ELISA - Enzyme-Linked Immunosorbent Assay

ELISA (Enzyme-Linked Immunosorbent Assay) Purpose • Quick test for a wide range of substances • Field test friendly •Disease detection •Chemical detection •Diagnostic tools Limitations • Results may not be absolute • Antibody must be available •Concentration may be unclear •False positive possible •False negative possible Materials Needed • Testing sample • Antibody (1 st , 2 nd ) / Antigen • Polystyrene microtiter plate • Blocking buffer • Washing buffer • Substrate • Enzyme Antibody Binding Domains Epitope and antibody Epitope: The part of the antigen that antibody recognizes Antibody Precipitation a) Contain antibody against pneumococcal polysaccharide on bacteria b) Polysaccharide solution added c) Insoluble antibody-antigen complex formed d) Precipitation occurs Polyclonal Antibody  Inject an animal with specific antigen Antigen  Antibodies are produced against different epitopes.  Repeat antigen inoculation to boost antibody production  Harvest serum from blood  Ex: Rabbit, goat, sheep Monoclonal Antibody  Inject an animal with specific antigen  Antibodies are produced against different epitopes.  Spleen of the animal is harvested for B cells.  Fusion of specific B cell with tumor cells to create hybridomas (immortal B cells)  Screen for antibody producing hybridomas  Culture hybridomas and purify specific antibodies Antibody Vocabulary  Primary antibody (1° Ab) ex: mouse anti-actin  Secondary antibody (2° Ab) ex: goat anti-mouse  Conjugated Enzyme ex: goat anti-mouse with HRP  Substrate (enzyme conversion) ex: DAB Antigen [...].. .ELISA for Antibody Detection (Enzyme-Linked Immunosorbent Assay) • • • • • • • • • • Capture of antigens in microplate Washes Block unbound sites in wells Washes Add antibody serum to wells Washs Add 2nd antibody (attach enzyme) Washes Color development Ex: HIV test Sandwich ELISA for Antigen Detection (Enzyme-Linked Immunosorbent Assay) • • • • • • • Capture 1st antibody... Color development • Ex: Pregnancy test Enzyme-Mediated Detection Enzyme Horseradish Peroxidase Substrate Abbrev Color Diaminobenzidine DAB Brown Enzyme Alkaline Phosphatase (AP) Substrate Abbrev Color Bromochloroindolylphosphate BCIP (AP substrate) Purple Nitro Blue Tetrazolium NBT (Enhance color) Horseradish Peroxidase Enzyme DAB Substrate DAB + H202 -HRP -> DAB ppt + H20 AP Enzyme BCIP &NBT Substrate . ELISA (Enzyme-Linked Immunosorbent Assay) Purpose • Quick test for a wide range of substances • Field test friendly •Disease. anti-actin  Secondary antibody (2° Ab) ex: goat anti-mouse  Conjugated Enzyme ex: goat anti-mouse with HRP  Substrate (enzyme conversion) ex: DAB Antigen ELISA for Antibody Detection (Enzyme-Linked. (attach enzyme) • Washes • Color development • Ex: HIV test Sandwich ELISA for Antigen Detection (Enzyme-Linked Immunosorbent Assay) • Capture 1 st antibody in microplate • Washes • Block unbound