough J. medical science and philosophy .2001 during immunohistochemistry technique is used; 22 (6): 39-40.
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15.Lassalle P, Molet S, ESM-1is a novel human endothelial cell-specificmolecule expressed in lung and regulated by cytokines.J Biol Chem.1996 such as Janin A; 271:20458.
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Claims (10)

1. ELISA kit that detects tumor markers endothelial cell specific molecule-1, described kit comprises: (1) coated antibody, described coated antibody are mouse-anti people ESM-1 antibody; (2) detect antibody, described detection antibody is biotinylation goat-anti people Endocan antibody; (3) enzyme len antibody, the anti-sheep IgG of the rabbit that described enzyme len antibody is a horseradish peroxidase-labeled; (4) people Endocan standard items; (5) chromogenic substrate of the enzyme of described enzyme len antibody.
2. ELISA kit as claimed in claim 1, described kit are used to detect the tumour of expressing ESM-1.
3. ELISA kit as claimed in claim 2, the tumour of wherein said expression ESM-1 are cancer of the stomach, kidney, breast cancer, glioma and non-small cell carcinoma.
4. ELISA kit as claimed in claim 3, described tumour are cancer of the stomach.
5. ELISA kit as claimed in claim 4, described kit are used for early diagnosis, monitoring therapeuticing effect or the prognosis evaluation of cancer of the stomach.
6. as each described ELISA kit in the claim 1 to 5, the working concentration of described coated antibody is 4 μ g/mL to 1 μ g/mL, the working concentration of described detection antibody is 10 μ g/mL to 0.1 μ g/mL, and the working concentration of described enzyme len antibody is 1 μ g/mL.
7. ELISA kit as claimed in claim 6, the working concentration of described coated antibody are 4 μ g/mL, and the working concentration of described detection antibody is 1 μ g/mL.
8. the application of endothelial cell specific molecule-1 in the preparation of lesion detection ELISA kit, described kit comprises: (1) coated antibody, described coated antibody are mouse-anti people ESM-1 antibody; (2) detect antibody, described detection antibody is biotinylation goat-anti people Endocan antibody; (3) enzyme len antibody, the anti-sheep IgG of the rabbit that described enzyme len antibody is a horseradish peroxidase-labeled; (4) people Endocan standard items; (5) chromogenic substrate of the enzyme of described enzyme len antibody.
9. application as claimed in claim 8, wherein said tumour are cancer of the stomach, kidney, breast cancer, glioma and non-small cell carcinoma.
10. application as claimed in claim 9, wherein said tumour are cancer of the stomach.
CN2010106167166A 2010-12-30 2010-12-30 ELISA (enzyme linked immunosorbent assay) kit for detecting endothelial cell specific molecule-1 (ESM-1) of tumour marker Pending CN102175850A (en)

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CN102998459A (en) * 2011-09-12 2013-03-27 香港中文大学 Biomarker for gastric cancer
CN104718455A (en) * 2012-09-07 2015-06-17 基诺麦因有限公司 Method and kit for detecting renal cancer blood biomarkers
CN105116152A (en) * 2015-08-10 2015-12-02 中国人民解放军第二军医大学 ELISA detection kit and detection method for human urine Endocan protein
CN113450873A (en) * 2021-05-14 2021-09-28 山东大学 Marker for predicting gastric cancer prognosis and immunotherapy applicability and application thereof

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102998459A (en) * 2011-09-12 2013-03-27 香港中文大学 Biomarker for gastric cancer
CN102998459B (en) * 2011-09-12 2015-09-02 香港中文大学 The biomarker of cancer of the stomach
CN104718455A (en) * 2012-09-07 2015-06-17 基诺麦因有限公司 Method and kit for detecting renal cancer blood biomarkers
CN104718455B (en) * 2012-09-07 2017-03-08 基诺麦因有限公司 The detection method of kidney blood biomarker (Biomarker) and kit
CN105116152A (en) * 2015-08-10 2015-12-02 中国人民解放军第二军医大学 ELISA detection kit and detection method for human urine Endocan protein
CN113450873A (en) * 2021-05-14 2021-09-28 山东大学 Marker for predicting gastric cancer prognosis and immunotherapy applicability and application thereof
CN113450873B (en) * 2021-05-14 2022-07-08 山东大学 Marker for predicting gastric cancer prognosis and immunotherapy applicability and application thereof

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