CN1746676A - A detection method for pancreatic cancer serum marker molecules - Google Patents
A detection method for pancreatic cancer serum marker molecules Download PDFInfo
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- CN1746676A CN1746676A CN 200410074306 CN200410074306A CN1746676A CN 1746676 A CN1746676 A CN 1746676A CN 200410074306 CN200410074306 CN 200410074306 CN 200410074306 A CN200410074306 A CN 200410074306A CN 1746676 A CN1746676 A CN 1746676A
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Abstract
本发明涉及一种胰腺癌血清标志分子的检测方法，通过定量免疫印记法检测人血清/血浆中凝溶胶蛋白浓度，根据所述凝溶胶蛋白含量降低的水平无创性地辅助早期发现胰腺癌。人血浆凝溶胶蛋白标准品，按照一定浓度梯度稀释后与待测血清样本总蛋白共同经SDS－PAGE分离，将分离的蛋白从凝胶上转移至PVDF膜上。含有样本的PVDF膜与抗人凝溶胶蛋白抗体做免疫印迹反应，印迹信号经化学发光、放大后使X光片曝光。印迹结果扫描后在图像分析软件辅助下，绘制凝溶胶蛋白标准品含量与印迹强度关系的标准曲线，并在标准曲线的线性范围内计算出同一凝胶上待测血清样品的凝溶胶蛋白含量。该方法简便、快捷，易于被患者接受，对辅助胰腺癌诊断有较高的敏感性和特异性。The invention relates to a method for detecting serum marker molecules of pancreatic cancer, which detects the concentration of gelsolin in human serum/plasma by quantitative immunoblotting, and noninvasively assists early detection of pancreatic cancer according to the reduced level of the gelsolin content. The human plasma gelsolin standard product is diluted according to a certain concentration gradient and separated by SDS-PAGE with the total protein of the serum sample to be tested, and the separated protein is transferred from the gel to the PVDF membrane. The PVDF membrane containing the sample is reacted with the anti-human gelsolin antibody for immunoblotting, and the blot signal is subjected to chemiluminescence and amplified to expose the X-ray film. After scanning the blot results, with the assistance of image analysis software, draw a standard curve of the relationship between the gelsolin standard content and the blot intensity, and calculate the gelsolin content of the serum sample to be tested on the same gel within the linear range of the standard curve. The method is simple, fast, easy to be accepted by patients, and has high sensitivity and specificity for auxiliary diagnosis of pancreatic cancer.
Description
Technical field
The present invention relates to a kind of detection method of pancreatopathy cancer serum mark molecule, this method is present in gelsolin in the blood serum (gelsolin) content by detection by quantitative, non-invasively the early detection of auxiliary cancer of pancreas.
Background technology
Cancer of pancreas is a kind of dangerous alimentary system malignant tumour, morbidity concealment, poor prognosis.Its M ﹠ M is the trend of rising over nearly 10 years, report according to Chinese disease surveillance indication mechanism, mortality ratio to China's cancer of pancreas in 2000 rose to 3.26/10 ten thousand by 2.18/10 ten thousand in 1991, and cancer of pancreas is the 5-7 position common cancer of China because of cancer mortality.In the U.S., newly-increased cancer of pancreas patient 30,700 people in 2003 because of dead 30,000 people of cancer of pancreas, occupy the 4th of malignant tumour death.Cancer of pancreas does not generally have symptom in early days.When tumour internal organs infiltration towards periphery, during symptoms such as appearance abdominal distension, backache even jaundice, the infringement or the transfer of local vascular, internal organs or lymph node appearred in most patients, lost the chance of radical resection, and chemotherapy, radiotherapy can not prolong life very effectively to the control of cancer of pancreas.At present, 5 total annual survival rates of cancer of pancreas are 3-5% only, the median survival time after making a definite diagnosis＜6 month.(after diameter＜3cm) carried out radical excision, yet 5 annual survival rates can reach 40% to the little cancer of pancreas of, no lymphatic metastasis in the pancreas for being confined to, and median survival time extends to 32 months.Therefore can realize that early diagnosis to cancer of pancreas is the key point in the cancer of pancreas control.
Seek the special biological marker molecule of cancer of pancreas is the important content of cancer of pancreas research always.Use surface enhanced laser desorption ionization mass-spectrometric techniques such as Rosty have found to be called liver cancer-small intestine-pancreas/pancreatitis associated protein I from pancreatic juice.This protein content obviously raises in the pancreatic juice of Pancreas cancer patients, and the susceptibility of its auxiliary diagnosis of pancreatic cancer and specificity are respectively 75% and 87%.Have certain danger owing to gather human body pancreatic juice, this method is not suitable for the routine clinical inspection.Kind surplus the blood serum tumor markers that is used for the cancer of pancreas auxiliary diagnosis at present has 10, wherein the most frequently used CA19-9, the susceptibility and the specificity of diagnosis cancer of pancreas are about 70% and 60%.But,, in the crowd, have 5%-10%Lewis blood group antigens negative patient not secrete CA19-9 approximately, so the application of CA19-9 auxiliary diagnosis cancer of pancreas has limitation because CA19-9 is Lewis blood group antigens signs.Seek specificity and the high blood serum tumour correlating markings thing molecule of susceptibility, early examine, early control necessary cancer of pancreas.
Summary of the invention
The object of the present invention is to provide a kind of detection method of pancreatopathy cancer serum mark molecule, this method can be measured gelsolin content in the human serum, serum gelsolin content obviously reduction can be used as a serologic marker molecule, auxiliary non-invasively diagnosis of pancreatic cancer, the early detection cancer of pancreas.
The objective of the invention is to realize by the following technical solutions: a kind of detection method of pancreatopathy cancer serum mark molecule, detect gelsolin concentration in the human serum by quantitative immune marking method, according to the auxiliary non-invasively early detection cancer of pancreas of level that described gelsolin content reduces, its concrete steps are:
A, the preoperative serum of collection patient are made the test serum sample after use 1 * phosphate buffer dilutes 50 times;
B, with the human plasma gelsolin standard items of concentration known, separate through sodium dodecyl sulfate-polyacrylamide gel electrophoresis jointly with described test serum sample according to the finite concentration gradient, wherein, the described human plasma gelsolin standard items that contain 4 continuous concentration of swimming lane on the glue that every contains described test serum sample at least;
C, will be transferred on the polyvinylidene fluoride film from described glue through the protein that separates by electrotransfer, this film is after the skim milk sealing, carry out immunoblotting reaction with anti-people's gelsolin antibody, reaction signal is through chemiluminescence, amplification, and in the darkroom, the X-ray sheet is exposed, form gelsolin trace X-ray sheet as a result;
D, use scanner are to the X-ray sheet scanning as a result of gelsolin trace, according to known gelsolin standard items cont signal intensity drawing standard curve, and in the range of linearity of typical curve, calculate in the above test serum sample of same glue unknown gelsolin content with image analysis software.
The invention has the beneficial effects as follows the quantitative immuning engram method that detects gelsolin (gelsolin) content among the patients serum by setting up, accurately measure serum gelsolin content.This method is easy, quick, the patient is easy to accept, and auxiliary diagnosis of pancreatic cancer is had higher susceptibility and specificity, also provides a kind of new, Non-Invasive test mode for cancer of pancreas people at highest risk examination.
Description of drawings
The present invention will be further described below in conjunction with drawings and Examples.
The computing method of Fig. 1 serum gelsolin content
The calculating of Fig. 2 serum gelsolin average content
The drafting of Fig. 3 serum gelsolin typical curve
Embodiment
A kind of detection method of pancreatopathy cancer serum mark molecule, detect gelsolin (gelsolin) concentration in the human serum by quantitative immune marking method, according to the auxiliary non-invasively early detection cancer of pancreas of level that described gelsolin content reduces, its concrete steps are:
A, collection patients serum: with vacuum test tube (the Greiner-Bio One that does not contain any anti-coagulants, Frickenhausen Germany) gathers 4 milliliters in patient's fasting blood in early morning sample, under 4 ℃ through 3000 rev/mins, centrifugal 15 minutes, in separation of serum on ice.Serum is sub-packed in a plurality of 0.5 milliliter centrifuge tubes, changes-80 ℃ of refrigerators immediately and preserve after quick-frozen on the dry ice.From refrigerator, take out during use, after melting naturally, make the test serum sample after diluting 50 times with 1 * phosphate buffer (phosphate buffered saline is called for short PBS) on ice;
B, with 1 * PBS dilution human plasma gelsolin standard items to 5 mcg/ml, make the human plasma gelsolin standard items of concentration known;
With the human plasma gelsolin standard items of concentration known and test serum sample jointly through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, be called for short SDS-PAGE) separate: protein standard substance and test serum sample are in 2 * sds gel sample loading buffer, after 100 ℃ of heating were handled in 10 minutes, add the SDS-PAGE well.The standard gelsolin swimming lane that contains by 4 kinds of continuous progressive concentrations on every glue (is that every swimming lane applied sample amount is respectively 1 mcg/ml; 2 mcg/ml; 3 mcg/ml; 3.5 mcg/ml) and several 1: 50 the dilution patients serum's sample to be measured (sample 5 microlitres on every swimming lane).Gel places on the vertical protein electrophorese instrument (mini-protein III, Bio-Rad Laboratories), under 100-120 volt constant-pressure conditions the protein of standard items and testing sample is being separated on the same glue with under the identical experiment condition;
The albumen that will separate behind C, the electrophoresis changes film instrument (mini-transblot cell from gel by electricity, Bio-Rad Laboratories) under 4 ℃, 110 volts constant-pressure conditions, is transferred to polyvinylidene fluoride film (polyvinylidene difluoride, be called for short pvdf membrane), changeed film 1 hour;
The pvdf membrane that contains sample sealed 3 hours in 4 ℃ through 10% skim milk (pacifying happy high calcium skimmed milk power green for a long time) of 1 * PBS dilution.On film, carry out immunoblotting reaction with anti-people's gelsolin antibody: the mouse anti human gelsolin monoclonal antibody (BD Biosciences Pharmingen) incubated at room 2-3 hour that adds dilution in 1: 1000; Film washing liquid is washed film, 5 minutes/time, washes altogether 3 times; Goat anti-mouse igg (the Santa Cruz Biotechnology) incubated at room 1 hour that adds the horseradish peroxidase-labeled of dilution in 1: 3000; Film washing liquid is washed film, 5 minutes/time, washes altogether 5 times; Add chemical illuminating reagent ECL (Santa CruzBiotechnology) and amplify gelsolin trace signal, (every film selects 3 kinds of different exposure time at least to X-ray sheet (Kodak X-Omat V.Film) exposure in the darkroom, exposure is more than 3 times), after development, photographic fixing, form gelsolin trace X-ray sheet as a result, see the A portion among Fig. 1.The gelsolin standard items (having continuous 4 variable concentrations on the glue at least) that demonstrate gradient dilution among the figure make Separation of Proteins with the test serum sample on same glue, and do the specific immunity engram analysis with gelsolin antibody.The result shows that the gelsolin molecular weight is 93kDa in the serum, and N113, N115 and N116 are the normal serum sample; P16, P57 and P118 are the pancreatopathy cancer serum sample.With transferrins (transferrin, molecular weight the are 80kDa) content of constant relatively expression in the human serum internal reference as the serum sample applied sample amount.
D, usefulness scanner (N-TEK NuScan 900) scanning gelsolin trace be the X-ray sheet as a result, uses image analysis software Quantity One 4.4.1 (Bio-Rad Laboratories) according to known gelsolin standard items cont signal intensity drawing standard curve.Have only when the standard items of the known content Western blot intensity corresponding with it is linear distribution (r＞0.94), otherwise can draw the typical curve of linear relationship between the two, if r≤0.94, the expression typical curve is insincere.Further in the range of linearity of typical curve, the Western blot intensity of test serum converses gelsolin content on the same glue of quantitative test.
Referring to shown in Figure 2, in order to reduce owing to the experimental error of time shutter difference to linear relationship influence between protein content and trace intensity, calculate gelsolin content under three different exposure time respectively, and average as the final content of gelsolin in the sample to be tested.
Referring to the B portion among Fig. 1, show protein content and corresponding trace signal intensity thereof among the figure according to the gelsolin standard items, typical curve with the drafting of Quantity One 4.4.1 software, and, in the range of linearity of typical curve, calculate gelsolin content in the test serum sample according to test serum sample gelsolin Western blotting signal intensity.Horizontal ordinate is Western blot intensity (pixel/square millimeter), and ordinate is protein concentration (mcg/ml).Std1-Std4 is the gelsolin standard items, and N113, N115 and N116 are the normal serum sample; P16, P57 and P118 are the pancreatopathy cancer serum sample.
Fig. 2 shows the computing method of gelsolin average content in the serum.A portion among the figure, B portion, C portion, D portion, E portion and F portion represent experimental result six times.Because the difference of time shutter has certain influence to the linear relationship between protein content and the Western blot intensity, in order to reduce experimental error, each experiment is got three kinds of different exposure time to X-ray sheet exposure (time shutter 1,2,3), calculate the average content of the serum sample gelsolin of three different exposure time respectively, as the final content of gelsolin in the testing sample.
The result judges: the content of gelsolin reduces in the test serum sample, when being lower than 191.15mg/L, points out this patient may suffer from cancer of pancreas, needs hig diligence.
In the present embodiment, need provide the data of the human plasma gelsolin of a concentration known as standard items, these data produce as follows:
With the human plasma gelsolin standard items of concentration known (1 mg/ml) (available from Cytoskeleton company, U.S. Denver, CO) be diluted to 5 mcg/ml with 1 * PBS, and increase progressively application of sample in gradient, make every swimming lane standard items applied sample amount be respectively 1 mcg/ml, 1.5 mcg/ml, 2 mcg/ml, 2.5 mcg/ml, 3 mcg/ml and 3.5 mcg/ml.The standard items point that in each batch experiment, contains 4 continuous concentration at least, the drawing standard curve.
Test serum (sample 5 microlitres on the every swimming lane) sample of human plasma gelsolin standard items and several 1: 50 dilution in 2 * sds gel sample loading buffer through 100 ℃ of heating after 10 minutes, the SDS-PAGE gel places vertical protein electrophorese instrument (mini-protein III, Bio-Rad Laboratories) on, the protein that makes standard items and sample to be tested under the 100-120 volt constant-pressure conditions on the same glue and under the identical experiment condition by the molecular weight size separation;
Change film instrument (mini-transblot cell, Bio-Rad Laboratories) under 4 ℃, 110 volts constant-pressure conditions by electricity, the albumen behind the electrophoresis is transferred to pvdf membrane (changeing film 1 hour) from gel.The pvdf membrane that contains sample sealed 3 hours in 4 ℃ through 10% skim milk (pacifying happy high calcium skimmed milk power green for a long time) of 1 * PBS dilution.The mouse anti human gelsolin monoclonal antibody (BD BiosciencesPharmingen) incubated at room 2-3 hour that adds dilution in 1: 1000; Film washing liquid is washed film, 5 minutes/time, washes altogether 3 times; Goat anti-mouse igg (the Santa Cruz Biotechnology) incubated at room 1 hour that adds the horseradish peroxidase-labeled of dilution in 1: 3000; Film washing liquid is washed film, 5 minutes/time, washes altogether 5 times; Add chemical illuminating reagent ECL (SantaCruz Biotechnology) and amplify gelsolin trace signal, (every film selects 3 kinds of different exposure time at least to X-ray sheet (KodakX-Omat K Film) exposure in the darkroom, exposure is more than 3 times), after development, photographic fixing, form gelsolin trace X-ray sheet as a result, referring to the A portion among Fig. 3.
Gelsolin trace result uses image analysis software Quantity One 4.4.1 (Bio-Rad Laboratories) according to known gelsolin standard items content and corresponding trace signal intensity drawing standard curve through scanner (N-TEK NuScan 900) scanning.Have only when the standard items of the known content Western blot intensity corresponding with it is linear distribution (r＞0.94), can draw the typical curve of linear relationship between the two.
A portion among Fig. 3 shows the Western blotting result after the gelsolin standard items increase progressively application of sample in gradient.Every swimming lane standard items applied sample amount is respectively 1 mcg/ml, 1.5 mcg/ml, 2 mcg/ml, 2.5 mcg/ml, 3 mcg/ml and 3.5 mcg/ml etc.; B portion among Fig. 3 show Western blot intensity that the protein concentration that uses Quantity One 4.4.1 software to draw the gelsolin standard items is corresponding with it typical curve (r=0.996).Horizontal ordinate is a Western blot intensity, i.e. mean pixel number (pixel/mm in every square millimeter of area
2), ordinate is a protein concentration, the proteinaceous micrograms of promptly every ml volumes (μ g/ml).
Used liquid formulations is as follows in the present embodiment:
1、1×PBS(pH7.4)：137mM?NaCl，2.68mM?KCl，10mM?Na
2HPO
4，1.76mM?KH
2PO
4。
2,2 * sds gel sample loading buffer: 100mM Tris-HCl, pH6.8,200mM DTT, 4%SDS, 0.2% bromophenol blue, 20% glycerine.
3, Tris-glycocoll electrophoretic buffer: 25mM Tris, 250mM glycocoll, pH8.3,0.1%SDS.
4, change film liquid: 25mM Tris, 192mM glycocoll, 20% methyl alcohol, pH8.3.
5, film washing liquid: 20mM Tris-HCl, pH7.5,200mM NaCl, 0.1%Tween-20.
The present invention sets up and detects pancreatopathy cancer serum marker molecule---the quantitative immuning engram method of gelsolin content new among the patients serum, and it is significant to the early detection cancer of pancreas accurately to measure serum gelsolin content.Detected the expression of blood plasma type gelsolin (gelsolin) in 65 routine ductal adenocarcinoma of pancreas, 34 other malignant tumours of routine digestive system (comprising the cancer of the esophagus 6 examples, cancer of the stomach 6 examples, colorectal cancer 5 examples, 12 carcinoma of the rectum, 5 examples, carcinoma of gallbladder 4 examples, cholangiocarcinoma 3 examples and islet-cell tumour 5 examples), 11 routine pancreas benign diseases (chronic pancreatitis 5 examples, false cyst 3 example and optimum adenoma 3 examples) and 54 routine normal human serums by the quantitative immuning engram method.The average content of gelsolin is 155.24 ± 44.29mg/L among the cancer of pancreas patients serum, significantly is lower than normal human serum gelsolin average content 224.44 ± 42.92mg/L; Other malignant tumor patient average serum gelsolin content of digestive system is 212.10 ± 60.35mg/L; Pancreas benign disease patients serum gelsolin content is 203.74 ± 26.70mg/L (P＜0.001).And serum gelsolin content is not subjected to other composition in the serum, and is as the influence of albumin, cholerythrin etc., more stable.Experimenter's operating characteristic (receiveroperating characteristic, ROC) curve display, with serum gelsolin concentration≤191.15mg/L is standard, susceptibility with serum gelsolin content detection cancer of pancreas is 78.5%, specificity is 72.7%, can be used as a serology auxiliary diagnosis marker molecule of cancer of pancreas.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009021360A1 (en) * | 2007-08-15 | 2009-02-19 | Mountgate Group Limited | Gelsolin binding agent compositions and uses of same |
| CN102798721A (en) * | 2012-05-28 | 2012-11-28 | 上虞常春生物技术有限公司 | Food allergen detection kit and food allergen detection method |
| CN101460849B (en) * | 2006-04-18 | 2013-02-13 | 株式会社英仙蛋白质科学 | Diagnostic agent and therapeutic agent for pancreatic cancer |
| CN106153917A (en) * | 2015-04-10 | 2016-11-23 | 中国医学科学院肿瘤医院 | Two groups of cancer of pancreas blood plasma diagnosis marker spectrums and application thereof |
| CN113917149A (en) * | 2021-09-30 | 2022-01-11 | 江苏扬新生物医药有限公司 | Application of gelsolin detection substance in preparation of uterine cancer evaluation detection reagent |
| CN115812151A (en) * | 2020-07-16 | 2023-03-17 | 免疫媒介有限公司 | Methods, arrays and their uses |
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2004
- 2004-09-08 CN CN 200410074306 patent/CN1746676A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101460849B (en) * | 2006-04-18 | 2013-02-13 | 株式会社英仙蛋白质科学 | Diagnostic agent and therapeutic agent for pancreatic cancer |
| WO2009021360A1 (en) * | 2007-08-15 | 2009-02-19 | Mountgate Group Limited | Gelsolin binding agent compositions and uses of same |
| WO2009024070A1 (en) * | 2007-08-15 | 2009-02-26 | Mountgate Group Limited | Detection and quantitation of urine gelsolin |
| US20110207152A1 (en) * | 2007-08-15 | 2011-08-25 | Enyun Shen | Gelsolin binding agent compositions and uses of same |
| US8367356B2 (en) * | 2007-08-15 | 2013-02-05 | Beijing Cotimes Biotech Co., Ltd. | Gelsolin binding agent compositions and uses of same |
| CN102798721A (en) * | 2012-05-28 | 2012-11-28 | 上虞常春生物技术有限公司 | Food allergen detection kit and food allergen detection method |
| CN106153917A (en) * | 2015-04-10 | 2016-11-23 | 中国医学科学院肿瘤医院 | Two groups of cancer of pancreas blood plasma diagnosis marker spectrums and application thereof |
| CN106153917B (en) * | 2015-04-10 | 2018-04-13 | 中国医学科学院肿瘤医院 | Two groups of cancer of pancreas blood plasma diagnosis marker spectrums and its application |
| CN115812151A (en) * | 2020-07-16 | 2023-03-17 | 免疫媒介有限公司 | Methods, arrays and their uses |
| CN113917149A (en) * | 2021-09-30 | 2022-01-11 | 江苏扬新生物医药有限公司 | Application of gelsolin detection substance in preparation of uterine cancer evaluation detection reagent |
| CN113917149B (en) * | 2021-09-30 | 2024-05-24 | 江苏扬新生物医药有限公司 | Application of gelsolin detector in preparation of uterine cancer assessment detection reagent |
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