细胞免疫学检测 ( cellular immunoassay ) 一、淋巴细胞表面标志物检测 (assay of...
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细胞免疫学检测 ( cellular immunoassay ) 一、淋巴细胞表面标志物检测 (Assay of membran molecules for lymphocytes ). 一、免疫细胞分离技术 (Technic of separation for immunocyte) 二、免疫细胞表面标志检测技术 ( Assay of membran molecules for ~ ) 三、免疫细胞功能检测技术 ( Assay for Immunocyte Tuntion ) 四、细胞因子与黏附分子检测技术 - PowerPoint PPT PresentationTRANSCRIPT
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cellular immunoassay
(Assay of membran molecules for lymphocytes
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(Technic of separation for immunocyte) Assay of membran molecules for Assay for Immunocyte Tuntion Assay for Cytokine and Adhesion molecule
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(Immune system)(Immune Organs)(Immunocyte)(Immunoglobulin) (Major Histocompatibility Complex; MHC)(Cytekine)(Leukocyte Differentiation Antigen and Adhesion molecule)
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(immune cells):
TBNK
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(Separation of immunocytes)
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(peripheral blood )(principle) /60010001(method) 1RT3060min 2RBC
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peripheral blood mononuclear cellsPBMC 20min
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-(ficoll-hypaque)(1.0770.001g/L)
1.0770.001/g/L
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Percoll()
(density)1.135g/L+PBS, (centrifugation)
(density gradient)
98%, 78%
, ,
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()(purificcation)PBMC, 0.83%
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1 specific membrane molecule/surface marker 2antigen of cluster differentiation CD1
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Precipitation of E rosetteErythrocyte Rosette forming cell; ERFC The sueface of T lymphocyte contains the receptor (CD2) of sheep erythrocyte ,which is called E receptor for short . T lympho-cytes can form E rosette with sheep erythro-cytes by its E receptor. Lymphocytes + sRBC , centrifugation----- T lympho-cytes() / B lympho-cytes
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2 BT T31 (affinity ) 2 immunomagnetic beads 3 (FCM)
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(magnetic beads):
; IgG(
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positive selection negtive selection
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(evalution)8099909599~100%FACS FACS FACSFACS 9599
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Attentions
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(principle)fluorescence activated cell sorterFACST
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(evalution) FACS
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(phagocytosis)Phagocytes ---- mononuclear macrophage neutrophileosinophilbasophilSeparation Mononuclear Percoll Macrophage Other
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Dendritic cellDendritic cellsDCsare the most potent antigen presenting cellAPCknownwhich have numerous specialized features that make them extremely efficient at capturing and presenting antigen and activating T cells and mediating immune response. T
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Dendritic cellDCs can be generatedin vitrofrom many different samples of human beings.DCs only constitue 0.51.0% of the peripheral blood mononuclear cells.DCs can be generated from monocyto/M + IL-4 and GM-CSFIdentifing DCs by CD marker
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(Assays for immunocytes)
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T lymphocytes T65%~70%Bone-marrow stem cellsbe derived(recycle)
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TSubsetTCRTTTCD4CD8 TCD4 TTh1Th2CD8 T(cytotoxicity)T(suppresion)TNK 1.1 T(lipid)MHC
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Type-I /2 T helperTh1,Th2Th1IL-2IFN-TNF-Th2IL-4 IL-5 IL-6 IL-10Th1Th2(regulation)(inhibition)(balance)(influence)
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T-cytotoxic cells, Tc/CTL1CD8+T(effect)(target cells)MHC I
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Tc/CTL2CTL(regulation)(tend to)IL-8IP-10IFN inducible protein 10IP-1(inflammatory reaction)CTLTc1Tc2
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T-suppresion cells, Ts(passive transfer test)(confirm)(may be)Tsspecificsurface marker
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B be derived BBBBplasma cellsIgBCRB-cell-receptor complex,BCR
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pro/preB rearrangeBCR BCR (B-cell-receptor) : B-cell surface immunoglobulin and its associated signalling molecules, CD79a and CD79b.
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immatureBSLpreBCR IgM IgMIgMBBCRBBrecognitionapoptosisBimmunological tolerance
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matureBIgMIgD(recognition of antigen)B
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activeBB710BIgBCR
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B Subset CD5BB1B2 B1 CD5 +Th
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B2 CD5 - Th
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Other immune cellsNKnatural killermononuclear phagocytegranulocytesmast
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NKnakedCD2CD16CD56CD69
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mononuclear phagocyte mononuclear macrophageMnon-specific specificantigen-presenting
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Different denomination Of mononuclear phagocyteBlood mononuclear cellsGeneral tissue macrophageMConnective tissue tissue cellsLiver Kupffers cellLung alveolar cellsSkin Langerhans cellNervous system micro-glia cells Abdomen abdomen() M
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Markers of mononuclear phagocyte MHC I II(co-stimulatory)CD40B7CD1CD2CD14CD64CD32CD16CD23CD35C3bCR3 C3bi
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Dendritic cellDCDC21MHCB72DC
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DC TDCBDCDCDC
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DCTCD1CD40MHCTDCTBDC
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Granulocyte and Mast cellsGranulocyteBMbone marrowFcRIgEIMHCB7CD40CD40L
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Assay for the surface markers of T lymphocyteTThTsTi(E-rosette)CD58(immunochemistry )(immunofluorescent)Tflow cytometry sorter, FCMS
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Assay for the surface markers of B lymphocyte
SmIgM (surface membrane )Fc(receptor)(complement)EAC sRBC+EA+C-- EACBCDFACS
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Assay for the surface markers of NK cellsNatural Killer(NK)cells express neither T cell nor B cell antigen receptors.CD16 and CD56 molecule are important markere of NK cells. ADCC()(antibody-dependent cellular cytotoxicity )
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Assays of Cellular immune Tunction
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Lymphocyte Transformation TestPrinciple: Antigens or mitogens (such as PHA and ConA ) are incubated with T lymphocytes in culture medium, allowing stimulation of T lymphocyte activity to transform into lymphoblasts.Methods: morphologic counting 3H-thymidine incorporation MTT colorimetry
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Lymphocyte proliferation rate: Blast cell count= ------------------------------- 100% Small lymphocyte + Blast cell count(200)
Reference value70%
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3H-thymidine incorporation
3H 3H TdR)+, DNA, DNA3H TdRSISIstimulating index= PHAcpm / cpm cpm
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MTT colorimetry
3-(4,5--2-)-2,5-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)MTT + / A570nmSI SI = A570nm / A570nm
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T cell-mediated cytotoxicityTTCTLTtarget cellTarget cellApoptosis is a highly ordered process in which cells are systematically disassembled.Morphology ElectrophoresisDNA FACS or ELISA
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Enzyme-linked immune spot,ELISPOT: , + (- ) -() + +,
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Skin test in vivo
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BIg IgBIgBIg
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Skin test in vivoIntradermal injection
Immediate (within 20 min)
Late (at 4-12 hours)
Delayed (24-48 hours)
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Assays for Phagocyte function Phagocyte function: chemotaxis taken into digest
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Chemotaxis of leucocyteChemotactic index (CI) = (A)/(B)
()
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(Phagocytic)(bactericidal)
+---, , phagocytic rate%= /100%phagocytic index%= /100%OthersNBT
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T1T2T3CD3/CD4/CD8CD4/CD8T
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B1B2BB
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NKNKMHCPhagocyte
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:1 2 3 4
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1 23TBNK 4T/B5