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