CN105467118A - Pancreatic cancer tumor marker and application method thereof - Google Patents
Pancreatic cancer tumor marker and application method thereof Download PDFInfo
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- CN105467118A CN105467118A CN201510539585.9A CN201510539585A CN105467118A CN 105467118 A CN105467118 A CN 105467118A CN 201510539585 A CN201510539585 A CN 201510539585A CN 105467118 A CN105467118 A CN 105467118A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 206010061902 Pancreatic neoplasm Diseases 0.000 title abstract description 14
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 title abstract description 14
- 208000008443 pancreatic carcinoma Diseases 0.000 title abstract description 9
- 201000002528 pancreatic cancer Diseases 0.000 title abstract description 8
- 239000000439 tumor marker Substances 0.000 title abstract 3
- 101000868967 Homo sapiens Corticosteroid-binding globulin Proteins 0.000 claims abstract description 63
- 102100032323 Corticosteroid-binding globulin Human genes 0.000 claims abstract description 51
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 15
- 238000008157 ELISA kit Methods 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 25
- 238000001514 detection method Methods 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 18
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims description 12
- 238000002965 ELISA Methods 0.000 claims description 12
- 239000012895 dilution Substances 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 12
- 102000049531 human SERPINA6 Human genes 0.000 claims description 12
- 238000013016 damping Methods 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000012160 loading buffer Substances 0.000 claims description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 102000008847 Serpin Human genes 0.000 claims description 3
- 108050000761 Serpin Proteins 0.000 claims description 3
- 108010011095 Transcortin Proteins 0.000 claims description 3
- 102000014034 Transcortin Human genes 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 230000003118 histopathologic effect Effects 0.000 claims description 2
- 229940005654 nitrite ion Drugs 0.000 claims description 2
- 210000005259 peripheral blood Anatomy 0.000 abstract description 9
- 239000011886 peripheral blood Substances 0.000 abstract description 9
- 230000001575 pathological effect Effects 0.000 abstract description 4
- 210000001519 tissue Anatomy 0.000 description 7
- 239000003862 glucocorticoid Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 206010052747 Adenocarcinoma pancreas Diseases 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 201000002094 pancreatic adenocarcinoma Diseases 0.000 description 2
- 210000004923 pancreatic tissue Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- -1 rare earth ions Chemical class 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000000407 Pancreatic Cyst Diseases 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
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Abstract
The invention relates to a pancreatic cancer tumor marker and an application method thereof. The pancreatic cancer tumor marker is SERPINA6. The SERPINA6 highly expresses in pancreatic cancer tumors, and is secreted to peripheral blood. The pancreatic cancer can be rapidly found and diagnosed through detecting the SERPINA6 expression level in pancreatic cancer pathological tissues or the concentration of the SERPINA6 in the peripheral blood. The invention also discloses a use method of an ELISA kit and a time-resolved fluorescent immunometric kit for detecting the SERPINA6.
Description
Technical field
The invention belongs to medical biotechnology research field, relate to a kind of pancreatic tumour mark and application process thereof.Shown pancreatic tumour mark is SERPINA6.SERPINA6 presents high expressed in pancreatic tumour tissue, and secretion is in peripheral blood.By detecting the SERPINA6 expression in cancer of pancreas pathological tissue or the SERPINA6 concentration in Peripheral Blood, can find rapidly and diagnosis of pancreatic cancer.
Background technology
Cancer of pancreas is the high tumour of a kind of high aggressive, grade malignancy, and the incidence of disease increases gradually in recent years.The complex treatment that treatment is mainly operation, chemotherapy and radiation combines of cancer of pancreas, its five year survival rate is less than 5%.Therefore, the Treatment and Prognosis of early diagnosis to cancer of pancreas seems particularly important, and wherein, it is diagnostic method that is the most frequently used, that the most easily promote that peripheral blood detects.Current clinical conventional diagnosis of pancreatic cancer mark as CA19-9, diagnosing tumour generation and monitor its recurrence in play important effect.But the specificity of these marks and susceptibility are often very low, can not meet clinical needs, especially in early days diagnostic value aspect, existing still lack a comparatively sensitive method.
SERPINA6 is one of member of serpin family, and also referred to as corticosteroid-binding globulin, it is main glucocorticoid transport protein.Serpin SERPINA6 is controlling to play key effect in the proteinase activity in different bioprocess.Combine more than 90% glucocorticoid and SERPINA6 in peripheral blood, after the carboxyl terminal of SERPINA6 is hydrolyzed, causes its conformation to change, thus promote glucocorticoid be released into tissue.Therefore, SERPINA6 can directly guide glucocorticoid to be assigned to the tissue site such as inflammation, tumour, plays an important role in physiology and disease occur.
By analyzing 40 routine Pancreatic Adenocarcinoma pathological sections, find that SERPINA6 presents high expressed in Pancreatic Adenocarcinoma, and periphery normal pancreatic tissue has no positive expression, also carry out the pancreatic tissue pathological immune histochemical staining of a routine liver cancer pancreas transfer in addition, had no its tumor group and be woven with positive staining.
In addition, operate time resolved immuno fluorometric detection method, during human peripheral blood is clear, SERPINA6 detects, and finds that the SERPINA6 concentration in Pancreas cancer patients peripheral blood is significantly higher than normal person, pancreatitis and pancreatic cyst patient.
Summary of the invention
The object of this invention is to provide a kind of pancreatic tumour mark, it is SERPINA6, and also referred to as corticosteroid-binding globulin, it is one of member of serpin family.SERPINA6 not only expresses and obviously increases in pancreatic tumour tissue, and as secreted protein, SERPINA6 can be secreted in peripheral blood, thus is detected in patients serum.
Invention also provides the application process that two kinds of SERPINA6 detect.A kind of is that SERPINA6 SABC in histopathologic slide detects, and another kind is the immunology detection of SERPINA6 in serum, and as ELISA, Time-resolved Fluoimmunoassay, chemoluminescence method, puts the method for exempting from.
The invention provides a kind of using method detecting the ELISA kit of SERPINA6.In addition, present invention also offers a kind of using method detecting the time resolution immunofluorescent reagent box of SERPINA6, this kit adopts time resolution immunofluorescence method, and having high special, high-sensitive advantage, is a kind of quantitative detecting method replacing radioimmunoassay at present.Its ultimate principle utilizes trivalent rare earth ions (as Eu
3+) as tracer, postpone to detect life-span longer specificity fluorescence excitation to get rid of the interference of non-specific and background fluorescence, namely measure the delayed fluorescence intensity that the rare earth ion in compound is excited, thus determine the concentration of testing protein.Use the TRF of fluorescence enhancement solution pik (pg, 10 can be detected
-12g/ml) albumen of level.
A kind of using method detecting the ELISA kit of SERPINA6.It is characterized in that: the ELISA kit of described detection SERPINA6 comprises following composition
(1) cleansing solution is the PBS containing 0.1%Tween-20;
(2) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) reaction terminating liquid is 2MH
2sO
4;
(4) be coated on the monoclonal mouse anti human SERPINA6 antibody in ELISA Plate, wrapping by concentration is 5 μ g/ml;
(5) detecting antibody is that the anti-human SERPINA6 of biotinylated rabbit resists more, and storing concentration is 50ng/ μ l;
(6) the affine mycin of the chain of horseradish peroxidase-labeled, storing concentration is 0.5 μ g/ μ l;
(7) standard items, recombined human SERPINA6 albumen, concentration is 100 μ g/ μ l;
Described using method is specially following steps
(1) in the ELISA Plate being coated with monoclonal mouse anti human SERPINA6 antibody, add the standard items 100 μ l/ hole of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 DEG C;
(2) discard sample and standard items, 300 μ l/ hole cleansing solutions wash three times;
(3) resist to concentration with the anti-human SERPINA6 of rabbit of sample loading buffer dilution detection antibody-biotin to be 75ng/ml, to add with 100 μ l/ holes the detection antibody diluted, hatch 1h for 37 DEG C more;
(4) discard detection antibody, 300 μ l/ hole cleansing solutions wash three times;
(5) with the affine mycin of chain of sample loading buffer dilution horseradish peroxidase-labeled, final concentration is 0.5 μ g/ml, and add ELISA Plate with 100 μ l/ holes, 37 DEG C of lucifuges hatch 0.5h;
(6) discard the affine mycin of chain of horseradish peroxidase-labeled, 300 μ l/ hole cleansing solutions wash four times;
(7) add TMB nitrite ion, room temperature lucifuge hatches 15min;
(8) in microplate reader, OD450nm value is read;
(9) utilize statistics mapping software according to the value drawing standard curve measured;
(10) concentration of the SERPINA6 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
Detect the using method of the time resolution immunofluorescent reagent box of SERPINA6, it is characterized in that: the time resolution immunofluorescent reagent box of described detection SERPINA6 comprises following composition
(1) bag is buffered liquid is PBS, and its composition is 140mMNaCl, 2.7mMKCl, 10mMNa
2hPO
4, 1.8mMKH
2pO
4, pH value is 7.4;
(2) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid containing 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH7.8) of 50mM, 0.05%Tween-20;
(4) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(5) fluorescence enhancement solution;
(6) the Tris-HCl damping fluid (pH7.8) that damping fluid is 50mM is detected, wherein containing 1%BSA, 1%bovineglobulin, 0.05%Tween40, diethylene triamine pentacetic acid (DTPA);
(7) coated antibody is monoclonal mouse anti human SERPINA6 antibody, and storing concentration is 1 μ g/ μ l;
(8) detecting antibody is that the anti-human SERPINA6 of biotinylated rabbit resists more, and storing concentration is 50ng/ μ l;
(9) Eu
3+the affine mycin of chain of mark, storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombined human SERPINA6 albumen, concentration is 100 μ g/ μ l;
Described using method is specially following steps
(1) diluting coated antibody-monoclonal mouse anti human SERPINA6 antibody to concentration with PBS is 4ug/ml, and add 96 hole ELISA Plate with 100 μ l/ holes, 4 DEG C are spent the night;
(2) discard coated antibody, 300 μ l/ hole cleansing solutions wash three times, add confining liquid 200 μ l/ hole, hatch 1h for 37 DEG C;
(3) discard confining liquid, 300 μ l/ hole cleansing solutions wash three times, add the standard items 100 μ l/ hole of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 DEG C;
(4) discard sample and standard items, 300 μ l/ hole cleansing solutions wash three times;
(5) resist to concentration with the anti-human SERPINA6 of rabbit of sample loading buffer dilution detection antibody-biotin to be 75ng/ml, to add with 100 μ l/ holes the detection antibody diluted, hatch 1h for 37 DEG C more;
(6) discard detection antibody, 300 μ l/ hole cleansing solutions wash three times;
(7) with detecting damping fluid dilution Eu
3+the affine mycin of chain of mark, final concentration is 500ng/ml, and add ELISA Plate with 100 μ l/ holes, 37 DEG C of lucifuges hatch 1h;
(8) Eu is discarded
3+the affine mycin of chain of mark, 300 μ l/ hole cleansing solutions wash four times
(9) fluorescence enhancement solution 200 μ l/ hole is added, room temperature lucifuge slow level shake 5min;
(10) read plate: setting excitation wavelength is 337nm, wavelength of transmitted light is 615nm, and time delay 200 microsecond measures the fluorescent value at 615nm place;
(11) utilize statistics mapping software according to the value drawing standard curve measured;
(12) concentration of the SERPINA6 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
Accompanying drawing explanation
Fig. 1. the SERPINA6 obviously raised in pancreatic tumour tissue expresses.
Fig. 2. in cancer of pancreas surrounding normal tissue, SERPINA6 has no positive expression.
Claims (4)
1. a pancreatic tumour mark, it is SERPINA6, and also referred to as corticosteroid-binding globulin, it is one of member of serpin family.
2.SERPINA6 is as a kind of pancreatic tumour mark, and its application process comprises two kinds: a kind of is that SERPINA6 SABC in histopathologic slide detects, and another kind is the SERPINA6 immunology detection in serum, as ELISA, Time-resolved Fluoimmunoassay, chemoluminescence method, puts the method for exempting from.
3. according to claim 2, detect the using method of the ELISA kit of SERPINA6, it is characterized in that: the ELISA kit of described detection SERPINA6 comprises following composition
(1) cleansing solution is the PBS containing 0.1%Tween-20;
(2) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) reaction terminating liquid is 2MH
2sO
4;
(4) be coated on the monoclonal mouse anti human SERPINA6 antibody in ELISA Plate, wrapping by concentration is 5 μ g/ml;
(5) detecting antibody is that the anti-human SERPINA6 of biotinylated rabbit resists more, and storing concentration is 50ng/ μ l;
(6) the affine mycin of the chain of horseradish peroxidase-labeled, storing concentration is 0.5 μ g/ μ l;
(7) standard items, recombined human SERPINA6 albumen, concentration is 100 μ g/ μ l;
Described using method is specially following steps
(1) in the ELISA Plate being coated with monoclonal mouse anti human SERPINA6 antibody, add the standard items 100 μ l/ hole of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 DEG C;
(2) discard sample and standard items, 300 μ l/ hole cleansing solutions wash three times;
(3) resist to concentration with the anti-human SERPINA6 of rabbit of sample loading buffer dilution detection antibody-biotin to be 75ng/ml, to add with 100 μ l/ holes the detection antibody diluted, hatch 1h for 37 DEG C more;
(4) discard detection antibody, 300 μ l/ hole cleansing solutions wash three times;
(5) with the affine mycin of chain of sample loading buffer dilution horseradish peroxidase-labeled, final concentration is 0.5 μ g/ml, and add ELISA Plate with 100 μ l/ holes, 37 DEG C of lucifuges hatch 0.5h;
(6) discard the affine mycin of chain of horseradish peroxidase-labeled, 300 μ l/ hole cleansing solutions wash four times;
(7) add TMB nitrite ion, room temperature lucifuge hatches 15min;
(8) in microplate reader, OD450nm value is read;
(9) utilize statistics mapping software according to the value drawing standard curve measured;
(10) concentration of the SERPINA6 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
4. according to claim 2, detect the using method of the time resolution immunofluorescent reagent box of SERPINA6, it is characterized in that: the time resolution immunofluorescent reagent box of described detection SERPINA6 comprises following composition
(1) bag is buffered liquid is PBS, and its composition is 140mMNaCl, 2.7mMKCl, 10mMNa
2hPO
4, 1.8mMKH
2pO
4, pH value is 7.4;
(2) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid containing 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH7.8) of 50mM, 0.05%Tween-20;
(4) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(5) fluorescence enhancement solution;
(6) the Tris-HCl damping fluid (pH7.8) that damping fluid is 50mM is detected, wherein containing 1%BSA, 1%bovineglobulin, 0.05%Tween40, diethylene triamine pentacetic acid (DTPA);
(7) coated antibody is monoclonal mouse anti human SERPINA6 antibody, and storing concentration is 1 μ g/ μ l;
(8) detecting antibody is that the anti-human SERPINA6 of biotinylated rabbit resists more, and storing concentration is 50ng/ μ l;
(9) Eu
3+the affine mycin of chain of mark, storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombined human SERPINA6 albumen, concentration is 100 μ g/ μ l;
Described using method is specially following steps
(1) diluting coated antibody-monoclonal mouse anti human SERPINA6 antibody to concentration with PBS is 4ug/ml, and add 96 hole ELISA Plate with 100 μ l/ holes, 4 DEG C are spent the night;
(2) discard coated antibody, 300 μ l/ hole cleansing solutions wash three times, add confining liquid 200 μ l/ hole, hatch 1h for 37 DEG C;
(3) discard confining liquid, 300 μ l/ hole cleansing solutions wash three times, add the standard items 100 μ l/ hole of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 DEG C;
(4) discard sample and standard items, 300 μ l/ hole cleansing solutions wash three times;
(5) resist to concentration with the anti-human SERPINA6 of rabbit of sample loading buffer dilution detection antibody-biotin to be 75ng/ml, to add with 100 μ l/ holes the detection antibody diluted, hatch 1h for 37 DEG C more;
(6) discard detection antibody, 300 μ l/ hole cleansing solutions wash three times;
(7) with detecting damping fluid dilution Eu
3+the affine mycin of chain of mark, final concentration is 500ng/ml, and add ELISA Plate with 100 μ l/ holes, 37 DEG C of lucifuges hatch 1h;
(8) Eu is discarded
3+the affine mycin of chain of mark, 300 μ l/ hole cleansing solutions wash four times
(9) fluorescence enhancement solution 200 μ l/ hole is added, room temperature lucifuge slow level shake 5min;
(10) read plate: setting excitation wavelength is 337nm, wavelength of transmitted light is 615nm, and time delay 200 microsecond measures the fluorescent value at 615nm place;
(11) utilize statistics mapping software according to the value drawing standard curve measured;
(12) concentration of the SERPINA6 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106908600A (en) * | 2017-03-20 | 2017-06-30 | 申冬昌 | One kind is used for detecting system lupus erythematosus（SLE kit) |
| CN106950374A (en) * | 2017-04-10 | 2017-07-14 | 南通大学附属医院 | Application of the albumen of Glypican 1 in diagnosis of pancreatic cancer, the detection method of positive excretion bulk concentration and application thereof |
| CN107033235A (en) * | 2017-06-14 | 2017-08-11 | 郑州大学第附属医院 | A kind of application of people tCD109 and its detection kit in diagnosis of pancreatic cancer |
| CN108929914A (en) * | 2018-08-30 | 2018-12-04 | 南通大学附属医院 | A kind of pancreatic cancer marker and its detection method |
| CN109856259A (en) * | 2018-12-29 | 2019-06-07 | 南通大学附属医院 | A kind of serum cancer of pancreas excretion body differential protein and its again verification process |
| WO2022075774A1 (en) * | 2020-10-08 | 2022-04-14 | 가톨릭대학교 산학협력단 | Biomarker for predicting metastasis of pancreatic cancer and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| WO2013152989A2 (en) * | 2012-04-10 | 2013-10-17 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
-
2015
- 2015-08-30 CN CN201510539585.9A patent/CN105467118B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| WO2013152989A2 (en) * | 2012-04-10 | 2013-10-17 | Eth Zurich | Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer |
Non-Patent Citations (5)
| Title |
|---|
| BRIAN GREENWAY ET AL.: "Oestrogen receptor proteins in malignant and fetal pancreas", 《BRITISH MEDICAL JOURNAL》 * |
| MULLER CA ET AL.: "Corticosteroid-binding globulin: a possible early predictor of infection in acute necrotizing pancreatitis", 《SCAND J GASTROENTEROL》 * |
| 张兵等: "丝氨酸蛋白酶抑制剂的研究进展", 《沈阳药科大学学报》 * |
| 邵晨等: "皮质醇结合球蛋白的生理功能及其编码基因突变", 《中国微生态学杂志》 * |
| 陈素娟等: "人皮质类固醇结合球蛋白放射免疫分析法", 《国外医学•内分泌学分册》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106908600A (en) * | 2017-03-20 | 2017-06-30 | 申冬昌 | One kind is used for detecting system lupus erythematosus（SLE kit) |
| CN106950374A (en) * | 2017-04-10 | 2017-07-14 | 南通大学附属医院 | Application of the albumen of Glypican 1 in diagnosis of pancreatic cancer, the detection method of positive excretion bulk concentration and application thereof |
| CN107033235A (en) * | 2017-06-14 | 2017-08-11 | 郑州大学第附属医院 | A kind of application of people tCD109 and its detection kit in diagnosis of pancreatic cancer |
| CN107033235B (en) * | 2017-06-14 | 2020-04-07 | 郑州大学第一附属医院 | Application of human tCD109 and detection kit thereof in pancreatic cancer diagnosis |
| CN108929914A (en) * | 2018-08-30 | 2018-12-04 | 南通大学附属医院 | A kind of pancreatic cancer marker and its detection method |
| CN109856259A (en) * | 2018-12-29 | 2019-06-07 | 南通大学附属医院 | A kind of serum cancer of pancreas excretion body differential protein and its again verification process |
| WO2022075774A1 (en) * | 2020-10-08 | 2022-04-14 | 가톨릭대학교 산학협력단 | Biomarker for predicting metastasis of pancreatic cancer and use thereof |
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