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):

TBNK

(Separation of immunocytes)

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

peripheral blood mononuclear cellsPBMC 20min

-(ficoll-hypaque)(1.0770.001g/L)

1.0770.001/g/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

DC TDCBDCDCDC

DCTCD1CD40MHCTDCTBDC

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