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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 )

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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 Presentation

Text of 细胞免疫学检测 ( cellular immunoassay ) 一、淋巴细胞表面标志物检测 (Assay of...

  • cellular immunoassay

    (Assay of membran molecules for lymphocytes

  • (Technic of separation for immunocyte) Assay of membran molecules for Assay for Immunocyte Tuntion Assay for Cytokine and Adhesion molecule

  • (Immune system)(Immune Organs)(Immunocyte)(Immunoglobulin) (Major Histocompatibility Complex; MHC)(Cytekine)(Leukocyte Differentiation Antigen and Adhesion molecule)

  • (immune cells):


  • (Separation of immunocytes)

  • (peripheral blood )(principle) /60010001(method) 1RT3060min 2RBC

  • peripheral blood mononuclear cellsPBMC 20min

  • -(ficoll-hypaque)(1.0770.001g/L)


  • Percoll()

    (density)1.135g/L+PBS, (centrifugation)

    (density gradient)

    98%, 78%

    , ,

  • ()(purificcation)PBMC, 0.83%

  • 1 specific membrane molecule/surface marker 2antigen of cluster differentiation CD1

  • 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

  • 2 BT T31 (affinity ) 2 immunomagnetic beads 3 (FCM)

  • (magnetic beads):

    ; IgG(

  • positive selection negtive selection

  • (evalution)8099909599~100%FACS FACS FACSFACS 9599

  • Attentions

  • (principle)fluorescence activated cell sorterFACST

  • (evalution) FACS

  • (phagocytosis)Phagocytes ---- mononuclear macrophage neutrophileosinophilbasophilSeparation Mononuclear Percoll Macrophage Other

  • 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

  • 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

  • (Assays for immunocytes)

  • T lymphocytes T65%~70%Bone-marrow stem cellsbe derived(recycle)

  • TSubsetTCRTTTCD4CD8 TCD4 TTh1Th2CD8 T(cytotoxicity)T(suppresion)TNK 1.1 T(lipid)MHC

  • Type-I /2 T helperTh1,Th2Th1IL-2IFN-TNF-Th2IL-4 IL-5 IL-6 IL-10Th1Th2(regulation)(inhibition)(balance)(influence)

  • T-cytotoxic cells, Tc/CTL1CD8+T(effect)(target cells)MHC I

  • Tc/CTL2CTL(regulation)(tend to)IL-8IP-10IFN inducible protein 10IP-1(inflammatory reaction)CTLTc1Tc2

  • T-suppresion cells, Ts(passive transfer test)(confirm)(may be)Tsspecificsurface marker

  • B be derived BBBBplasma cellsIgBCRB-cell-receptor complex,BCR

  • pro/preB rearrangeBCR BCR (B-cell-receptor) : B-cell surface immunoglobulin and its associated signalling molecules, CD79a and CD79b.

  • immatureBSLpreBCR IgM IgMIgMBBCRBBrecognitionapoptosisBimmunological tolerance

  • matureBIgMIgD(recognition of antigen)B

  • activeBB710BIgBCR

  • B Subset CD5BB1B2 B1 CD5 +Th

  • B2 CD5 - Th

  • Other immune cellsNKnatural killermononuclear phagocytegranulocytesmast

  • NKnakedCD2CD16CD56CD69

  • mononuclear phagocyte mononuclear macrophageMnon-specific specificantigen-presenting

  • 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

  • Markers of mononuclear phagocyte MHC I II(co-stimulatory)CD40B7CD1CD2CD14CD64CD32CD16CD23CD35C3bCR3 C3bi

  • Dendritic cellDCDC21MHCB72DC



  • Granulocyte and Mast cellsGranulocyteBMbone marrowFcRIgEIMHCB7CD40CD40L

  • Assay for the surface markers of T lymphocyteTThTsTi(E-rosette)CD58(immunochemistry )(immunofluorescent)Tflow cytometry sorter, FCMS

  • Assay for the surface markers of B lymphocyte

    SmIgM (surface membrane )Fc(receptor)(complement)EAC sRBC+EA+C-- EACBCDFACS

  • 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 )

  • Assays of Cellular immune Tunction

  • 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

  • Lymphocyte proliferation rate: Blast cell count= ------------------------------- 100% Small lymphocyte + Blast cell count(200)

    Reference value70%

  • 3H-thymidine incorporation

    3H 3H TdR)+, DNA, DNA3H TdRSISIstimulating index= PHAcpm / cpm cpm

  • MTT colorimetry

    3-(4,5--2-)-2,5-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)MTT + / A570nmSI SI = A570nm / A570nm

  • T cell-mediated cytotoxicityTTCTLTtarget cellTarget cellApoptosis is a highly ordered process in which cells are systematically disassembled.Morphology ElectrophoresisDNA FACS or ELISA

  • Enzyme-linked immune spot,ELISPOT: , + (- ) -() + +,

  • Skin test in vivo

  • BIg IgBIgBIg

  • Skin test in vivoIntradermal injection

    Immediate (within 20 min)

    Late (at 4-12 hours)

    Delayed (24-48 hours)

  • Assays for Phagocyte function Phagocyte function: chemotaxis taken into digest

  • Chemotaxis of leucocyteChemotactic index (CI) = (A)/(B)


  • (Phagocytic)(bactericidal)

    +---, , phagocytic rate%= /100%phagocytic index%= /100%OthersNBT

  • T1T2T3CD3/CD4/CD8CD4/CD8T

  • B1B2BB

  • NKNKMHCPhagocyte

  • :1 2 3 4

  • 1 23TBNK 4T/B5