ELISA

Enzyme-Linked Immunosorbent Assay

Based on antibodies

• What is an antibody?

• What types of antibodies exist?

• What kinds of antibodies are used in our lab today?

What does an antibody look like?

-Immunoglobulin domains

-Complementarity-determining

Regions (CDRs)

-Hinge

-Fc= Fragment crystalline

-Fab= Fragment antigen binding

-F(ab)’2= Protease digestion still

useful to bind antigen

Polyclonal Antibodies

Monoclonal antibodies

Antibody-based assays

Ag Ag

Types of immunodetection systems

1. Direct immunodetection Primary antibody conjugated with enzyme system

2. Indirect immunodetection Secondary antibody conjugated with enzyme system

3. Sandwich indirect immunodetection Antigen applied in soluble form

4. Indirect immunodetection with biotin linkers Biotinylated primary antibodies

Ag Ag AgAg

HRPHRP

HRPHRP HRPHRP

HRPHRP

Ag Ag

HRP

Streptavidin

HRP

HRP HRP

HRP

Simple ELISA protocol(For screening monoclonal antibodies)

1. Coat antigen onto microplate

2. Allow protein adsorption and block unoccupied sites with neutral protein

3. Add antibody solution (hybridoma supernatant) into each well

4. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate

5. Read absorbance in spectrophotometer with appropriate filter and quantitate relative antigen levels

Sandwich ELISA protocol

1. Coat primary antibody onto microplate

1a. Allow antibody adsorption and block unoccupied sites with neutral protein (BSA)

2. Add antigen to be detected in biological (clinical) material into each well

4. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate

5. Read absorbance in ELISA spectrophotometer with appropriate filter and quantitate relative antigen levels

3. Add second primary antibody against antigen into each well

Today’s assay

Ag Ag

Types of immunodetection systems

1. Direct immunodetection Primary antibody conjugated with enzyme system

2. Indirect immunodetection Secondary antibody conjugated with enzyme system

3. Sandwich indirect immunodetection Antigen applied in soluble form

4. Indirect immunodetection with biotin linkers Biotinylated primary antibodies

Ag Ag AgAg

HRPHRP

HRPHRP HRPHRP

HRPHRP

Ag Ag

HRP

Streptavidin

HRP

HRP HRP

HRP

NPTII

Two neomycin phosphotransferase genes are used in selection of transformed organisms: the neomycin phosphotransferase I (nptI) gene and the neomycin phosphotransferase II (nptII) gene. The second one is the more widely used. It was initially isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. The gene codes for the aminoglycoside 3'-phosphotransferase (denoted aph(3')-II or NPTII) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as:

* kanamycin * neomycin * geneticin (G418), and * paromomycin.