CN203275415U - Protein chip for detecting esophageal squamous carcinoma marker and kit thereof - Google Patents
Protein chip for detecting esophageal squamous carcinoma marker and kit thereof Download PDFInfo
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Abstract
The utility model discloses a protein chip for screening specific proteins of esophageal squamous carcinoma with high throughput and multiple indexes, and further discloses a kit matched with the chip for use. 55 kinds of differential proteins of the esophageal squamous carcinoma in the serum or the plasma are used as esophageal squamous carcinoma markers and proteins to prepare the protein chip in a contrast manner. The protein chip consists of a substrate, and protein detection indexes and contrast detection index coatings distributed on the substrate in arrays. The kit is internally provided with a detection liquid matched with an experiment agent. The protein chip and the kit disclosed by the utility model are applied to determining protein maps of normal crowds, precancerous lesions crowds with esophageal squamous and crowds with the esophageal squamous, and provide a meaning capable of performing early stage screening of the esophageal squamous and diagnosis and illness monitoring in middle and later stages.
Description
Technical field
The invention belongs to the biochip technology field, particularly a kind of protein-chip.Use this protein-chip and kit examination esophageal squamous cell carcinoma differential protein mark thereof.
Background technology
The cancer of the esophagus is one of the 8th modal malignant tumour in the world, is considered to the malignant tumor of digestive tract that second is difficult to cure after cancer of pancreas.China is again Incidence of esophageal cancer and mortality ratio higher country in the world, account for global more than 50%, the annual cancer of the esophagus case that 250,000 new diagnosis are approximately arranged, the incidence of disease occupies the 4th after being only second to cancer of the stomach, lung cancer, liver cancer, and the mankind's life and health in serious threat.The cancer of the esophagus is divided into again esophageal squamous cell carcinoma and gland cancer, and the ratio of Chinese esophageal squamous cell carcinoma is up to more than 90%.The research of the cancer of the esophagus has very important significance to reducing M ﹠ M.At present cancer of the esophagus examination and diagnosis are still take endoscopy of esophagus and mucous membrane of esophagus iodine staining as main, there is certain drawback, as higher in cost, checking efficiency is low, the intrusive mood inspection makes patient compliance poor, also be subject to the impact of doctor's the human factors such as technical merit, be not suitable for early screening and clinical detection.Along with research advances to biology field, go out aberrant gene from gene level research examination, carry out gene sequencing, find the mutational site, then to seek responsive medicine be a treatment approach, but this process is more loaded down with trivial details.Along with going deep into genome understanding, it is found that medicine normally acts on protein rather than the gene itself of gene code, although human genome has comprised whole hereditary information of our healths, but will through a series ofly transcribe, translation process realizes protein expression, the performance of the various functions of human body is all completed by protein.Protein is executor and the agent of vital movement, life quintessence can more deep, be more pressed close in research to the protein group, and this has expedited the emergence of a new scientific domain--protein science: specialize in human body a large amount of unknown protein and their multifarious morphology and functions.Surface-enhanced laser desorption ionization time of flight mass spectrometry (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, SELDI-TOF-MS) technology is a kind of proteomics research technology, by serum and weak cation rabphilin Rab chips incorporate, read the haemocyanin collection of illustrative plates with mass spectrometer, with the protein fingerprint analysis software, the analysis of mass spectrometric data is obtained protein fingerprint.Serum protein fingerprint of esophagus cancer is comprised of the protein of 12 different mass-to-charge ratioes (M/Z).But the mass-to-charge ratio of the detected protein of this kind method multiple proteins often combines, can not determine it is any protein, and to have be much the protein of mankind's the unknown, and follow-up individualized treatment and new drug development are brought difficulty, is not that the people adopts gradually.Select the known albumen research of the mankind to find the differential protein mark by prestissimo.Protein is divided into again histone and haemocyanin, and haemocyanin has sampling easily, and sample preparation is simple, can reflect that the instant physiological and pathological of health changes, and is suitable as the mark of tumor screening, diagnosis and state of illness monitoring.The genesis of the cancer of the esophagus is the variation of normal cell process polygenes, multi-step, develops into gradually the cancer cell with malignant proliferation characteristic and constantly shifts the process of sending out.Change has all occured in the expression of the several genes that esophageal cancer cell is relevant to propagation, apoptosis, differentiation and transfer, and this kind variation finally shows as expression and the Functional change of range protein in cell, thereby affects the vital movement of cell.The protein that changes in Carcinogenesis and little peptide can be discharged in blood or other body fluid through tumour cell metabolism, secretion.Therefore, by the change of research cancer of the esophagus serum proteins, might find the protein relevant or special to the cancer of the esophagus or peptide matters, i.e. oesophagus cancer-serum protein marker.But the content of haemocyanin is all low-down, and namely, there is certain difficulty in low-abundance protein so haemocyanin detects.
traditional method of protein detection mainly contains enzyme linked immunosorbent assay (ELISA), radioimmunoassay for detecting (RIA), exempt to put determination method (IR-MA) and fluorescence immunoassay (FIA) etc., enzyme linked immunosorbent assay is present clinical detection method with the most use, but enzyme-linked immunologic adsorption test method need to add the incubation time that half an hour to one hour is arranged when being examined sample and enzyme labelled antibody, detection also must be completed after the plate process just can add reaction substrate to develop the color through washing for several times in the centre on microplate reader, step is comparatively loaded down with trivial details time-consuming, and it is low that existence detects positive rate, the deficiencies such as easy pollution, and once can only detect a kind of index, according to these methods, every part of serum is carried out the manpower that is determined at of multinomial mark, all unrealistic on material resources and financial resources.People wish to have a kind of quick, easy, cheap, and the diagnostic method that can detect simultaneously the many index of many person-portions improves efficient and the accuracy of detection.The appearance of protein chip technology makes high flux, studies serum photeomics on a large scale and become possibility.Protein chip only needs to add successively confining liquid on reaction plate, is examined two anti-getting final product of sample, cleansing solution and colloid gold label.Liquid is examined antibody in sample and the second antibody of colloid gold label and is just automatically completed absorption in the process of automatically diafiltration, and second antibody does not need substrate develop the color colour generation, directly observed result because of colloid gold label.Protein-chip is a kind ofly protein example is detected, identifies, analyzes and diagnoses by immunology principle at solid substrate surface fixing protein or polypeptide microarray, can be applicable to the association areas such as life science, medicine and pharmacology.Existing protein chip, expensive, generally only contain one or more for detection of the marker protein of humoral specimen, the mark that can be applied at present clinical detection is very single, the main EGFR of detection acceptor comes auxiliary group of diagnosis of esophageal cancer, false positive rate is higher, and sensitivity, accuracy is on the low side, poor repeatability.Still there is no at present special cancer of the esophagus examination and the widespread use of diagnosis protein chip.For example, application for a patent for invention number is 200510116733.2, the applying date is 2005.10.28, open day is 2007.05.02, publication number is CN1955737A's " a kind of protein chip for screening precancerous lesion of cancer of esophagus " application for a patent for invention, and the protein chip of this invention has only comprised 16 kinds of protein markers, and only has this albumen of TP53 to overlap, two kinds of target antibody differences that protein chip detects, the conclusion that it draws and meaning are also different; Utility model application number is 200820059666.4, the applying date is 2008.06.13, Granted publication day is 2009.05.27, Granted publication number is " a kind of male multi-tumor marker detection protein chip and kit thereof " utility model patent of CN201247246Y, its chip only contains 7 kinds of marks, and the mark that can be used for detecting the cancer of the esophagus only has 3 kinds: CYFRA21-1, SCC-AG, CEA.These three kinds of marks are low for the diagnostic accuracy of the cancer of the esophagus, and repeatability is low, and false positive is high, and only limits to detect male esophageal cancer.though the research of 50 multiple protein is arranged in prior art, " Use of novel autoantibody and cancer-related protein arrays for the detection of esophageal adenocarcionma in seru " (Thorac Cardiovasc Surg as people such as U.S. Kilic A, 2008) protein chip of research relates to 53 kinds of tumor associated antigens, but it only limits to detect for early stage adenocarcinoma of esophagus, the selected protein diversity of selected protein marker and the present invention is very large, the present invention be directed to esophageal squamous cell carcinoma, the albumen that overlaps only has: IL-8, EGFR, CA19-9, CA72-4, 4 kinds of MMP-7 etc.In a word, prior art lack a kind of can the high flux examination and detect protein chip that esophageal squamous cell carcinoma, price can be accepted for people, can penetration and promotion in crowd's health check-up.
Summary of the invention
The present invention aims to provide a kind of protein chip that detects the haemocyanin mark for esophageal squamous cell carcinoma, comprises by substrate and array being distributed in on-chip Protein Detection index and control test index coating forms.Protein Detection index coating comprises multiple esophageal squamous cell carcinoma mark, and control test index coating comprises sample contrast mark, positive control mark, negative control mark, reagent contrast mark.Substrate is glass chip or plastic base, and every kind of tumor markers is point sample repeatedly, and every kind of contrast mark is point sample repeatedly, each array repeatedly, a plurality of esophageal squamous cell carcinoma marker protein chips are on 1 substrate.Comparatively be typically every kind of tumor markers and repeat 3 point samples, every kind of contrast mark repeats 3 point samples, and each chip array repeats 16 times, and 16 esophageal squamous cell carcinoma marker protein chips are on a substrate.
the selected albumen of protein chip of the present invention is known esophageal squamous cell carcinoma associated protein mark, and the target protein that detects is clear and definite, application is strong, and special in the especially Chinese esophageal squamous cell carcinoma occurred frequently of the cancer of the esophagus, selected mark also belongs to the mark of squama cancer, select the contrast of 55 kinds of esophageal squamous cell carcinoma protein markers and albumen to make protein chip, to the three class crowds that the esophageal squamous cell carcinoma district occurred frequently is detected by gastroscope and the histopathology diagnosis is made a definite diagnosis, that is: normal population, the serum of each 150 examples of Precancerous Esophageal Squamous Cell Carcinoma crowd and esophageal squamous cell carcinoma crowd or the differential protein of blood plasma, thereby establish this three classes crowd's esophageal squamous cell carcinoma associated protein collection of illustrative plates, finding out the developing differential protein mark of esophageal squamous cell carcinoma is: CA72-4, CTAG1B, Cyfra21-1, GAGE7, SERPINB4 is totally 5 kinds of albumen, be used for the esophageal squamous cell carcinoma early screening, in, late period, diagnosis and state of illness monitoring provided a kind of detection method.A kind of means are provided for seeking sensitive drug simultaneously.
Esophageal squamous cell carcinoma marker protein chip of the present invention, be distributed in on-chip Protein Detection index and control test index coating forms by substrate and array, with the physics fixing means, namely be coated with the protein such as point sample antigen on the glass substrate of azobenzene polymer.With after light irradiation, azobenzene polymer produces the checker of cis-isomer-trans-isomer, and protein is absorbed in azobenzene polymer inside gradually.After stopping illumination, azobenzene polymer is frozen into solid again, and protein has just realized that physics is fixed on above substrate, and substrate is glass chip or plastic base.the Protein Detection index comprises: AnnexinI, the Annexin II, BIRC5, CA19-9, CA72-4, CCNB1, CCND1, CCNE1, CDC25A, CDC25B, CDK1, CDK2, CDK4, CDK6, CDKN1A, CDKN1B, CDKN2A (A), CDKN2A (B), CDKN2A (C), Clu, CTAG1B, Cyfra21-1, E2F1, EGFR (A), EGFR (B), ERBB2, GAGE7, GJA8, HAVCR1, HOOK2, ID-1, IGF2BP3, IL-8, MGMT, MMP-7, MSH2, MVD, MYC, NFKB1, pol β, PRDX6, PTK2, PTK2abnova, RAR-b, S100A4, SERPINB4, Smad3, SOD2, SQSTM1, SURF-1, TACSTD2 (A), TACSTD2 (B), TGF-b1, TP53, TP63 is totally 55 kinds of tumor markerses, contrast with sample, positive control, negative control and reagent contrast forming array formula point sample coating, dot matrix is on a substrate simultaneously, each array repeats 16 groups.
A preference of above-mentioned substrate is the aldehyde radical slide.
Above-mentioned control test coating comprises: sample contrast, positive control, negative control and reagent contrast.The sample contrast is people's serum antigen; Positive control is the human IgG antibody; Negative control is antigen point sample dilution used; The reagent contrast is biotin labeled human IgG antibody.
In each array on the said chip coating, each Protein Detection index has 3 to repeat a little, and the contrast index also has 3 to repeat a little.What and size of array is big or small with what, substrate of protein marker and size point sample is directly relevant.
The preference of above-mentioned Protein Detection index and detection arrays is: when Protein Detection index point diameter is 0.01mm, and dot spacing 0.01mm, array size is 0.56mm * 0.6mm, and 16 arrays can be set on the nitrocellulose filter protein chip.
The protein chip kit that detects esophageal squamous cell carcinoma comprises: infiltrate (3), sample dilution (4), gold cross-linking reagent dilution (5), concentrated cleaning solution (6) detects liquid A (7), detects liquid B (8), bovine serum albumen solution (9), gold cross-linking reagent (10), biotin labeled human IgG antibody (11), the golden cross-linking reagent (12) of antibiotin mark.Each liquid is bottled, is fixed in packing box with the papery masterplate.Each several part is fixed in a packing box by the papery masterplate, does not limit modes of emplacement, supports the use during inspection.
The sample dilution 4 of mentioned reagent box is a kind of protein stabiliser (Gentel Dilution Buffer TM, production number: No.B255); Can be formed by 3%BSA and 0.05% washing agent (pH=7.3) configuration.
Above-mentioned concentrated cleaning 6 (10 * Gentel Wash Buffer TM, production number: No.2-1016)) is a kind of neutral buffered liquid, by washing agent formulated (pH=6.8).
Above-mentioned biotin labeled human IgG antibody 11 preparations are by 20mg/mL bovine serum albumin diluted to 100 times, further by sample diluted to 50, and 000 times.
The golden cross-linking reagent 12 of above-mentioned antibiotin mark is by 200mg SilverQuant Reagent C (Gentel, production number: No.10-2135) with 200mL SilverQuant Conjugate Buffers (Gentel, production number: No.10-2134) be mixed with golden cross-linking reagent, further use again the golden cross-linking reagent (Gentel of antibiotin, SilverQuant Anti-biotin Gold Conjugate, production number: No.10-1031) formulated.
Above-mentioned bovine serum albumen solution 9 is by 20mg/mL bovine serum albumin formulated (pH=7.4)
Above-mentioned detection liquid A, detection liquid B refer to matching used two kinds of reagent, a kind of silver nitrate reagent that contains, and another kind contains citric acid.
A chip can detect 16 samples simultaneously, not only has higher sensitivity, simultaneously greatly reduce costs and improve detection efficiency, has overcome the drawback that protein chip in the past is expensive, be difficult to popularize.
Description of drawings
Fig. 1 is the outward appearance floor map of installation and detection chip device.4 chips are arranged, and 1 represents a chip; Every chip comprises 16 arrays, and 2 represent an array, and an array can detect a sample.When Protein Detection index point diameter is 0.01mm, dot spacing 0.01mm, an array size is 0.56mm * 0.6mm, the size of a substrate is 25mm * 75mm.
Fig. 2 is the sample application array schematic diagram.Formed by 14 * 15 point samples.
Fig. 3 is the mark point sample location drawing of sample application array schematic diagram.4 angle A1-A3, A13-A15, N1-N3, N13-N15 are the sample contrast, respectively have 3 to repeat point sample, B1-D1, K15-M15 are positive controls, respectively have 3 to repeat point sample, E1-G1 is negative control, has 3 to repeat point sample, K1-M1, B15-D15 are the reagent contrasts, respectively have 3 to repeat point sample, all the other each row are respectively Protein Detection index point sample AnnexinI, the Annexin II, BIRC5, CA19-9, CA72-4, CCNB1, CCND1, CCNE1, CDC25A, CDC25B, CDK1, CDK2, CDK4, CDK6, CDKN1A, CDKN1B, CDKN2A (A), CDKN2A (B), CDKN2A (C), Clu, CTAG1B, Cyfra21-1, E2F1, EGFR (A), EGFR (B), ERBB2, GAGE7, GJA8, HAVCR1, HOOK2, ID-1, IGF2BP3, IL-8, MGMT, MMP-7, MSH2, MVD, MYC, NFKB1, pol β, PRDX6, PTK2, PTK2abnova, RAR-b, S100A4, SERPINB4, Smad3, SOD2, SQSTM1, SURF-1, TACSTD2 (A), TACSTD2 (B), TGF-b1, TP53, maximum 4 indexs of the every row of TP63, respectively there are 3 to repeat point sample.
Fig. 4 is the kit schematic diagram.3 represent infiltrate; 4 representative sample dilutions; 5 represent golden cross-linking reagent dilution; 6 represent concentrated cleaning solution; 7 representatives detect liquid A; 8 representatives detect liquid B; 9 represent bovine serum albumen solution; 10 represent golden cross-linking reagent; 11 represent biotin labeled human IgG antibody; 12 represent the golden cross-linking reagent of antibiotin mark.
Fig. 5 is for detecting the serum result schematic diagram, and wherein sample contrast, positive control and reagent control signal are stronger, the negative control signal a little less than, reliable experiment result; 13 is that the CA72-4 signal is stronger, all the other index signals a little less than.
Fig. 6-Figure 10 is above-mentioned 55 kinds of albumen index haemocyanin collection of illustrative plates schematic diagram of normal population, Precancerous Esophageal Squamous Cell Carcinoma crowd and esophageal squamous cell carcinoma patient three class crowds; The white histogram represents the normal population protein graphical spectrum; The grid histogram represents Precancerous Esophageal Squamous Cell Carcinoma crowd protein graphical spectrum; Black represents the esophageal squamous cell carcinoma protein graphical spectrum.
Embodiment
Embodiment 1:
A kind of preparation that detects the protein chip of esophageal squamous cell carcinoma
Its step is mainly: (1) prepares the matrix nitrocellulose filter; (2) design chips is determined arrangement mode and the point sample position of dot matrix; (3) spotting needle sucking-off antigen adopt to spray point sample, and the electronic voltage spray technique is simultaneously on specking to a glass substrate, (4) assembling nitrocellulose filter, and seal, dry, packing, preserve.
The preparation method of above-mentioned protein chip for detection of the multiple serum antibody mark of esophageal squamous cell carcinoma specifically comprises the following steps:
1. the source of esophageal squamous cell carcinoma marker detection antigen-antibody
(1) detect determining of index antigen:
All antigens are available from Aureal Dongyuan County, Beijing bio tech ltd (OriGene), inferior novacine skill company (Abnova) and MyBioSource company.
(2) detect determining of indicator antibody:
The detection antibody that adopts is to being Mouse Hybridoma Cells monoclonal antibody pair, and its accuracy of detection and accuracy obviously are better than polyclonal antibody.
2. the point sample preparation detects the film chip of index coating
SmartArrayerTM 48 micro-array chip point sample instrument speckings, spotting needle is on 384 orifice plate sucking-off antigen speckings and nitrocellulose filter, and latticed form is 14 * 15 rectangular arrays, as shown in Figure 2.When spot diameter is 0.01mm, dot spacing 0.01mm, the antigen array size is 0.56mm * 0.6mm, pad pasting after point sample, sealing; At last after chip is dry, packing in 4 ℃ of preservations.
3. protein chip assembling
With having the vessel of separating conversion zone, conversion zone is fixed, assembling mode is first chip groove screw to be unscrewed, taking out chip front side upwards is placed in the chip groove, screwing screw is fixed in the chip groove chip, the packing ring heavy wall is sticked in opposite side equipment the thin-walled contact chip, the chip groove is placed on packing ring, the screw fixed equipment, (specifically seeing the SIMplex64Multi-Array System explanation of Gentel, production number 4-1025).After assembling is completed, schematic diagram as shown in Figure 1.
Embodiment 2:
The preparation of kit
A kind of for detection of the serum of esophageal squamous cell carcinoma and/or the kit of blood plasma marker thing, as shown in Figure 4, by sample dilution 4, concentrated cleaning solution 6, bovine serum albumen solution 9, biotin labeled human IgG antibody 11, the golden cross-linking reagent 12 of antibiotin mark detects liquid AB(7,8).Each liquid is bottled, and 4 ℃ of low temperature are preserved, and support the use during inspection.
Concentrated cleaning solution 6 is a kind of neutral buffered liquid (10 * Gentel Wash Buffer TM, production numbers: No.2-1016); Can formulated by washing agent (pH=6.8).Provide 250mL, one bottle.Be diluted in the 225mL ultrapure water mixing during use with 25mL.
Bovine serum albumen solution 9 is by 20mg/mL bovine serum albumin formulated (pH=7.4).
Biotin labeled human IgG antibody 11 is two anti-, and is formulated by following methods: by 20mg/mL bovine serum albumin diluted to 100 times, and further by sample diluted to 50,000 times.Provide 50ul, a pipe.
The golden crosslinked examination 12 of antibiotin mark comprises SilverQuant Reagent C (Gentel, production number: No.10-2135) 200mg, SilverQuant Conjugate Buffers (Gentel, production number: No.10-2134) 200mL, SilverQuant Anti-biotin Gold Conjugate (Gentel, production number: No.10-1031) 100uL.Formulated by following methods: 200mg SilverQuant Reagent C (Gentel, production number: No.10-2135) join 200mL SilverQuant Conjugate Buffers (Gentel, production number: be mixed with golden cross-linking reagent No.10-2134), get the golden cross-linking reagent (Gentel that 8mL adds the 80uL antibiotin again, production number: No.10-1031), mixing.
Detecting liquid A, B uses the SilverQuant Reagent A (production number 10-2132) of Gentel company and SilverQuant Reagent B (production number 10-2112) as supporting detection liquid.Each 1 bottle of A liquid, B liquid, every bottle of 10mL.
Embodiment 3:
The application of protein chip and kit and detection
the application of the protein chip for detection of esophageal squamous cell carcinoma mark antigen of the present invention, main adopt Ag-Ab-two anti--esophageal squamous cell carcinoma mark antigen in nm of gold combination-deposition of silver method detection human blood, method is that human serum is added on chip surface, antibody in sample is respectively at the corresponding antigen combination that is fixed on chip, add two anti-reactions after drying, washing away unconjugated two resists, add golden cross-linking reagent, close with two resistive connections, wash away unconjugated nm of gold, add silver nitrate, the short deposition of silver of nm of gold is in gold surface, scan and collect signal, the gray scale of sample point is directly proportional to the concentration of Serum Antibody.
In the application of above-mentioned protein chip kit for detection of esophageal squamous cell carcinoma mark antigen, the concrete using method of its antigen-antibody reaction and detection is as follows:
To the chip of specking antigen, positive control, negative control, reagent contrast with the sealing of 3%BSA solution, room temperature 2 hours, sealing substrate surface non-specific site;
2. after the drying confining liquid, room temperature was placed 4 hours, carried out drying;
3. at the conversion zone of chip, add the human serum sample through the 1:500 dilution; Every hole adds 75ul, is placed in 22 ℃ of isothermal reactions, oscillating reactions 1 hour, and wash 5 times with 1 times of cleansing solution;
4. throw away liquid, every hole adds two anti-75ul, at room temperature, is placed in 22 ℃, isothermal reaction instrument, hatches albumen 1 hour with 160 rev/mins of rotating speeds on the earthquake device.And the damping fluid with 1 times of dilution washs 5 times;
5. throw away liquid, the aliquot Silver Quant reagent C (Gentel production number 10-2135) of 200 milligrams are joined in the affine damping fluid of Silver Quant of 200 milliliters (Gentel, production number 10-2134).Get the golden cross-linking reagent that 8mL adds the 80uL antibiotin again (Gentel, production number: No.10-1031), mixing.Every hole adds 75 μ L gold affinity reagent, and under room temperature, 45 minutes (160 rev/mins) are cultivated in concussion;
6. throw away liquid, the Silver Quant reagent A (Gentel, production number 10-2132) of Gentel company and Silver Quant reagent B (production number 10-2112) are mixed, add on chip on each conversion zone, every array adds 100 μ L, oxidation stain;
7. scan and collect signal with Gentel detection system (Gentel Proteomics Multi-System TM production number 10-1030), chip scanning figure sees that Fig. 1 is a chip, Fig. 2 is an array, and (AthenaQuant Analysis Software v1.6) analyzes scanning result according to esophageal squamous cell carcinoma chip analysis software.
8. use the method to detect each examples up to a hundred of normal population, Precancerous Esophageal Squamous Cell Carcinoma crowd and esophageal squamous cell carcinoma new cases of esophageal squamous cell carcinoma district occurred frequently, Fig. 6-Figure 10 is three class crowds' esophageal squamous cell carcinoma generation associated protein collection of illustrative plates.Ordinate represents molecular weight of albumen, and unit is dalton, and horizontal ordinate is albumen, totally 55 kinds.Three class crowds' protein graphical spectrum has significant difference, can be used to examination and diagnosis person under inspection, picture and this three albuminoids collection of illustrative plates that the person under inspection draws are compared, and carry out statistical study, thereby which kind of crowd judgement belongs to.
Protein chip of the present invention is a kind of just can detect a plurality of samples simultaneously, thereby reduction testing cost, the sensitivity that detects and accuracy also improve greatly, and the while reaches the purpose of examination and diagnosis according to esophageal squamous cell carcinoma generation different times protein graphical spectrum and differential protein mark.
Through comparison, randomly draw in the esophageal squamous cell carcinoma district occurred frequently through endoscope and histopathologic diagnosis and be diagnosed as each 10 examples of esophageal squamous cell carcinoma patient, Precancerous Esophageal Squamous Cell Carcinoma patient and normal person, take a blood sample and carry out mark by other people, not allowing experimental implementation person know.Then each group blood sample is upset by the operator and carried out the blind method detection of protein-chip, do not know which class crowd this blood sample belongs to when detecting, analyzing data and diagnosis.Detected protein content and collection of illustrative plates and the esophageal squamous cell carcinoma that draws before, Precancerous Esophageal Squamous Cell Carcinoma and normal person all kinds of crowd's protein content mean value and protein graphical spectrum are compared, and diagnostic criteria is that protein content and certain class crowd protein content average comparison no difference of science of statistics are regarded as such crowd.Check with mark again after making diagnosis, determine whether diagnosis is correct.This result detects cancer 9 examples altogether, front 11 examples of cancer, and normal 9 examples, wherein cancer does not detect 1 example, and accuracy is 90%; Error detection 1 example before cancer, accuracy is 90%, and the normal person does not detect 1 example, and accuracy is 90%, therefore this protein chip rate of correct diagnosis is 90%.Table 2 illustrates that for crowd's albumen average and esophageal squamous cell carcinoma, Precancerous Esophageal Squamous Cell Carcinoma and normal person's albumen average of sampling observation compare indifference (p＞0.05) crowd of sampling observation belongs to respectively esophageal squamous cell carcinoma, Precancerous Esophageal Squamous Cell Carcinoma and normal population.
Table 1. sampling observation crowd's albumen average and esophageal squamous cell carcinoma, Precancerous Esophageal Squamous Cell Carcinoma and normal person's albumen average be (p) relatively
The present invention is directed to the protein chip of having contained the detection of phase esophageal squamous cell carcinoma morning, noon and afternoon, 55 kinds of tumor associated antigens that esophageal squamous cell carcinoma is relevant are placed on a chip, the albumen kind increases greatly, all albumen are available from Origene, Abnova, MyBioSource company, comprise 3 native proteins (Cyfra21-1, CA72-4, CA19-9), 46 purification of recombinant proteins, 2 recombinant protein dissolved matters (CTAG1B, GAGE7); Selected according to the albumen existence form cyclin, haemocyanin, the autoantibody that extensively exist in blood.
The employing nitrocellulose filter is that surface chemistry (Nitrocellulose-based SurfaceChemistry) the technology ankyrin of medium can keep the Activity and stabill of protein, reduce non-specific binding, adopt the short deposition of silver method of gold grain, this method makes remolding sensitivity ELISA method improve 9 times, reappearance is also high 9 times than fluorescent technique, can improve accuracy and the science of detection, reduce false positive, diagnosis rate can reach more than 90%.Adopt Silver
Detection platform scan chip in about 10 microns resolution, susceptibility with height, can analyze simultaneously high density and high flux picture format, greatly improved detection efficiency and reduced cost, 16 arrays are arranged on every chip of this chip, each array has 192 antigens, a chip can detect 16 samples simultaneously, can place simultaneously 4 chips on chip detecting equipment, can detect simultaneously 64 samples, greatly improve detection efficiency.And detection method commonly used can't provide enough sensitivity and precision quantitative test multiple proteins as fluorescence and colorimetric detection.The present invention can find fast, thereby checking and selection markers improve clinical detection speed, can also quantitatively detect protein content in microarray, is conducive to the staging diagnosis in disease progression.
Claims (8)
1. protein-chip that detects the esophageal squamous cell carcinoma mark, comprise: be distributed in on-chip Protein Detection index and control test index coating forms by substrate and array, it is characterized in that: Protein Detection index coating comprises multiple esophageal squamous cell carcinoma mark, and control test index coating comprises sample contrast mark, positive control mark, negative control mark, reagent contrast mark.
2. a kind of protein-chip that detects the esophageal squamous cell carcinoma mark according to claim 1, it is characterized in that: Protein Detection index coating comprises at least: AnnexinI, the Annexin II, BIRC5, CA19-9, CA72-4, CCNB1, CCND1, CCNE1, CDC25A, CDC25B, CDK1, CDK2, CDK4, CDK6, CDKN1A, CDKN1B, CDKN2A (A), CDKN2A (B), CDKN2A (C), Clu, CTAG1B, Cyfra 21-1, E2F1, EGFR (A), EGFR (B), ERBB2, GAGE7, GJA8, HAVCR1, HOOK2, ID-1, IGF2BP3, IL-8, MGMT, MMP-7, MSH2, MVD, MYC, NFKB1, pol β, PRDX6, PTK2, PTK2abnova, RAR-b, S100A4, SERPINB4, Smad3, SOD2, SQSTM1, SURF-1, TACSTD2 (A), TACSTD2 (B), TGF-b1, TP53, TP63 is totally 55 kinds of tumor markerses,
Sample contrast mark behaviour serum antigen, the positive control mark is the human IgG antibody, and the negative control mark is antigen point sample dilution used, and reagent contrast mark is biotin labeled human IgG antibody.
3. a kind of protein-chip that detects the esophageal squamous cell carcinoma mark according to claim 2, it is characterized in that: substrate is glass chip or plastic base, every kind of tumor markers is point sample repeatedly, every kind contrasts repeatedly point sample of mark, each array repeatedly, a plurality of esophageal squamous cell carcinoma marker protein chips are on 1 substrate.
4. a kind of protein-chip that detects the esophageal squamous cell carcinoma mark according to claim 3, it is characterized in that: every kind of tumor markers repeats 3 point samples, every kind of contrast mark repeats 3 point samples, each chip array repeats 16 times, and 16 esophageal squamous cell carcinoma marker protein chips are on a substrate.
5. a kind of protein-chip that detects the esophageal squamous cell carcinoma mark according to claim 4, it is characterized in that: when Protein Detection index point diameter is 0.01mm, dot spacing 0.01mm, an array size is 0.56mm * 0.6mm, the size of a substrate is 25mm * 75mm.
6. protein chip kit that detects the esophageal squamous cell carcinoma mark, its feature comprises: infiltrate (3), sample dilution (4), gold cross-linking reagent dilution (5), concentrated cleaning solution (6) detects liquid AB(7,8), bovine serum albumen solution (9), gold cross-linking reagent (10), biotin labeled human IgG antibody (11), the golden cross-linking reagent (12) of antibiotin mark; Each liquid is bottled, is fixed in packing box with the papery masterplate, and each several part is fixed in a packing box by the papery masterplate, does not limit modes of emplacement, supports the use during inspection.
7. a kind of protein chip kit that detects the esophageal squamous cell carcinoma mark according to claim 6, it is characterized in that: described bovine serum albumen solution (9) is formulated by 20 mg/mL bovine serum albumins, and the ph value is 7.4.
8. a kind of protein chip kit that detects the esophageal squamous cell carcinoma mark according to claim 7, it is characterized in that: detection liquid AB(7,8) refer to matching used two kinds of reagent, detect liquid A(7) be a kind of silver nitrate reagent that contains, detect liquid B(8) be a kind of citric acid reagent that contains.
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| CN 201320127642 CN203275415U (en) | 2013-03-20 | 2013-03-20 | Protein chip for detecting esophageal squamous carcinoma marker and kit thereof |
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| CN 201320127642 CN203275415U (en) | 2013-03-20 | 2013-03-20 | Protein chip for detecting esophageal squamous carcinoma marker and kit thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149369A (en) * | 2013-03-20 | 2013-06-12 | 江苏元化生命科技有限公司 | Protein chip for detecting esophageal squamous carcinoma marker and kit box of protein chip |
| CN108715845A (en) * | 2018-05-15 | 2018-10-30 | 郑州大学 | A kind of construction method of epithelium of esophagus tissue pol β specific knockdown mouse esophageal precancerous lesion models |
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| CN113777311A (en) * | 2021-09-16 | 2021-12-10 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
-
2013
- 2013-03-20 CN CN 201320127642 patent/CN203275415U/en not_active Withdrawn - After Issue
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149369A (en) * | 2013-03-20 | 2013-06-12 | 江苏元化生命科技有限公司 | Protein chip for detecting esophageal squamous carcinoma marker and kit box of protein chip |
| CN110740733A (en) * | 2017-06-16 | 2020-01-31 | 豪夫迈·罗氏有限公司 | Methods of diagnosis and treatment of IRAK 4-mediated conditions and disorders |
| CN108715845A (en) * | 2018-05-15 | 2018-10-30 | 郑州大学 | A kind of construction method of epithelium of esophagus tissue pol β specific knockdown mouse esophageal precancerous lesion models |
| CN108715845B (en) * | 2018-05-15 | 2021-09-24 | 郑州大学 | Construction method of esophagus epithelial tissue pol beta specificity knockout mouse esophagus precancerous lesion model |
| CN113180595A (en) * | 2021-03-25 | 2021-07-30 | 河北工程大学 | Detection system for determining professional fatigue degree of key industry based on human saliva protein |
| CN113777311A (en) * | 2021-09-16 | 2021-12-10 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN113777311B (en) * | 2021-09-16 | 2023-08-01 | 郑州大学 | ELISA kit for auxiliary diagnosis of esophageal squamous carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
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