CN1811452A - Method for detecting B type hepatitis virus specificity T cell by flow cytometer cell factor detecting method - Google Patents
Method for detecting B type hepatitis virus specificity T cell by flow cytometer cell factor detecting method Download PDFInfo
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- CN1811452A CN1811452A CN 200610037814 CN200610037814A CN1811452A CN 1811452 A CN1811452 A CN 1811452A CN 200610037814 CN200610037814 CN 200610037814 CN 200610037814 A CN200610037814 A CN 200610037814A CN 1811452 A CN1811452 A CN 1811452A
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Abstract
The present invention relates to a flow cytometry cell factor detection method for detecting hepatitis B virus specific T cell. Said invention utilizes flow cytometry cell factor detection method to detect whole blood, and adopts the following steps: under the environment of having cell factor blocking agent brefeldin adding stirmulus antigen and synergic stimulant in whole blood to make antigen stimulation, after 6 hr of stimulation, washing, identifying lymphocyte and schizolyzing red blood cell, washing and breaking membrane, finally, using fluorescently-labelled antibody to fluorescently stain the cell factor in the cell, after the staining process is completed, utilizing flow cytometer to make analysis.
Description
Technical field
Flow cytometry cytokines measurement method detects the method for hepatitis B specific T-cells, belongs to immune analysis detection technique field.
Background technology
After hepatitis type B virus (HBV) infects, the immune response ability that a principal element that determines the degree that is in a bad way and the course of disease to lapse to is a body.Wherein in patient's body hepatitis B specific T-cells (cytotoxic Tlympho-cyte, CTL call the HBV specific CTL in the following text) remove viral aspect the performance important function.CD4 in the T cell
+Cell can be divided into specificity T auxiliary cell (Th) under antigenic stimulus, Th can promote the HBV specific CTL to produce, and to keeping HBV specific CTL activity, producing continuation cellular immunity and play an important role.CD8 in the T cell
+Cell can be divided into the HBV specific CTL under antigenic stimulus.Studies show that the HBV specific CTL plays a crucial role in control HBV infects: in (1) defense system at body, CTL is the principal element of pathogen in the scavenger-cell; (2) all with the shortage of HBV specific CTL, the control of virus infections and HBV specific CTL quantity and activity are proportionate in the HBV persistent infection; (3) can activate the HBV specific CTL by methods such as dna vaccination immunity, DC vaccine therapies, and then remove virus infections; (4) directly adoptive immunity input HBV specific CTL can be removed infective virus.
Studies show that the reaction of HBV specific CTL is strong among the patient that acute HBV infects, but after clinical recovery and serum conversion take place, very low-level HBVDNA is still sustainable to exist decades.Long-standing micro-HBV DNA is relevant with long-standing HBV specific CTL.Compare with acute self limiting HBV infection, the chronic hepatitis B patient by the 51Cr method for releasing, is difficult to detect the reaction of HBV specific CTL in the peripheral blood.By same technology, virus replication is active, the HBV specific CTL lacks in the tangible chronic HBV infection blood samples of patients of liver inflammation, and HBV specific CTL frequency is lower in the hepatic tissue; And the chronic HBV infection person that virus replication is inactive, the liver inflammation is slight, HBV specific CTL frequency is higher in blood and the hepatic tissue.Foreign study is also found: chronic hepatitis B is the spontaneous or interferon-induced patient who partially or completely recovers of experience, can be formed on intensity and specificity aspect and be similar to CTL that the acute hepatitis b patient the has reaction to hepatitis B.Patient,, find that HBV specific CTL frequency raises significant difference significantly by 51Cr method for releasing and Te-tramer technology through lamivudine therapy.The HBV specific C D8 of this raising
+Cytoactive can cause CD4
+Cell activity is recovered, and causes the decline of HBV virus load simultaneously.Therefore Lamivudine can make HBV specific CTL functional rehabilitation to chronic hepatitis B patient's treatment.Behind lamivudine therapy,, HBV specific CTL function is restored by obtaining the specificity of multiple epitope.Above results suggest, the immunization therapy of reacting for hepatitis B by raising HBV specific CTL provides possibility.
CTL detection method commonly used has: the isotope cytotoxicity analysis, as 51Cr method for releasing, 125I-UdR method for releasing and tritiated uridine method for releasing etc.; Unicellular level determination CTL activity, as the PCR of cell within a cell factor dyeing, special TXi Baoshouti (TCR), limiting dilution analysis method (limiting dilution analysis, LDA) etc.Because the ratio of specific CTL in peripheral blood lymphocyte is very low, detects these low-level specific CTLs and needs extremely sensitive method.Traditional 51Cr discharges analytic approach and LDA method, and indirect detection antigentic specificity CTL needs a large amount of CTLs action effect cells, must increase earlier external, and the whole process cycle is long, susceptibility is low, time-consuming, effort, is difficult to widespread use.
Enzyme linked immunological spotting method (the Enzyme-linked immunospot assay of the detection IFN-that developed recently gets up cytokine secretion cell quantities such as (IFN-γ), Elispot) and the tetramer detection method (Te-tramer) that detects T cell-specific antigen receptor quantity have very high sensitivity because of it, can detect (15～20)/10
6The low-frequency degree specific CTL, more and more come into one's own and use more widely.But in practice, also come with some shortcomings.The condition of ELISPOT detection positive cell is more strict, and stimulation time is long, needs constantly to replenish the cell growth substance; The Te-tramer detection specificity, but do not act on the T cell.
Utilization flow cytometry cytokines measurement method (cytokine flow cytometry, CFC) be meant flow cytometer cell intrinsic factor detection method, the characteristics of this method are the functions of utilizing the multiparameter of flow cytometer to analyze simultaneously, accurately recognition objective cell and detect the secretion situation of its intrinsic factor.
Do not see that utilization flow cytometry cytokines measurement method detects the report of replying of HBV specific CTL in chronic hepatitis B (CHB) patient's peripheral blood and the hepatic tissue.
Summary of the invention
The purpose of this invention is to provide the method that a kind of flow cytometry cytokines measurement method detects the hepatitis B specific T-cells, is a kind of highly sensitive, and detected object is unrestricted, the detection that is used for the HBV specific CTL that cost is low.
Technical scheme of the present invention: (cytokine flowcytometry CFC) detects whole blood utilization flow cytometry cytokines measurement method.Under the environment that cell factor sealer brefeldin A is arranged, in whole blood, add stimulator antigen and collaborative stimulant carries out antigenic stimulus.Usually washing after six hours that stimulate, identification lymphocyte and splitting erythrocyte.Wash subsequently and rupture of membranes.Use the fluorescent-labeled antibody pair cell inner cell factor to carry out fluorescence labeling dyeing, up flow type cell instrument analysis after finishing at last.
Our detection scheme is based on the detection of using collaborative stimulant (CD28/CD49d), polychrome reagent (CD8PerCP-Cy5.5) detection and two kinds of cell factors (INF-r and CD69).
Instrument: Beckman coulter-3XL flow cytometer, CO2gas incubator (company of HRCP-Haier), hydro-extractor.
Main agents
1. cell factor sealer: brefeldin A (Brefeldin A), Sigma company product.
2. collaborative stimulant: CD28/CD49d, Sigma company product.
3. stimulator antigen: HBcAg specific polypeptide 18-27, (amino acid sequence: FLPSDFFPSV), Shanghai lumber Bioisystech Co., Ltd is synthetic, and sells to the public.
4. lymphocyte CD 8 probes: CD8PerCP-Cy5.5, (the plain mark CD8 of isothiocyanic acid-CY5.5 composite fluorescence antibody), BD company product.
5. hemolysin: Lysing Solution, BD company product.
6. rupture of membranes agent: permeabilizing solution, BD company product.
7. r-interferon antibody fluorescence labeling: IFN-r FITC, BD company product.
8. lymphocyte CD 69 cell activation probe: CD69-PE, (the CD69 antibody of PE mark), BD company product.
9. negative control agent: IgG2a FITC/IgG1 PE, BD company product.
10. phosphate buffer (PBS, pH7.0), autogamy.
Testing process
1. antigenic stimulus: measure adding whole blood 100 μ l in the pipe at fluidic cell, brefeldin A 10 μ l, CD28/CD49d 2.5 μ l and HBcAg specific polypeptide 1 μ l will measure pipe and place CO2gas incubator, 5%CO
2, propagation 6 hours under 37 ℃ of conditions.
2. washing: the PBS liquid 2ml with precooling washs 1 time, and centrifugal 1500rpm/ branch is abandoned supernatant.Remove remaining brefeldin A, CD28/CD49d and HBcAg specific polypeptide, stay lymphocyte and red blood cell.
3. lymphocyte identification: add CD8PerCP-Cy5.510 μ l, CD69 PE 10 μ l lucifuges 30 minutes, identification CD8 is positive and by the antigen activated lymphocytes.
4. splitting erythrocyte: add hemolysin 2ml, lucifuge 15 minutes.
5. centrifugal, washing: centrifugal 1500rpm/ branch, abandon supernatant, the PBS liquid 2ml repeated washing of usefulness precooling 2 times is removed red blood cell.
6. rupture of membranes: add rupture of membranes agent 0.5ml, lucifuge 10 minutes.Lymphocytic cell membrane is punched, make to guarantee that simultaneously intracellular matter does not leak by the cytokine antibodies cell of can freely coming in and going out.
7. washing: the PBS liquid with precooling washs 1 time, and centrifugal 1500rpm/ branch is abandoned supernatant.Remove the rupture of membranes agent.
8. dyeing: add IFN-r FITC 10 μ l, lucifuge 30 minutes makes intracytoplasmic gamma interferon be dyeed by anti-gamma interferon fluorescence antibody mark.
9. cells were tested by flow cytometry.Regulate the magnitude of voltage of flow cytometer forward direction (FS), side direction (SS) two parameters, most lymphocytes are presented in the two-parameter point diagram of FS/SS.Make up CD8PerCP-Cy5.5 and CD69PE histogram, identify the CD8 that has been activated in the drawings
+/ CD69
+Cell.Make up IFN-rFITC one-parameter histogram, and set back, negative zone and detect positive sample, as positive particle occurs, just be identified as the T cell that the hepatitis B antigen peptide is had the anti-property of specificity
Beneficial effect of the present invention:
CFC and ELISPOT be relatively: the CFC method is meant flow cytometer cell intrinsic factor detection method, and the characteristics of this method are the functions of utilizing the multiparameter of flow cytometer to analyze simultaneously, accurately recognition objective cell and detect the secretion situation of its intrinsic factor.CFC and ELISPOT detect cell factor and are based on individual cells.Both differences are the technology (flow cytometer is a multiparameter) of (1) signal output; (2) time length of Ci Jiing and condition (stimulated PBMCs 24 hours in ELISPOT film bottom usually; CFC detects the polypropylene tube moderate stimulation whole blood of usefulness or PBMCs6 hour).(3) the detected positive cell of CFC is the three-to-four-fold of ELISPOT.This may be partly because the condition of ELISPOT detection positive cell be more strict.
CFC and MHC-Ig tetramer (or dipolymer) dyeing are compared: MHC-peptide section tetramer and MHC-Ig dipolymer are the strong instruments of visual inspection T cells with antigenic specificity.Tetramer and dipolymer and CFC detect the difference of observing: (1) tetramer and dipolymer detect the reaction of T cell-specific only by a peptide-MHC antigenic determinant, but CFC can be used to measure the reaction of complicated antigen; (2) tetramer and dipolymer detect specificity, but does not act on the T cell, otherwise CFC detects the cell factor that the antigen-specific sexual cell produces.
The present invention sets up novel immuno analytical method, be used for the detection of HBV specific CTL, obviously be better than 51Cr determination method, limiting dilution assay (LAD), the MHC-tetramer (dimer) method, the inventive method is not only highly sensitive, detected object is unrestricted, cost is low, and directly the expression of the functional cell factor of detection specificity CTL can be widely used in clinical detection.
Embodiment
Embodiment 1
Get normal control's whole blood 100 μ l, by specification said determination method is carried out detecting operation.Recording HBV specific CTL value is 0.2%.
Embodiment 2
Get acute hepatitis B patient whole blood 100 μ l, by specification said determination method is carried out detecting operation.Recording HBV specific CTL value is 1.8%.
Embodiment 3
Get chronic hepatitis B patient whole blood 100 μ l, by specification said determination method is carried out detecting operation.Recording HBV specific CTL value is 0.8%.
Claims (1)
1, a kind of flow cytometry cytokines measurement method detects the method for hepatitis B specific T-cells, it is characterized in that using flow cytometry cytokines measurement method that whole blood is detected, under the environment that cell factor sealer brefeldin A is arranged, in whole blood, add stimulator antigen and collaborative stimulant carries out antigenic stimulus, stimulate washing after six hours, identification lymphocyte and splitting erythrocyte, wash subsequently and rupture of membranes, use the fluorescent-labeled antibody pair cell inner cell factor to carry out fluorescence labeling dyeing, up flow type cell instrument analysis after finishing at last;
1. antigenic stimulus: measure adding whole blood 100 μ l in the pipe at fluidic cell, brefeldin A 10 μ l, CD28/CD49d 2.5 μ l and HBcAg specific polypeptide 1 μ l will measure pipe and place CO2gas incubator, breed 6 hours under 5%CO2,37 ℃ of conditions;
2. washing: the PBS liquid 2ml with precooling washs 1 time, and centrifugal 1500rpm/ branch is abandoned supernatant, removes remaining brefeldin A, CD28/CD49d and HBcAg specific polypeptide, stays lymphocyte and red blood cell;
3. lymphocyte identification: add CD8PerCP-Cy5.5 10 μ l, CD69 PE10 μ l lucifuge 30 minutes, identification CD8 is positive and by the antigen activated lymphocytes;
4. splitting erythrocyte: add hemolysin 2ml, lucifuge 15 minutes;
5. centrifugal, washing: centrifugal 1500rpm/ branch, abandon supernatant, the PBS liquid 2ml repeated washing of usefulness precooling 2 times is removed red blood cell;
6. rupture of membranes: add rupture of membranes agent 0.5ml, lucifuge 10 minutes is punched to lymphocytic cell membrane, makes to guarantee that simultaneously intracellular matter does not leak by the cytokine antibodies cell of can freely coming in and going out;
7. washing: the PBS liquid with precooling washs 1 time, and centrifugal 1500rpm/ branch is abandoned supernatant, removes the rupture of membranes agent;
8. dyeing: add IFN-r FITC, lucifuge 30 minutes makes intracytoplasmic gamma interferon be dyeed by anti-gamma interferon fluorescence antibody mark;
9. cells were tested by flow cytometry: the magnitude of voltage of regulating flow cytometer forward direction FS, side direction SS two parameters, most lymphocytes are presented in the two-parameter point diagram of FS/SS, make up CD8PerCP-Cy5.5 and CD69PE histogram, identify the CD8 that has been activated in the drawings
+/ CD69
+Cell makes up IFN-rFITC one-parameter histogram, and sets back, negative zone and detect positive sample, as positive particle occurs, just be identified as the T cell that the hepatitis B antigen peptide is had the anti-property of specificity.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469310A (en) * | 2007-12-28 | 2009-07-01 | 北京百奥科生物技术有限公司 | Method for enriching target cells from biology specimen and method for removing leucocyte |
| CN101445784B (en) * | 2008-12-31 | 2010-09-15 | 浙江工业大学 | Eupenicillium brefeldianum variety ZJB082702 and application thereof in preparation of Brefeldin A by fermentation |
| CN104969056A (en) * | 2013-02-01 | 2015-10-07 | 贝克顿迪金森公司 | Methods and systems for assessing sample behavior in a flow cytometer |
| CN107478563A (en) * | 2017-07-19 | 2017-12-15 | 浙江省人民医院 | A kind of method based on the double cell factors of Flow cytometry tuberculosis specificity |
| CN109294987A (en) * | 2018-11-05 | 2019-02-01 | 中山大学附属第医院 | Non-therapeutic method for recovering immune function of infiltrating T lymphocytes |
| CN113917159A (en) * | 2021-09-30 | 2022-01-11 | 杭州联科生物技术股份有限公司 | Flow detection method of lymphocyte subpopulation |
-
2006
- 2006-01-13 CN CN 200610037814 patent/CN1811452A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469310A (en) * | 2007-12-28 | 2009-07-01 | 北京百奥科生物技术有限公司 | Method for enriching target cells from biology specimen and method for removing leucocyte |
| CN101445784B (en) * | 2008-12-31 | 2010-09-15 | 浙江工业大学 | Eupenicillium brefeldianum variety ZJB082702 and application thereof in preparation of Brefeldin A by fermentation |
| CN104969056A (en) * | 2013-02-01 | 2015-10-07 | 贝克顿迪金森公司 | Methods and systems for assessing sample behavior in a flow cytometer |
| CN107478563A (en) * | 2017-07-19 | 2017-12-15 | 浙江省人民医院 | A kind of method based on the double cell factors of Flow cytometry tuberculosis specificity |
| CN109294987A (en) * | 2018-11-05 | 2019-02-01 | 中山大学附属第医院 | Non-therapeutic method for recovering immune function of infiltrating T lymphocytes |
| CN113917159A (en) * | 2021-09-30 | 2022-01-11 | 杭州联科生物技术股份有限公司 | Flow detection method of lymphocyte subpopulation |
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