Materi kuliah tentang tentang enzyme-linked immuno absorbent assay.

1. ELISA
Enzyme-Linked Immunosorbent
Assay

2. Based on antibodies
• What is an antibody?
• What types of antibodies exist?
• What kinds of antibodies are used in our lab
today?

3. 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

4. Polyclonal Antibodies
Rat
Rabbit
Sheep
Goat
Rat
Rabbit
Sheep
Goat

5. Monoclonal
antibodies
Rat

6. Antibody-based assays

7. 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
HRP
HRP
HRP
HRP HRP
HRP
HRP
HRP
Ag Ag
HRP
Streptavidin
HRP
HRP HRP
HRP

8. 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

9. 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

10. Today’s assay

11. 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
HRP
HRP
HRP
HRP HRP
HRP
HRP
HRP
Ag Ag
HRP
Streptavidin
HRP
HRP HRP
HRP

12. 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.