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Principles of the Elisa Technique

By Maria da Costa

Competitive EIA

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Competitive Elisa (1)

Competitive Elisa (2)

Microwell coated with anti-IgG antibodies

Add Standard or Sample

anti-IgG antibodies

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Competitive Elisa (3)

Competitive Elisa (4)

EE EE EE

Add Enzyme Conjugate

EE

EE EE EE

Add Antibody Solution

EE

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Competitive Elisa (5)

Competitive Elisa (6)

EE

EE

Reaction starts

EE

EE

EE

Wash 3x

EE

EE

Equilibrium between bound and unbound analyte of standards or samples and enzyme-conjugated

EE

Removal of excessive reagents

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Competitive Elisa (6)

Competitive Elisa (7)

After 3x Washing

EE EE

S

EE

P

EE

Add Substrate/Chromogen

Total removing of unbound reagents

Substrate (S) develops a colored Product (P) in the presence of enzyme

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Competitive Elisa (8)

FAST test standard curve

Add Stop Solution

EE EE

Reaction is stopped and the developed color is measured visually or by means of a spectrophotometer

The intensity of the color inversely proportional to the concentration of the sample/standard - the more color, the lower the concentration in the sample/standard

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Long test standard curve

Sandwich EIA

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (1)

Sandwich Elisa (2)

Micro well coated with antigen specific antibodies

Add Standard or Sample from 30 to 60 min

Required incubation time

antigen specific antibodies

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (3)

Sandwich Elisa (3)

Required incubation time from 30 to 60 min There will be an equilibrium of bound and unbound antigen

Wash 3x

Removal of unbound antigen of standard or sample

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (4)

Sandwich Elisa (4)

Add antibody-enzyme conjugate

Add antibody-enzyme conjugate

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (4)

Sandwich Elisa (4)

Add antibody-enzyme conjugate

Add antibody-enzyme conjugate

There will be a binding to the antigen-antibody complex

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (5)

Sandwich Elisa (6)

Wash 3x

After 3x Washing

Removal of unbound antibody-enzyme conjugate

Total removing of unbound antibody-enzyme conjugate

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich Elisa (7)

Sandwich Elisa (8)

S

P

Add Substrate/Chromogen

Add Stop Solution

Substrate (S) develops a colored Product (P) in the presence of enzyme

Reaction is stopped and the developed color is measured by means of a spectrophotometer

The intensity of the color is directly proportional to the concentration of the sample/standard - the more color, the higher is the concentration in the sample/standard

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

Sandwich standard curve

DaCosta //

R Biopharm AG ­ Dolivostrasse 10 ­ D-64293 Darmstadt

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Microsoft PowerPoint - Elisa tests.ppt

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