CN109324186B - Excretion body protein CD82, GPC1 combine CA19-9 for cancer of pancreas early diagnosis and curative effect monitoring - Google Patents
Excretion body protein CD82, GPC1 combine CA19-9 for cancer of pancreas early diagnosis and curative effect monitoring Download PDFInfo
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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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention relates to technical field of biomedical detection, specifically a kind of multi objective joint-detection, detection kit and detection method for cancer of pancreas early diagnosis and curative effect monitoring.The index includes excretion body CD82 expressing quantity, excretion body GPC1 expressing quantity and CA19-9 content.The present invention combines newest excretion body protein marker and detects, and compensates for the single lower defect of Indexs measure sensibility and specificity of CA19-9, keeps diagnostic result more accurate effective.This method provides new method for cancer of pancreas early diagnosis and curative effect monitor and detection, to realize cancer of pancreas early diagnosis, early treatment, improves diagnosis and treatment efficiency, extends patient survival and improvement prognosis lays the foundation.
Description
Technical field
Invention is related to technical field of biomedical detection, specifically, being a kind of by Exo-CD82+(excretion body CD82 egg
White expression quantity), Exo-GPC1+(excretion body GPC1 expressing quantity) and CA19-9, detection be used in combination, for cancer of pancreas early stage
Diagnosis and curative effect monitor and detection kit and its detection method.
Background technique
Cancer of pancreas is one of most common malignant tumour in the whole world, has high lethality, survival rate is only 3% within 5 years.
In the past 40 years, the survival rate of Pancreas cancer patients is not improved.And China's pancreas cancer morbidity is in rising trend, falls ill within 2015
Number about 9.01 ten thousand people.Since cancer of pancreas lacks specific tumour marker, only relies on traditional iconography means and be difficult to reality
The early diagnosis of existing cancer of pancreas and large-scale Mass screening directly result in 80% Pancreas cancer patients and have occurred to turn when making a definite diagnosis
It moves, wherein only 15% patient is able to carry out operative treatment, and Postoperative recurrent rate is high.Therefore, it is early to be suitable for clinical cancer of pancreas
Phase diagnosis and curative effect monitor and detection method are urgently developed.
Currently, diagnosis of pancreatic cancer relies primarily on imageological examination, CA19-9 can not represent the cancer of pancreas state of an illness completely；Exo-
GPC1+ also only exists in conceptual phase for cancer of pancreas early diagnosis.And it early find early treatment this improves patient's prognosis and clinical
Therapeutic effect raising is most important, and cancer of pancreas early diagnosis and curative effect monitoring can provide individualized treatment scheme in time for patient and mention
For decision-making foundation, patient survival is directly affected.Therefore there is an urgent need to one kind in diagnosis of pancreatic cancer treatment can be early
Phase detects the detection technique of cancer of pancreas or prognosis.
Summary of the invention
It is an object of the invention to effectively carry out cancer of pancreas early diagnosis and curative effect monitor and detection, provide a species specificity and
The all fabulous cancer of pancreas early diagnosis of sensibility and curative effect monitor and detection kit and its detection method.
The first aspect of the present invention provides Exo-CD82+(excretion body CD82 expressing quantity) joint CA19-9 or its
Detection reagent or Exo-CD82+(excretion body CD82 expressing quantity) joint Exo-GPC1+(excretion body GPC1 protein expression
Amount) and CA19-9 or its detection reagent purposes, be used to prepare diagnosis of pancreatic cancer and/or curative effect monitor and detection kit.
In another preferred example, the diagnosis includes prejudging.
In another preferred example, described to be diagnosed as early diagnosing.
In another preferred example, the detection sample of the detection kit includes blood sample.
In another preferred example, the blood sample is selected from the group: blood, blood plasma, serum or combinations thereof.
In another preferred example, the blood sample is pretreated blood sample.
In another preferred example, the blood sample is the blood plasma after being centrifuged.
In another preferred example, the pretreatment obtains plasma sample comprising steps of separate to whole blood.
In another preferred example, the plasma sample is centrifugated after being collected by EDTA heparin tube or heparin sodium heparin tube.
In another preferred example, the detection sample of the detection kit is peripheral blood sample.
In another preferred example, the detection sample of the detection kit is serum sample.
In another preferred example, the detection reagent includes: to detect the reagent of CA19-9 content, is detected outside Exo-GPC1+(
Secrete body GPC1 expressing quantity) reagent, detect Exo-CD82+(excretion body CD82 expressing quantity) reagent.
In another preferred example, the detection reagent includes: to detect the reagent of CA19-9 content, is detected outside Exo-GPC1+(
Secrete body GPC1 expressing quantity) reagent, detect Exo-CD82+(excretion body CD82 expressing quantity) reagent, BCA detect egg
White Concentration Reagent box.
In another preferred example, the detection kit by detection pancreatic disease patients serum in CA19-9 content and
Excretion body CD82 expressing quantity, or pass through CA19-9 content, excretion body GPC1 albumen table in detection pancreatic disease patients serum
Up to amount and excretion body CD82 expressing quantity, it to be used for diagnosis of pancreatic cancer and/or curative effect monitor and detection.
In another preferred example, the detection kit by detection pancreatic disease patients serum in CA19-9 content,
Exo-GPC1+(excretion body GPC1 expressing quantity) and Exo-CD82+(excretion body CD82 expressing quantity), it is examined for cancer of pancreas
Disconnected and/or curative effect monitor and detection.
In another preferred example, the detection kit by detection pancreatic disease patients serum in CA19-9 content and
Exo-CD82+ is used for diagnosis of pancreatic cancer and/or curative effect monitor and detection.
In another preferred example, the detection kit by detection pancreatic disease patients serum in CA19-9 content and
Exo-GPC1+ is used for diagnosis of pancreatic cancer and/or curative effect monitor and detection.
In another preferred example, the risk for being diagnosed as assessment patient's cancer of pancreas illness, predicts whether as cancer of pancreas.
In another preferred example, the kit is a kind of noninvasive liquid biopsy detection kit.
In another preferred example, the excretion body is isolated and purified using density-gradient centrifugation method and supercentrifugation.
In another preferred example, the detection reagent is selected from the group: reagent, the detection Exo-CD82 of detection CA19-9 content
+ reagent, detect Exo-GPC1+ reagent, or combinations thereof.
In another preferred example, the detection reagent is selected from the group: the reagent of CA19-9 content, detection in detection serum
The reagent of Exo-GPC1+, the reagent for detecting Exo-CD82+, or combinations thereof.
In another preferred example, the detection reagent is to detect the reagent of CA19-9 content in serum, and use streaming
Cell art method detects the reagent combination of Exo-CD82+ and Exo-GPC1+.
In another preferred example, the kit includes:
(1) reagent of CA19-9 content and detection Exo-CD82+(excretion body CD82 expressing quantity in serum are detected)
Reagent, or,
(2) it is outer that the reagent of CA19-9 content in serum, the reagent for detecting excretion body CD82 expressing quantity and detection are detected
Secrete the reagent of body GPC1 expressing quantity.
In another preferred example, the kit includes the reagent of CA19-9 content and detection Exo- in detection serum
The reagent of CD82+.
In another preferred example, the kit includes the reagent of CA19-9 content in detection serum, detects Exo-
The reagent of CD82+, and the reagent of detection Exo-GPC1+.
In another preferred example, the kit includes the examination of the reagent and detection Exo-GPC1+ of detection Exo-CD82+
Agent.
In another preferred example, the kit includes the reagent of CA19-9 content in detection serum, and uses streaming
The reagent of cell art method detection Exo-CD82+.
In another preferred example, the kit includes the reagent of CA19-9 content in detection serum, and uses streaming
The reagent of cell art method detection Exo-CD82+ and Exo-GPC1+.
In another preferred example, the Exo-CD82+(excretion body CD82 expressing quantity) using between specific antibody
Connect calibration, Flow cytometry.
In another preferred example, the Exo-CD82+(excretion body CD82 expressing quantity), Exo-GPC1+(excretion body
GPC1 expressing quantity) use specific antibody indirect calibration, Flow cytometry.
In another preferred example, the Exo-CD82+(excretion body CD82 expressing quantity) using microballoon coupling excretion
The flow cytometry detection of antibody label specific proteins after body.
In another preferred example, the Exo-CD82+(excretion body CD82 expressing quantity) and Exo-GPC1+(excretion
Body GPC1 expressing quantity) using the flow cytometry detection of antibody label specific proteins after microballoon coupling excretion body.
In another preferred example, the CA19-9 is used as positive control in kit.
In another preferred example, the Exo-CD82+ is used as positive control in kit.
In another preferred example, the Exo-GPC1+ is used as positive control in kit.
The second aspect of the present invention provides the kit of a kind of diagnosis of pancreatic cancer and/or curative effect monitor and detection, the examination
Agent box includes:
(i) CA19-9 detection system, including the reagent for detecting CA19-9 content in serum；
(ii) optionally excretion body isolates and purifies system, and it includes separating for excretion body that the excretion body, which isolates and purifies system,
The reagent of purifying；
(iii) Exo-CD82+ detection system or Exo-CD82+ and Exo-GPC1+ detection system, the detection system packet
The reagent for detecting Exo-CD82+ is included, or for detecting Exo-CD82+(excretion body CD82 expressing quantity) and Exo-GPC1
The reagent of+(excretion body GPC1 expressing quantity)；
(iv) label or specification, the label or specification indicate the kit for diagnosis of pancreatic cancer and/or treatment
Imitate monitor and detection.
In another preferred example, it is described detection Exo-CD82+(excretion body CD82 expressing quantity) reagent be using stream
Formula cell art detect Exo-CD82+(excretion body CD82 expressing quantity) reagent.
In another preferred example, the detection Exo-CD82+(excretion body CD82 expressing quantity) and Exo-GPC1+(outside
Secrete body GPC1 expressing quantity) reagent be using Flow cytometry Exo-CD82+(excretion body CD82 expressing quantity)
With Exo-GPC1+(excretion body GPC1 expressing quantity) reagent.
In another preferred example, the kit includes the reagent of CA19-9 content in detection serum, and uses streaming
Cell art detects the reagent of excretion body GPC1 expressing quantity, excretion body CD82 expressing quantity.
In another preferred example, the following contents is indicated in the label or specification:
CA19-9 content is X3 in test object peripheral blood, and Exo-GPC1+ content is X1, and Exo-CD82+ content is X2, will
X3, X1 and X2 numerical value substitute into logistic regression equation shown in Formulas I:
Ln [p/ (1-p)]=13.047*X1+4.344*X2+0.017*X3-5.797 (I)；
Sample P value is calculated, and is compared with diagnostic points (P=0.2282), P > 0.2282, interpretation is that cancer of pancreas is positive, P
≤ 0.2282 interpretation is feminine gender；
CA19-9 testing result X3 unit is U/mL, Exo-CD82+(X2) and testing result Exo-GPC+(X1) be small
Number, showed in the form of % (such as a certain pattern detection result: CA19-9 content: 9.36U/mL, Exo-CD82+:9.95%, Exo-
GPC+:3.62%, then X1=0.0362, X2=0.0995, X3=9.36 substitute into Formulas I and obtain P=- 0.2192, P < 0.2282, the sample
Interpretation is feminine gender).
In another preferred example, the kit includes the reagent of CA19-9 content in detection serum, and uses streaming
The reagent of cell art detection excretion body CD82 expressing quantity.
In another preferred example, the following contents is indicated in the label or specification:
CA19-9 content is X3 in test object peripheral blood, and Exo-CD82+ content is X2, X3 and X2 numerical value is substituted into formula II
Shown in logistic regression equation:
Ln [p/ (1-p)]=8.187*X2+0.021*X3-4.999 (II)；
Sample P value is calculated, and is compared with diagnostic points (P=0.5828), P > 0. 5828, interpretation is that cancer of pancreas is positive, P
≤ 0. 5828 interpretation is feminine gender；
CA19-9 testing result X3 unit is U/mL, Exo-CD82+(X2) testing result be decimal, showed in the form of %.
In another preferred example, the test object is pancreatic disease patient.
In another preferred example, the test object is doubtful pancreatic disease patient.
In another preferred example, in the described detection serum reagent of CA19-9 content be using UniCel DxI 800 from
The reagent of CA19-9 content in the dynamic luminous Access immunoassay system detection serum of particulate.
In another preferred example, the reagent of CA19-9 content includes: in the detection serum
A0) R1:Access CA19-9 kit；
B0) R1a: it is coated with the goat-anti CA19-9 monoclonal antibody magnetic-particle of biotin labeling；
C0) R1b: the goat-anti CA19-9 monoclonal antibody of alkali phosphatase enzyme mark；
D0) R2: neutral detergent solution；
E0 it includes S1, S2, S3, S4, S5, S6 that) S1~S6:Access CA19-9, which calibrates product, wherein the CA19-9 contained is dense
Degree respectively is 0,30,90,300,900,2000U/ml.
In another preferred example, the Access CA19-9 kit is purchased from Beckman (Access GI Monitor
- 2 packs of 50 tests/pack Cat#387687)。
In another preferred example, the reagent isolated and purified for excretion body includes:
A) density gradient Jie -1(Density gradient medium-1)；
B) density gradient Jie -2(Density gradient medium-2)；
C) density gradient Jie -3(Density gradient medium-3)；
D) density gradient Jie -4(Density gradient medium-4)；
E) optionally PBS buffer solution (PBS Buffer).
In another preferred example, the use Flow cytometry excretion body GPC1 expressing quantity and excretion body
The reagent of CD82 expressing quantity includes:
N) microballoon (preferably invitrogen microballoon)；
O) BSA powder (Bull Serum Albumin)；
P) glycine (Glycine)；
Q) GPC1 antibody (GPC1 antibody, Cat # PA5-28055)；
R) CD82 antibody (CD82 antibody, Cat # PA5-13228)；
S) rabbit-anti fluorescence secondary antibody (anti-rabbit antibody FITC 488nm, Cat # A-11078)；
T) optionally antibody diluent (antibody Diluent).
In another preferred example, the reagent packet using Flow cytometry excretion body CD82 expressing quantity
It includes:
N) microballoon (preferably invitrogen microballoon)；
O) BSA powder (Bull Serum Albumin)；
P) glycine (Glycine)；
R) CD82 antibody (CD82 antibody, Cat # PA5-13228)；
S) rabbit-anti fluorescence secondary antibody (anti-rabbit antibody FITC 488nm, Cat # A-11078)；
T) optionally antibody diluent (antibody Diluent).
In another preferred example, the reagent packet using Flow cytometry excretion body GPC1 expressing quantity
It includes:
N) microballoon (preferably invitrogen microballoon)；
O) BSA powder (Bull Serum Albumin)；
P) glycine (Glycine)；
Q) GPC1 antibody (GPC1 antibody, Cat # PA5-28055)；
S) rabbit-anti fluorescence secondary antibody (anti-rabbit antibody FITC 488nm, Cat # A-11078)；
T) optionally antibody diluent (antibody Diluent).
In another preferred example, in the reagent, specific proportion is as follows:
CD82 primary antibody solution concentration 0.3-1.8ul antibody stoste (Cat # PA5-13228): 20ul antibody diluent；
GPC1 primary antibody solution concentration 0.8-2.0ul antibody stoste (Cat # PA5-28055): 20ul antibody diluent；Rabbit-anti fluorescence two
Antiantibody is 6-9ug/ml using concentration；Wherein, antibody diluent is the PBS solution containing 2%BSA.
In another preferred example, the kit includes:
A) density gradient Jie -1(Density gradient medium-1)；
B) density gradient Jie -2(Density gradient medium-2)；
C) density gradient Jie -3(Density gradient medium-3)；
D) density gradient Jie -4(Density gradient medium-4)；
E) optionally PBS buffer solution (PBS Buffer)；
F) 0.22 μm of filter (0.22 μm of fillter)；
G) optionally syringe；
H) optionally ultracentrifugation pipe；
I) optionally steel syringe needle (preferably growing thin steel syringe needle)；
J) optionally dehydrated alcohol；
K) BSA normal concentration protein solution；
L) BCA working solution A liquid；
M) BCA working solution B liquid；
N) invitrogen microballoon (invitrogen beads)；
O) BSA powder (Bull Serum Albumin)；
P) glycine (Glycine)；
Q) optional GPC1 antibody (GPC1 antibody)；
R) CD82 antibody (CD82 antibody)；
S) rabbit-anti fluorescence secondary antibody (anti-rabbit antibody FITC 488nm)；
T) optionally antibody diluent (antibody Diluent)；
U) optionally for the reagent of CA19-9 content in detection serum.
The third aspect of the present invention, a kind of method for providing diagnosis of pancreatic cancer and/or curative effect monitoring, comprising steps of
(1) a sample is provided, the sample includes the blood sample for carrying out human peripheral blood；
(2) CA19-9 content in serum, unit U/mL are detected；
(3) Exo-CD82+(excretion body CD82 expressing quantity is detected), or detection Exo-CD82+(excretion body CD82 albumen
Expression quantity) and Exo-GPC1+(excretion body GPC1 expressing quantity)；
(4) according to the Logistic regression equation of two index joint-detections, by CA19-9 content, Exo-CD82+ numerical value
(X3, X2) substitutes into equation shown in formula II, calculates P value；
Ln [p/ (1-p)]=8.187*X2+0.021*X3-4.999 (II)；
P > 0. 5828, interpretation are that cancer of pancreas is positive or prognosis mala, the interpretation of P≤0. 5828 are negative or prognosis bona；
Or
According to the Logistic regression equation of three index joint-detections, by CA19-9 content, Exo-CD82+ and Exo-GPC1
+ numerical value (X3, X2, X1) substitutes into equation shown in Formulas I, calculates P value；
Ln [p/ (1-p)]=13.047*X1+4.344*X2+0.017*X3-5.797 (I)；
P > 0.2282 is judged as that cancer of pancreas is positive or prognosis mala, P≤0.2282 are judged as that cancer of pancreas is negative or prognosis is good
It is good.
In another preferred example, described to be diagnosed as early diagnosing.
In another preferred example, the method is shone using the automatic particulate of UniCel DxI 800, and Access is immune to be surveyed
Determine CA19-9 content in system detection serum.
In another preferred example, the method uses Flow cytometry Exo-GPC1+(excretion body GPC1 protein expression
Amount) and Exo-CD82+(excretion body CD82 expressing quantity).
In another preferred example, described to use Flow cytometry Exo-CD82+(excretion body CD82 expressing quantity)
Include the following steps:
(3a1) isolates and purifies excretion body；
(3b1) carries out the method progress amplification of signal of immunofluorescence label using microballoon coupling excretion body and to excretion body；
(3c1) is strong to Exo-CD82+ fluorescence using Flow Cytometry methods to the excretion body for having carried out immunofluorescence label
Degree is detected；
(3d1) analyzes Exo-CD82+ numerical value.
In another preferred example, described to use Flow cytometry Exo-CD82+(excretion body CD82 expressing quantity)
With Exo-GPC1+(excretion body GPC1 expressing quantity) include the following steps:
(3a2) isolates and purifies excretion body；
(3b2) carries out the method progress amplification of signal of immunofluorescence label using microballoon coupling excretion body and to excretion body；
(3c2) uses Flow Cytometry methods to Exo-CD82+, Exo- the excretion body for having carried out immunofluorescence label
GPC1+ fluorescence intensity is detected；
(3d2) analyzes Exo-CD82+, Exo-GPC1+ numerical value.
In another preferred example, the step (3a1), isolating and purifying to be combined with density gradient centrifugation in (3a2)
Ultracentrifugal method carries out excretion body and isolates and purifies.
In another preferred example, the method uses kit as according to the second aspect of the invention.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 embodiment sample flow cytometer detection Exo-CD82+, Exo-GPC1+ result analysis chart.
The ROC that Fig. 2 Healthy People and Pancreas cancer patients peripheral blood CA9-9 content, Exo-GPC1 and Exo-CD82 carry out is tied
Fruit analysis.
Specific embodiment
The present inventor after extensive and in-depth study, passes through detection Pancreas cancer patients group peripheral blood and health group peripheral blood
In CA19-9 content and Exo-GPC1+ and Exo-CD82+ it is horizontal, for the first time it was unexpectedly observed that in Pancreas cancer patients excretion body
CD82 expressing quantity is higher than Healthy People, is in opposite trend with expression quantity in tissue.Further, inventors have found that Exo-
CD82+, Exo-GPC1+ and CA19-9 joint-detection have been obviously improved the sensitivity and specificity of cancer of pancreas early diagnosis detection.
Specifically, individually using AUC=0.724 when CA19-9 differentiating pancreatic cancer patient and Healthy People in the present invention；Exo-CD82+ is mono-
AUC=0.852 when solely using；AUC=0.885 when Exo-GPC1+ is used alone；Triple combination uses AUC=0.942, sees Fig. 2.This
Invention has a clear superiority in terms of cancer of pancreas early diagnosis, early diagnoses detection for cancer of pancreas and curative effect monitoring provides one kind
Powerful noninvasive method.Exo-CD82+, Exo-GPC1+ and CA19-9 joint-detection are early diagnosed for cancer of pancreas at present
And the detection method of curative effect monitoring is reported there has been no pertinent literature and clinical application.The present invention by detection CA19-9 content,
Exo-GPC1+ and Exo-CD82+ provides new means for cancer of pancreas auxiliary diagnosis for cancer of pancreas early diagnosis detection, with
Just it realizes that clinical cancer of pancreas is early found, is treated in time, prevent disease progression, extend patient survival, improve patient's prognosis.
The present invention is completed on this basis.
Term
As used herein, term " optional ", " optional " or " optionally " mean event or situation described later
It can occur but be not required to occur.For example, " optionally excretion body isolates and purifies system " refers to that " excretion body, which isolates and purifies, is
System " can have but be not required, and can be can arbitrarily be used for the system that excretion body isolates and purifies.
CA19-9 has been widely used in the detection of cancer of pancreas as a kind of tumor marker.Most of Pancreas cancer patients
Levels of serum CA 19-9 is apparently higher than normal person, especially cancer of pancreas end-stage patients positive rate up to 75%, is important auxiliary
Diagnosis index.However, CA19-9 content does not increase significantly in Early pancreatic carcinoma patient.Liver and gallbladder system cancer, gastric cancer, colorectal cancer are suffered from
The CA19-9 level of person and pancreas benign disease patient can also increase.So CA19-9 index is not solely used for cancer of pancreas morning
Phase diagnosis.
Exo-CD82+、Exo-GPC1+
In the present invention, " Exo-CD82+ " is equal with both " excretion body CD82 expressing quantity/content " meaning, can be mutual
Change use.
In the present invention, " Exo-GPC1+ " is equal with both " excretion body GPC1 expressing quantity/content " meaning, can be mutual
Change use.
Excretion body is concerned as emerging liquid biopsy biomarker.Contain tumour on the excretion body in tumour source
Cell specific markers have enrichment to tumour cell relevant information, are of great significance in lesion detection.According to report
Road, Exo-GPC1+ (excretion body GPC1 expressing quantity) can be used for distinguishing chronic pancreatitis and Pancreas cancer patients, have high
Specificity and sensibility.Exo-GPC1+ is extremely low in Healthy People and expression quantity in Patients With Chronic Pancreatitis, in Pancreas cancer patients
One of height expression, prompt Exo-GPC1+ or can be used as diagnosis of pancreatic cancer index.It uses and receives in 106950374 A of patent CN
Rice plasma enhancing scattering method detection GPC1 protein positive excretion bulk concentration is used for diagnosis of pancreatic cancer, as the result is shown the party
Method has preferable diagnosis effect to cancer of pancreas advanced stage, but poor to cancer of pancreas early diagnosis effect.
CD82 albumen is one of TM4SF transmembrane protein family member, is primarily involved in the adjustment of hyperplasia, tumour cell
Transfer regulation has inhibiting effect to the generation of cancer transfer, low in multiple cancerous tissues (low expression in cancerous tissue, but there has been no grind
Study carefully the correlation of CD82 expressing quantity and cancer in report excretion body) expression, it is expected to the molecule as cancer diagnosis and prognosis
Marker.It is in phase that CD82 expressing quantity, which is higher than expression quantity in Healthy People, with tissue, in Pancreas cancer patients excretion body in the present invention
Anti- trend, reason is still in further probing into.Nevertheless, Exo-CD82+ is early diagnosed for cancer of pancreas and curative effect monitoring
There has been no correlative studys.
ROC curve
As used herein, term " ROC curve " is Receiver operating curve, is with false positive probability (1- specificity)
For horizontal axis, true positives probability (susceptibility) be coordinate diagram composed by the longitudinal axis and subject under the conditions of particular stimulation due to adopting
The curve drawn with the Different Results that different judgment criterias obtains.Select optimal diagnosis threshold value.ROC curve is closer to a left side
The accuracy at upper angle, test is higher.Point near the ROC curve in the upper left corner is the least best threshold value of mistake, false sun
Property and false negative sum it is minimum.Two or more comparison of different diagnostic tests to disease identification ability.To same
When two or more diagnostic method of kind disease is compared, the ROC curve of each test can be plotted in same coordinate,
Intuitively to differentiate between good and evil, the work of subject representated by the ROC curve close to the upper left corner is most accurate.It also can be by calculating separately
Area (AUC) under the ROC curve of each test is compared, the AUC of any test is maximum, then any test is examined
Disconnected value is best.
Diagnosis of pancreatic cancer and/or the method for curative effect monitoring
A kind of method that the present invention provides diagnosis of pancreatic cancer and/or curative effect monitoring, comprising steps of
(1) a sample is provided, the sample includes the blood sample for carrying out human peripheral blood；
(2) CA19-9 content in serum, unit U/mL are detected；
(3) Exo-CD82+(excretion body CD82 expressing quantity is detected), or detection Exo-CD82+(excretion body CD82 albumen
Expression quantity) and Exo-GPC1+(excretion body GPC1 expressing quantity)；
(4) according to the Logistic regression equation of two index joint-detections, by CA19-9 content, Exo-CD82+ numerical value
(X3, X2) substitutes into equation shown in formula II, calculates P value；
Ln [p/ (1-p)]=8.187*X2+0.021*X3-4.999 (II)；
P > 0. 5828, interpretation are that cancer of pancreas is positive or prognosis mala, the interpretation of P≤0. 5828 are negative or prognosis bona；
Or
According to the Logistic regression equation of three index joint-detections, by CA19-9 content, Exo-CD82+ and Exo-GPC1
+ numerical value (X3, X2, X1) substitutes into equation shown in Formulas I, calculates P value；
Ln [p/ (1-p)]=13.047*X1+4.344*X2+0.017*X3-5.797 (I)；
P > 0.2282 is judged as that cancer of pancreas is positive or prognosis mala, P≤0.2282 are judged as that cancer of pancreas is negative or prognosis is good
It is good.
One typical the method for the present invention includes:
The present invention checks Healthy People, Pancreas cancer patients, and acquires peripheral blood.It is automatic using UniCel DxI 800
Particulate shines Access immunoassay system to isolated serum progress CA19-9 detection.Using density gradient centrifugation
Blood plasma excretion body is isolated and purified with ultracentrifugal method, microballoon is coupled the excretion body after isolating and purifying, uses indirect immunofluorescence
Excretion body GPC1 albumen, excretion body CD82 albumen is marked in method, measures Exo-GPC1+(excretion using flow cytometry
Body GPC1 expressing quantity) and Exo-CD82+(excretion body CD82 expressing quantity).
The present invention passes through CA19-9 content, Exo- in patients serum before 26 Healthy Peoples of detection, 24 treatment of pancreatic cancer
GPC1+(excretion body GPC1 expressing quantity) and Exo-CD82+(excretion body CD82 expressing quantity), it is returned by logistic
And ROC curve analysis is found, CA19-9, Exo-CD82+ and Exo-GPC1+ Combining diagnosis cancer of pancreas significant effect AUCCombining diagnosis=
0.942,P<0.01.Effect is better than CA19-9 and individually diagnoses, AUCCA19-9=0.7340,P<0.01；Exo-CD82+ and Exo-GPC1
+ independent diagnosis effect (AUCCD82=0.8518,P<0.01；AUCGPC1=0.8846,P< 0.01) it is individually diagnosed also superior to CA19-9.
Its susceptibility of CA19-9, Exo-CD82+, Exo-GPC1+ joint-detection is 95.8%, and specificity is 65.38%, high degree
Improve the recall rate of Pancreas cancer patients.Present invention firstly discovers that Exo-CD82+, Exo-GPC1+ and CA19-9 Combining diagnosis pair
The predicting function of cancer of pancreas early diagnosis.
The kit of diagnosis of pancreatic cancer and/or curative effect monitor and detection
The present invention also provides a kind of diagnosis of pancreatic cancer and/or the kits of curative effect monitor and detection.
In general, kit of the present invention includes:
(i) CA19-9 detection system, including the reagent for detecting CA19-9 content in serum；
(ii) optionally excretion body isolates and purifies system, and it includes separating for excretion body that the excretion body, which isolates and purifies system,
The reagent of purifying；
(iii) Exo-CD82+ detection system or Exo-CD82+ and Exo-GPC1+ detection system, the detection system packet
The reagent for detecting Exo-CD82+ is included, or for detecting Exo-CD82+(excretion body CD82 expressing quantity) and Exo-GPC1
The reagent of+(excretion body GPC1 expressing quantity)；
(iv) label or specification, the label or specification indicate the kit for diagnosis of pancreatic cancer and/or treatment
Imitate monitor and detection.
Main advantages of the present invention include:
(1) the method for the present invention has been obviously improved the sensitivity and specificity of cancer of pancreas early diagnosis detection；
(2) novel excretion body protein marker is used for cancer of pancreas early diagnosis and prognostic monitoring for the first time by the present invention.
(3) convection type detection data of the present invention carries out extreme point removal processing, keeps the analysis of flow cytometer detection data more objective
Specification.Energy batch execution data simultaneously is suitble to clinical expansion.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
The work such as part such as U.S. Sambrook. J " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, and 2002
Year) described in condition, or according to the normal condition proposed by manufacturer (such as product manual).Unless otherwise stated, otherwise
Percentage and number are calculated by weight.Experimental material used in following embodiment and reagent unless otherwise instructed can be from commercially available
Channel is obtained or can be prepared by literature method.
Embodiment 1: kit of the invention is prepared
Kit forms of the invention are as follows:
A) CA19-9 detection includes reagent
1) (Access CA19-9 kit is purchased from Beckman (Access GI to R1:Access CA19-9 kit
50 tests/pack Cat#387687 of Monitor -2 packs of)): 50 test/boxes；
2) R1a: it is coated with the goat-anti CA19-9 monoclonal antibody magnetic-particle of biotin labeling；
3) R1b: the goat-anti CA19-9 monoclonal antibody of alkali phosphatase enzyme mark；
4) R2: neutral detergent solution.
5) it includes S1, S2, S3, S4, S5, S6 that S1~S6:Access CA19-9, which calibrates product:, wherein the CA19-9 contained is dense
Degree respectively is 0,30,90,300,900,2000U/ml.
B) GPC1, CD82 expressing quantity detection reagent in excretion body:
1) excretion body separates: density gradient Jie -1(Density gradient medium-1) 6ml
2) density gradient Jie -2(Density gradient medium-2) 6ml
3) density gradient Jie -3(Density gradient medium-3) 6ml
4) density gradient Jie -4(Density gradient medium-4) 6ml
5) PBS buffer solution (PBS Buffer) 100ml
6) 0.22 μm of filter (0.22 μm of fillter) 5
7) ultracentrifugation pipe (5.9mL) 5
8) length is 1, steel syringe needle thin
9) 5ml syringe 15
10) BSA normal concentration protein solution (0,0.05,0.1,0.25,0.5,1.0 mg/ml)
11) BCA working solution A liquid 50ml
12) BCA working solution B liquid 2ml
13) it is coupled: 100 μ l of invitrogen microballoon (invitrogen beads)
14) BSA powder (Bull Serum Albumin) 2.0g
15) glycine (Glycine) 1g
16) 8 μ L of GPC1 antibody (GPC1 antibody)
17) 5 μ L of CD82 antibody (CD82 antibody)
18) rabbit-anti fluorescence secondary antibody (anti-rabbit antibody FITC 488nm) 2 μ L
19) antibody diluent (antibody Diluent) 1ml.
Embodiment 2: the detection method of the kit of embodiment 1
1, it acquires blood sample and sample prepares:
Since peripheral blood has the advantages that draw materials convenience, non-invasive, continuous detection, examined using peripheral blood detection cancer of pancreas
Disconnected biomarker can find the generation of cancer of pancreas early, and the early diagnosis of clinical cancer of pancreas has been increased to a new water
It is flat.
Blood sample is collected in Datong with coal hospital, the people's hospital of Heibei, Shanghai City Sixth People's Hospital: 24
Healthy volunteer, 26 Pancreas cancer patients.Sample collection: the peripheral blood for collecting about 3 ~ 6 patients ml is put into promoting vacuum and coagulates in pipe,
It overturns for several times sufficiently to combine, stands about half an hour, 1600g is centrifuged 10min, and supernatant is transferred to new pipe.The secondary progress of supernatant
12000g is centrifuged 10min, takes supernatant, is added in new centrifuge tube and obtains serum.It is anti-that collection 2-8ml peripheral blood is put in dedicated EDTA
In solidifying pipe (pipe lid is purple), gently overturns 8 times, prevent grumeleuse, obtain no grumeleuse, the whole blood without haemolysis, 3000g is centrifuged in 2h
5 minutes, supernatant was transferred to new pipe.
, CA19-9 content detection in serum sample:
Contained using CA19-9 in the luminous Access immunoassay system detection serum of the automatic particulate of UniCel DxI 800
Amount, specific steps are as follows:
1) enter test request screen from main menu.
2) to each sample, the position on a specimen holder, input sample information and the test name that need to be detected are set.
3) sample cell (cup) is put into the position set in specimen holder.
4) operation key (Run) is pressed to start to detect.
5) calibration needed for instrument can remind operator's operation.
6) system will automatically calculate testing result.
, GPC1, CD82 expressing quantity detection in blood plasma excretion body:
Excretion body is carried out using density gradient centrifugation and ultracentrifugal method to isolate and purify, and is coupled excretion body using microballoon
Method increase detection particle volume be convenient for Flow cytometry application.With GPC1 antibody (GPC1-antibody),
Excretion body surface face GPC1 albumen and CD82 albumen after CD82 antibody (CD82-antibody) specificity capture coupling；Finally use rabbit
Anti- fluorescence secondary antibody (anti-rabbit antibody FITC 488nm) carries out antibody dyeing to GPC1 and CD82 primary antibody.Using
Flow cytometry excretion body surface face GPC1 and CD82 protein fluorescence value.Testing result is through the analysis software processing of extreme value removal rate
Carrying out analysis to sample positive rate afterwards can be obtained the expression quantity of Exo-GPC1, Exo-CD82 albumen.Testing result substitutes into
Logistic regression equation obtains sample P value, is compared with Cutoff value, and P > cutoff value is judged as cancer of pancreas, and P≤
Cutoff value is judged as health.
Specific steps include:
(1) blood plasma differential centrifugation: for plasma sample under the conditions of 4 °C, 10000g is centrifuged 30min；With pipettor by upper layer blood
Slurry is transferred in new centrifuge tube A, separately prepares a new centrifuge tube B, and blood plasma in A pipe is filtered through 0.22 μm of filter and is managed into B.
(2) prepared by density gradient media: ultracentrifugation pipe C flag sample number, replaces 1ml syringe with long fine steel needle head
Included syringe needle, is slowly injected into centrifuge tube bottom for 1ml density gradient media 1；1ml density gradient media 2 is slow from centrifuge tube bottom
Injection holds up medium 1；1ml density gradient media 3 is slowly injected into from centrifuge tube bottom, holds up medium 2 and 1；By 1ml density level bands
Degree medium 4 is slowly injected into from centrifuge tube bottom, holds up medium 3,2 and 1.
(3) plasma sample is loaded: the blood plasma in B pipe being slowly added in ultracentrifugation pipe with 5ml syringe, if sample size
Deficiency supplements residual volume with PBS, ultracentrifugation pipe volume is full of.Centrifugation channel closure is carried out using thermoplastic envelope instrument.Note: add
Avoid flow velocity is too fast sample is made to break up density gradient media layering when sample.
(4) it isolates and purifies: rotor parameter being set according to the Ultracentrifuge rotor of use, using 150,000g in 4 °C of items
10-20h is centrifuged under part.Supernatant is abandoned after centrifugation, is suspended and is precipitated with 2ml PBS, and excretion body suspension 5mL syringe is shifted
Into new ultracentrifugation pipe (D), 2ml PBS washs ultracentrifugation pipe C, and cleaning solution is transferred to together in D pipe, and residual volume is used
PBS is full of, and thermoplastic envelope instrument is sealed.150,000g are centrifuged 2-4h under the conditions of 4 °C, abandon supernatant after centrifugation, according to heavy
Shallow lake amount is added appropriate PBS and suspends (100-700 μ L), and is transferred in new 1.5ml centrifuge tube (E).
(5) excretion liquid solution concentration mensuration: according to BCA kit specification prepare protein concentration gradient titer (0,
0.05,0.1,0.25,0.5,1.0mg/ml).According to A liquid: B liquid=1ml:20 μ L proportional arrangement dyeing liquor is (current existing
Match, be protected from light storage).20 μ L protein standard liquid are got involved in ELISA Plate (A1-A6), are added Duplicate Samples (B1-B6), 20 μ L excretions
Liquid solution is added in ELISA Plate (A7), is added Duplicate Samples (B7).200 μ L dyeing liquors are added in every hole.It is protected from light in 37 °C of insulating boxs
It is incubated for 30min, reads the absorbance under 562nm using microplate reader.Calibration curve equation is calculated according to protein standard liquid, by sample
Pipe OD value substitutes into calibration curve equation, calculates the volume of excretion body sample protein concentration values and 30 μ g excretion liquid solutions.
(6) microballoon is coupled excretion body:
A) preparation of reagents:
Reaction terminating liquid: Gly containing 100mM, the PBS solution of 2% BSA；2ml
Cleaning solution: the PBS solution containing 2% BSA；1ml
Confining liquid: the PBS solution containing 10%BSA；0.8ml
B) operating procedure:
Take 2 1.5ml centrifuge tubes labeled as No. 0 pipe, No. 1 pipe；Invitrogen microballoon (beads) ultrasound 5s is vortexed mixed
It is even.No. 0 pipe, each 10 μ l beads of addition of No. 1 pipe, 90 μ l PBS are added in No. 0 pipe, so that final volume is 100 μ l；No. 1 pipe is added
30 μ g excretion liquid solutions, addition PBS to total volume are 100 μ l.Room temperature rotation mixes 15min；Above-mentioned solution is diluted to PBS
400 μ l are vortexed and mix, and room temperature rotation is incubated for 30min；14,800g centrifugation 5min take out supernatant reservation and do BCA detection (reference
BCA detection kit)；Precipitating is suspended with 1mL reaction terminating liquid, is vortexed and is mixed, and room temperature rotation is incubated for 30min；Contain 2% with 400 μ L
The PBS solution of BSA is washed microballoon 1 time (14800g, 5min)；PBS confining liquid suspended microspheres with 400 μ L containing 10% BSA precipitate,
It mixes, room temperature rotation is incubated for 30min；14800g is centrifuged 5min, abandons supernatant；Microballoon 1 is washed with 400 PBS solutions of the μ L containing 2%BSA
Secondary (14800g, 5min)；Precipitating is suspended 4 DEG C with the PBS solution of 100 μ L, 2% BSA and saves or directly carry out subsequent experimental.
C) excretion body coupling amount is analyzed:
Standard curve is drawn according to the protein concentration of standard items and OD, lists calibration curve equation and R2Value；
Calibration curve equation y=ax+b；Excretion body coupling amount (see Table 1) on microballoon is calculated according to calibration curve equation.
Excretion body coupling amount testing result example on 1 microballoon of table:
| Sample | OD | Supernatant concentration (mg/mL) after coupling | (ug) is measured in coupling | Coupling efficiency | Stoste additive amount (ug) |
| No. 0 pipe | 0.0932 | ||||
| No. 1 pipe | 0.1285 | 0.0269 | 17.52 | 61.99% | 28.27 |
Supernatant concentration=(sample OD-background pipe OD)/a after coupling；Supernatant concentration=(No. 1 pipe OD-No. 0 after being coupled
Pipe OD)/a
Supernatant concentration * 400 after amount=stoste additive amount-coupling in coupling (reaction system is 400uL when coupling)
Amount/stoste additive amount in coupling efficiency=coupling；Coupling efficiency >=40%, that is, subsequent experimental can successfully be carried out by being coupled；
If coupling efficiency is too low, it is proposed that re-start coupling.
(7) excretion body surface face GPC1, CD82 Protein Detection (by taking 1 sample as an example):
A) preparation of reagents:
Cleaning solution: 100mgBSA+5mLPBS
GPC1 primary antibody solution :+20 μ l cleaning solution of 1.5 μ l GPC1, mono- antigen liquid (Cat # PA5-28055)；
CD82 primary antibody solution :+20 μ l cleaning solution of 1 μ l CD82, mono- antigen liquid (Cat # PA5-13228)；
By every 2 μ l loss calculation of pipe；
Two corresponding anti-solution: taking rabbit-anti fluorescence secondary antibody (anti-Rabbit antibody FITC 488nm), (stoste is diluted to
100 μ g/mL) 30 μ l+300 μ l cleaning solutions, it mixes well, is kept in dark place；
B) operating procedure:
3 1.5ml centrifuge tubes are taken, 0, GPC1, CD82 is labeled as；By the sample (about 100 μ l of every pipe) after coupling, it is diluted to
500 μ l are mixed；Microballoon after each corresponding addition of pipe 50 μ l coupling, 14800g are centrifuged 5min；No. 0 pipe adds 20 μ l cleaning solutions；
Each pipe of GPC1 group adds 20 μ l GPC1 primary antibody solution, and CD82 pipe adds 20uLCD82 primary antibody solution；Wink is from 15s, turn up
14800g；Then sufficient vortex mixes, and stationary incubation 30min in 37 DEG C of insulating boxs, every 10min mix primary；14800g centrifugation
5min abandons supernatant；Cleaning solution is washed 1 time；20 μ l two corresponding anti-solutions are added in each pipe, and wink is from 15s, turn up 14800g；It mixes well, 37
It stands to be protected from light in DEG C insulating box and is incubated for 30min, every 10min is mixed 1 time；14800g is centrifuged 5min and abandons supernatant, and cleaning solution is washed 1 time,
PBS is washed 1 time.It is suspended and is precipitated with 100 μ l PBS, be vortexed and mix, flow cytometer is detected (to be used with reference to BD flow cytometer
Method).
C) flow cytometer detection interpretation of result:
The positive rate of Exo-CD82+, Exo-GPC1+ are analyzed.By flow cytometer showed software obtain Exo-GPC1,
The positive rate of Exo-CD82, convection type result carries out finishing analysis in Excel, and flow cytometer detection result histogram copies (such as Fig. 1
It is shown).
Collect 26 Healthy Peoples and 24 Pancreas cancer patients peripheral bloods, in aforementioned manners detect serum in CA19-9 content and
Exo-GPC1, Exo-CD82 expression quantity.
Embodiment 3: to Healthy People and Pancreas cancer patients peripheral blood CA19-9 content, Exo-CD82+ and Exo-GPC1+ table
Logistic regression analysis and ROC curve analysis are carried out up to amount
The present invention have detected CA19-9 content and Exo-GPC1+ in 26 Healthy Peoples and 24 Pancreas cancer patients serum,
Exo-CD82+ expression quantity is returned by logistic and ROC curve analysis is found, CA19-9, Exo-GPC1+ and Exo-CD82+
Combining diagnosis cancer of pancreas effect AUC the most significantCombining diagnosis=0.942,P<0.01.Three index joint-detection prediction effects are better than any
Two indexes Exo-CD82 combine Exo-GPC1(AUC=0.8892), Exo-CD82 combine CA19-9(AUC=0.9212), Exo-
GPC1 combines CA19-9(AUC=0.9267) detection effect.Three index joint-detection prediction effects are better than independent index Exo-CD82
(AUC=0.8518), Exo-GPC1(AUC=0.8846), CA19-9(AUC=0.7340).CA199 is individually diagnosed, AUCCA19-9=
0.7340,P<0.01；Independent diagnosis effect (the AUC of Exo-GPC1, Exo-CD82 expression quantityGPC1=0.8846,P<0.01；AUCCD82=
0.8518,P< 0.01) (table 2) is individually diagnosed also superior to CA19-9.
The ROC of 2 Healthy People of table and Pancreas cancer patients peripheral blood CA19-9, Exo-CD82+ and Exo-GPC1+ Combining diagnosis
Analysis
Embodiment 4:Exo-CD82+, Exo-GPC1+ and CA19-9 expression quantity associated detecting method is for cancer of pancreas early stage
Diagnose the verifying of detection
The present invention verifies the diagnostic points of enrolled 26 Healthy Peoples, 24 Pancreas cancer patients Combining diagnosis, and will
CA19-9 content (X3), Exo-GPC1+(X1) and Exo-CD82+(X2) numerical value substitute into logistic regression equation, calculate sample P
Value.The ROC analysis measurement of CA19-9, Exo-CD82+ and Exo-GPC1+ associated detecting method shows: i.e. according to " outstanding mounting index "
Sensibility-(1- specificity), which is exactly optimal dividing value, available " outstanding mounting index " maximum value for R=
0.95, i.e. CA19-9, Exo-GPC1+, Exo-CD82+ joint-detection susceptibility is 95%；With this condition, Healthy People and pancreas
The best dividing value of cancer patient is P=0.2282, i.e., the described sample P value is compared with diagnostic points (P=0.2282), P > 0.2282,
Interpretation is that cancer of pancreas is positive, and the interpretation of P≤0.2282 is feminine gender.This diagnostic method verification result false negative rate is only 4.17%, false sun
Property rate be 34.62%(statistical result be shown in Table 3 and table 4).Extremely low false negative rate may insure this diagnostic method in adjuvant clinical pancreas
Sensitivity when gland cancer early diagnoses, high degree improve the recall rate of Pancreas cancer patients, reduce clinical cancer of pancreas rate of missed diagnosis,
Receive correct therapeutic scheme in time for patient and help is provided, extends patient survival and prognosis.Present invention firstly discovers that CA19-
9, the predicting function that Exo-CD82, Exo-GPC1 Combining diagnosis early diagnose cancer of pancreas.
3 Exo-CD82+, Exo-GPC1+ and CA19-9 associated detecting method judging result of table is compared with clinical diagnosis result
4 Exo-CD82+, Exo-GPC1+ and CA19-9 joint-detection of table is for cancer of pancreas early diagnosis verifying
In addition, the present invention joins enrolled 26 Healthy Peoples, 24 Pancreas cancer patients using Exo-CD82+ and CA19-9
Conjunction is detected, and CA19-9 content (X3) and Exo-CD82+(X2) numerical value are substituted into the logistic regression equation of table 6, calculating
Sample P value.The ROC analysis measurement of CA19-9 and Exo-CD82+ associated detecting method shows: i.e. sensitive according to " outstanding mounting index "
Property-(1- specificity), the index value place of being maximized be exactly be exactly optimal dividing value, available " outstanding mounting index " maximum value for R=
0.762, i.e. CA19-9, Exo-CD82+ joint-detection susceptibility is 76.2%；With this condition, Healthy People and Pancreas cancer patients
Best dividing value is P=0.5828, i.e., the described sample P value is compared with diagnostic points (P=0.5828), and P > 0.5828, interpretation is pancreas
Gland cancer is positive, and the interpretation of P≤0.5828 is feminine gender.This diagnostic method verification result false negative rate is only 20.83%, and false positive rate is
0%(statistical result is shown in Table 5 and table 6).The result shows that the joint of this two indexes can also obtain good effect for clinical diagnosis
Fruit the results are shown in Table 5 and table 6.
5 Exo-CD82+ and CA19-9 associated detecting method judging result of table is compared with clinical diagnosis result
6 Exo-CD82+ and CA19-9 joint-detection of table is for cancer of pancreas early diagnosis verifying
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1.(1) the purposes of excretion body CD82 expressing quantity detection reagent joint CA19-9 detection reagent, or
(2) excretion body CD82 expressing quantity detection reagent joint excretion body GPC1 expressing quantity detection reagent and CA19-9 inspection
The purposes of test agent, which is characterized in that be used to prepare diagnosis of pancreatic cancer and/or curative effect monitor and detection kit.
2. purposes according to claim 1, which is characterized in that the detection kit passes through detection pancreatic disease patient
CA19-9 content and excretion body CD82 expressing quantity in serum, or contained by CA19-9 in detection pancreatic disease patients serum
Amount, excretion body GPC1 expressing quantity and excretion body CD82 expressing quantity, for diagnosis of pancreatic cancer and/or curative effect monitoring inspection
It surveys.
3. purposes according to claim 1, which is characterized in that the excretion body uses density-gradient centrifugation method and hypervelocity
Centrifugal process isolates and purifies.
4. purposes according to claim 1, which is characterized in that the detection reagent is selected from the group: CA19- in detection serum
The reagents of 9 contents, the reagent for detecting excretion body GPC1 expressing quantity, the reagent for detecting excretion body CD82 expressing quantity or
A combination thereof.
5. purposes according to claim 1, which is characterized in that the kit includes:
(1) reagent of the reagent of CA19-9 content and detection excretion body CD82 expressing quantity in serum is detected, or,
(2) reagent of CA19-9 content in serum, the reagent for detecting excretion body CD82 expressing quantity and detection excretion body are detected
The reagent of GPC1 expressing quantity.
6. purposes according to claim 1, which is characterized in that the excretion body CD82 expressing quantity, excretion body
GPC1 expressing quantity uses specific antibody indirect calibration, Flow cytometry.
7. the kit of a kind of diagnosis of pancreatic cancer and/or curative effect monitor and detection, which is characterized in that the kit includes:
(i) CA19-9 detection system, including the reagent for detecting CA19-9 content in serum；
(ii) optional excretion body isolates and purifies system, and it includes isolating and purifying for excretion body that the excretion body, which isolates and purifies system,
Reagent；
(iii) excretion body CD82 protein expression amount detection systems or excretion body CD82 expressing quantity and excretion body GPC1 albumen
Amount detection systems are expressed, the detection system includes the reagent for detecting excretion body CD82 expressing quantity, or for detecting
The reagent of excretion body CD82 expressing quantity and excretion body GPC1 expressing quantity；
(iv) label or specification, the label or specification indicate the kit and supervise for diagnosis of pancreatic cancer and/or curative effect
Control detection.
8. kit according to claim 7, which is characterized in that the reagent of CA19-9 content is in the detection serum
Using the examination of CA19-9 content in the luminous Access immunoassay system detection serum of the automatic particulate of UniCel DxI 800
Agent.
9. kit according to claim 7, which is characterized in that the kit includes:
A) density gradient Jie -1；
B) density gradient Jie -2；
C) density gradient Jie -3；
D) density gradient Jie -4；
E) optional PBS buffer solution；
F) 0.22 μm of filter；
G) optional syringe；
H) optional ultracentrifugation pipe；
I) optional steel syringe needle；
J) optional dehydrated alcohol；
K) BSA normal concentration protein solution；
L) BCA working solution A liquid；
M) BCA working solution B liquid；
N) invitrogen microballoon；
O) BSA powder；
P) glycine；
Q) optional GPC1 antibody；
R) CD82 antibody；
S) rabbit-anti fluorescence secondary antibody；
T) optional antibody diluent；
U) for detecting the reagent of CA19-9 content in serum.
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| CN114164268A (en) * | 2021-07-14 | 2022-03-11 | 中山大学孙逸仙纪念医院 | Application of hnRNPA1 in diagnosis, prognosis and treatment of pancreatic cancer |
| CN117330481B (en) * | 2023-11-27 | 2024-04-09 | 南京联笃生物科技有限公司 | Flow detection method for exosomes and application thereof |
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| JP5808349B2 (en) * | 2010-03-01 | 2015-11-10 | カリス ライフ サイエンシズ スウィッツァーランド ホールディングスゲーエムベーハー | Biomarkers for theranosis |
| CN102426256A (en) * | 2011-08-16 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | Carbohydrate antigen 19-9 (CA19-9) quantitative determination kit and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3771487A1 (en) * | 2019-07-29 | 2021-02-03 | Bundesrepublik Deutschland, Vertreten durch den Bundesminister für Wirtschaft und Energie, dieser Vertreten durch den Präsidenten der | Method for the simultaneous determination of various analytes in an environmental sample and / or an agricultural raw material based on core-shell microparticles |
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