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 suited to diagnosis at the screening level, surveillance where larger scale sample research
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

Introduction
Why ELISA? Versatile -Passive attachment to solid phase -Easy separation of bound &unbound reactants Simple - Microtiter plate - Kit feasible
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

Introduction
Summer School
Sensitive Quantifiable - color , read by eye - Multichannel spectrophotometer - Data can be stored - Data can be analyzed statistically

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

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.
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

ELISA Methods
Summer School
Direct ELISA Indirect ELISA Sandwich ELISA

Influenza Unit, Pasteur Institute of Iran

summer 2011

Direct ELISA
Summer School

Ag

Wash

Ab Wash specific for Ag Substrate

(conjugate)

Influenza Unit, Pasteur Institute of Iran

summer 2011

Indirect ELISA
Summer School

Ag

Ab

Wash

Substrate
Ab conjugated
(antispecies)
Influenza Unit, Pasteur Institute of Iran summer 2011

Sandwich ELISA
Summer School
-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

Influenza Unit, Pasteur Institute of Iran

summer 2011

Direct Sandwich ELISA

Summer School

Ab

Ag
Ab conj
(same on s.p or different species)

Substrate

Influenza Unit, Pasteur Institute of Iran

summer 2011

Indirect Sandwich ELISA

Summer School

Ab

Ag

Ab
(different species) Anti-species conj

Substrate

Influenza Unit, Pasteur Institute of Iran

summer 2011

General information
Summer School
Solid Phase -Microtiter 96-well plate - Polyvinyl chloride(flexible) - Polystyrene (inflexible)

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

General information
Summer School
Washing - Separate bound and unbound - Washing buffer - At least 3 times for every well - Washing methods Dilution buffer

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 ,
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

General information
Summer School
Enzyme Conjugates - Horseradish Peroxidase Plus Hydrogen Peroxide Substrate such as OPD, TMB,ABTS, - AlkalinePhosphatase Plus p-Nitrophenylphosphate

Influenza Unit, Pasteur Institute of Iran

summer 2011

General information
Summer School
Substrate - Substrate concentrations must be optimized - Certain solutions can be made and stored e.g.OPD

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
Influenza Unit, Pasteur Institute of Iran summer 2011

Summer School

General information
Summer School
Reading - Spectrophotometric Reading - Optic Density

Influenza Unit, Pasteur Institute of Iran

summer 2011

Thanks