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

Types of immunodetection systems

1. Direct immunodetection
Primary antibody conjugated with enzyme system

2. Indirect immunodetection
Secondary antibody conjugated with enzyme system
HRP HRP

HRP

HRP

Ag

Ag

Ag

Ag

3. Sandwich indirect immunodetection

Antigen applied in soluble form

4. Indirect immunodetection with biotin linkers

Biotinylated primary antibodies

HRP

HRP

HRP

HRP
HRP HRP

HRP

HRP

HRP Ag Ag

Ag

Ag

Streptavidin

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

3. Add second primary antibody

against antigen 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

Todays assay

Types of immunodetection systems

1. Direct immunodetection
Primary antibody conjugated with enzyme system

2. Indirect immunodetection
Secondary antibody conjugated with enzyme system
HRP HRP

HRP

HRP

Ag

Ag

Ag

Ag

3. Sandwich indirect immunodetection

Antigen applied in soluble form

4. Indirect immunodetection with biotin linkers

Biotinylated primary antibodies

HRP

HRP

HRP

HRP
HRP HRP

HRP

HRP

HRP Ag Ag

Ag

Ag

Streptavidin

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.