清华大学：现代生命科学实验教学中心《生物化学基础实验》教学课件（PPT讲稿）Enzyme-linked immunosorbent assay（ELISA）

Enzyme-linkedimmunosorbentassay(ELISA)ELISA

Enzyme-linked immunosorbent assay (ELISA) ELISA

ImmunoassayThe technique of immunoassay usinglabelledreagents for detectingantigensandantibodiesare exquisitelysensitiveandextremelyeconomicalinthereagents(immunoassayforantibody)Solid-phaseassaysforantibodiesemployingligandslabelledwithradioisotopesorenzymes(enzyme-linkedimmunosorbentassay;ELisA)aremost widelyusedofallimmunologicalassays

Immunoassay ⚫ The technique of immunoassay using labelled reagents for detecting antigens and antibodies are exquisitely sensitive and extremely economical in the reagents(immunoassay for antibody). ⚫ Solid-phase assays for antibodies employing ligands labelled with radioisotopes or enzymes(enzyme-linked immunosorbent assay; ELISA) are most widely used of all immunological assays

Enzyme-linkedimmunosorbentassay(ELISA)·lnthissystem,ligandisamoleculewhichcan detect the antibody andis covalentlycoupledtoanenzymesuchasperoxidase.The amountoftestantibodyismeasuredbyassessingthemountofcoloured end-productbyoptical densityscanningoftheplate

Enzyme-linked immunosorbent assay(ELISA) ⚫ In this system, ligand is a molecule which can detect the antibody and is covalently coupled to an enzyme such as peroxidase.The amount of test antibody is measured by assessing the mount of coloured end-product by optical density scanning of the plate

Atypicaltitrationcurve·Antibodytitrescanonlybedetectedcorrectly within the linear range.Typicallythe plateaubindingis 20-100timesthebackground.Thesensitivityofthetechniqueisusually about1-50ng/mlofspecificantibody

A typical titration curve ⚫ Antibody titres can only be detected correctly within the linear range. Typically the plateau binding is 20-100 times the background. The sensitivity of the technique is usually about 1-50ng/ml of specific antibody

ProcedureofELiSAIncubate microtitreplatewellwithantigen;Washoffunbound antigen;Incubate withantibodyWash off unbound antibody;Incubate with labelledanti-immunoglobulin;Washoff unbound labelledantibody;Count/incubate with enzyme substratesolution

Procedure of ELISA ⚫ Incubate microtitre plate well with antigen; ⚫ Wash off unbound antigen; ⚫ Incubate with antibody; ⚫ Wash off unbound antibody; ⚫ Incubate with labelled anti-immunoglobulin; ⚫ Wash off unbound labelled antibody; ⚫ Count/incubate with enzyme substrate solution

KidsofTestingofSARSvirusTherecombinationalproteinofSARSvirusHuman serum(containingtheantibodyofSARSvirus?)(differentdilutionofserum)Polyclonal antibody againstHumanlgG-HRP(horseradishperoxidase)Substratesolution,OD490nm

Kids of Testing of SARS virus ⚫ The recombinational protein of SARS virus ⚫ Human serum(containing the antibody of SARS virus?) (different dilution of serum) ⚫ Polyclonal antibody against HumanIgG-HRP (horseradish peroxidase) ⚫ Substrate solution, OD490nm

Kidsof TestingofHBsAgMonoclonalantibodyagainstHBsAg(anti-HBsAgantibody)Humanserum(containing HBsAg?)Polyclonal antibodyagainstHBsAg-HRP(horseradishperoxidase)(anti-HBsAg-HRP)Substratesolution,OD450nm

Kids of Testing of HBsAg ⚫ Monoclonal antibody against HBsAg(anti￾HBsAg antibody) ⚫ Human serum(containing HBsAg?) ⚫ Polyclonal antibody against HBsAg￾HRP(horseradish peroxidase)(anti￾HBsAg-HRP) ⚫ Substrate solution, OD450nm

OurTestofELISAAntigen:HumanIgG(5μg/well)·Firstantibody:Rabbitanti-humanIgGantiserum(serumdilution)Secondaryantibody:Goatanti-rabbitIgGHRP(1:20,000)

Our Test of ELISA ⚫ Antigen: Human IgG (5mg/well). ⚫ First antibody: Rabbit anti-human IgG antiserum (serum dilution). ⚫ Secondary antibody:Goat anti-rabbit IgG￾HRP (1:20,000)

IMMUNOLOGICALTECHNIQUES1.For recognizingand quantifyingantigensintissuesorfluidsmanyimmunologicaltechniques utilizethe exquisitespecificity of theantigen-antibodybond.2. Cell populations canbe identifiedandcharacterizedby theirsurface markers,usingthetechniques ofimmunofluorescence orimmunohistochemistry

IMMUNOLOGICAL TECHNIQUES ⚫ 1. For recognizing and quantifying antigens in tissues or fluids many immunological techniques utilize the exquisite specificity of the antigen-antibody bond. ⚫ 2. Cell populations can be identified and characterized by their surface markers, using the techniques of immunofluorescence or immunohistochemistry

IMMUNOLOGICALTECHNIQUES.3.Cellpopulationscanbeisolatedaccordingtotheirsurfacemarkers,bytechniqueswhichincludefluorescence-activated cellsorting(FACS),panninganddensity-dependentcentrifugations.4.Theprincipleassaysforlymphocytefunctionare by antibody or cytokine production,byproliferationinresponsetoantigen,orbycytotoxicity

IMMUNOLOGICAL TECHNIQUES ⚫ 3. Cell populations can be isolated according to their surface markers, by techniques which include fluorescence-activated cell sorting (FACS), panning and density-dependent centrifugations. ⚫ 4. The principle assays for lymphocyte function are by antibody or cytokine production, by proliferation in response to antigen, or by cytotoxicity