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In the name of God. Summer School. Influenza Unit, Pasteur Institute of Iran summer 2011. ELISA. Dr V.Mazaheri. Introduction . Enzyme Linked Immunosorbent Assay Immunologic test Enzyme as a reporter label Qualify &Quantify
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Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
ELISA Dr V.Mazaheri
Introduction • Enzyme Linked Immunosorbent Assay • Immunologic test • Enzyme as a reporter label • Qualify &Quantify • suited to diagnosis at the screening level, surveillance where larger scale sample research Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Introduction • Many of the accepted standard assays in many scientific fields are ELISA-based • In conjunction with molecular method for identifying and characterizing disease agents and to assess disease prevalence • 2 Research groups in Sweden and Netherlands • Literature search from 1970s Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Introduction Why ELISA? Versatile -Passive attachment to solid phase -Easy separation of bound &unbound reactants Simple • Microtiter plate • Kit feasible Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Introduction Sensitive Quantifiable - color , read by eye • Multichannel spectrophotometer • Data can be stored • Data can be analyzed statistically Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Brief definition of terms Solid phase Usually a microtiter plate well Adsorption The process of adding an antigen or antibody, diluted in buffer Washing with a buffered solution to separate bound (reacted) from unbound (unreacted) reagents Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Brief definition of terms Antigens A protein or carbohydrate Antibodies Produced in response to antigenic stimuli Antispecies antibodies E.g. guinea pig serum injected into a rabbit elicits the production of rabbit anti-guinea pig antibodies. Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Brief definition of terms Enzyme A substance that can react at low concentration as a catalyst to promote a specific reaction Enzyme conjugate An enzyme that is attached irreversibly to a protein, usually an antibody Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Brief definition of terms Substrate A chemical compound with an enzyme reacts specifically (a color reaction) Stopping The process of stopping the action of an enzyme on a substrate Reading Measurement of color produced Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
ELISA Methods Direct ELISA Indirect ELISA Sandwich ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Direct ELISA Summer School Ag Wash Ab specific for Ag (conjugate) Wash Substrate Influenza Unit, Pasteur Institute of Iran summer 2011
Indirect ELISA Summer School Ab Wash Ag Substrate Ab conjugated (antispecies) Influenza Unit, Pasteur Institute of Iran summer 2011
Sandwich ELISA -Measures the amount of antigen between two layers of antibodies - The antigens must be contain at least two antigenic sites - Valuable when the concentration of antigens is low Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
Direct Sandwich ELISA Summer School Ab Ag Substrate Ab conj (same on s.p or different species) Influenza Unit, Pasteur Institute of Iran summer 2011
Indirect Sandwich ELISA Ab Ag Ab (different species) Summer School Substrate Anti-species conj Influenza Unit, Pasteur Institute of Iran summer 2011
General information Solid Phase -Microtiter 96-well plate - Polyvinyl chloride(flexible) • Polystyrene (inflexible) Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Coating - Diffusion coefficient of the attaching molecule - Ratio of the surface area being coated to the volume of the coating solution - Concentration of the substance being adsorbed - Temperature - Time of adsorption - Coating buffer Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Washing • Separate bound and unbound • Washing buffer • At least 3 times for every well • Washing methods Dilution buffer Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Blocking Conditions and Nonspecific Reactions - To prevent nonspecific adsorption of proteins to wells from samples added after the coating of the solid phase before, during, or as a combination of both times • High background color • Blocking agents: BSA, Nonfat dried milk , Gelatin,Detergents ,… Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Enzyme Conjugates • Usually antibodies are conjugated to enzymes • The species in which the antiserum is produced can be important - Different sources and batches of conjugates from the same manufacturer may vary - The linkage of an enzyme to an Ag or Ab affect the specificity of an assay Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Enzyme Conjugates • Horseradish Peroxidase Plus Hydrogen Peroxide Substrate such as OPD, TMB,ABTS,… • AlkalinePhosphatase Plus p-Nitrophenylphosphate Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Substrate • Substrate concentrations must be optimized • Certain solutions can be made and stored e.g.OPD Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Stopping Reactions -Molar concentrations of strong acids or strong bases stop enzymic activity by quickly denaturing enzymes • Wavelength & Color • TMB(Blue) Yellow,450 nm • OPD(Green/Orange) Orange/Brown,492 nm Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
General information Reading • Spectrophotometric Reading • Optic Density Summer School Influenza Unit, Pasteur Institute of Iran summer 2011
