ELISA - Enzyme-Linked Immunosorbent Assay

ELISA
(Enzyme-Linked Immunosorbent Assay)

Purpose
•
Quick test for a wide range of substances
•
Field test friendly
•Disease detection
•Chemical detection
•Diagnostic tools

Limitations
•
Results may not be absolute
•
Antibody must be available
•Concentration may be unclear
•False positive possible
•False negative possible

Materials Needed
•
Testing sample
•
Antibody (1
st
, 2
nd
) / Antigen

•
Polystyrene microtiter plate
•
Blocking buffer
•
Washing buffer
•
Substrate
•
Enzyme

Antibody Binding Domains

Epitope and antibody
Epitope: The part of the antigen that antibody recognizes

Antibody Precipitation
a) Contain antibody against pneumococcal
polysaccharide on bacteria
b) Polysaccharide solution added
c) Insoluble antibody-antigen complex formed
d) Precipitation occurs

Polyclonal Antibody

Inject an animal with
specific antigen
Antigen

Antibodies are produced

against different epitopes.

Repeat antigen inoculation
to boost antibody production

Harvest serum from blood

Ex: Rabbit, goat, sheep

Monoclonal Antibody

Inject an animal with specific
antigen

Antibodies are produced against
different epitopes.

Spleen of the animal is harvested
for B cells.

Fusion of specific B cell with
tumor cells to create hybridomas
(immortal B cells)

Screen for antibody producing
hybridomas

Culture hybridomas and purify
specific antibodies

Antibody Vocabulary

Primary antibody (1° Ab)
ex: mouse anti-actin

Secondary antibody (2° Ab)
ex: goat anti-mouse

Conjugated Enzyme
ex: goat anti-mouse with HRP

Substrate (enzyme conversion)
ex: DAB
Antigen

ELISA for Antibody Detection
(Enzyme-Linked Immunosorbent Assay)
•
Capture of antigens in microplate
•
Washes
•
Block unbound sites in wells
•
Washes
•
Add antibody serum to wells
•
Washs
•

Add 2
nd
antibody (attach enzyme)
•
Washes
•
Color development
•
Ex: HIV test

Sandwich ELISA for Antigen Detection
(Enzyme-Linked Immunosorbent Assay)
• Capture 1
st
antibody in microplate
•
Washes
•
Block unbound sites in wells
•
Washes
• Add antigen serum to wells
• Washes
•
Add labeled 2
nd
antibody specific to antigen of interest (with
substrate)
• Washes
• Color development

• Ex: Pregnancy test

Enzyme-Mediated Detection
Enzyme
Horseradish Peroxidase
Substrate Abbrev. Color
Diaminobenzidine DAB Brown
Enzyme
Alkaline Phosphatase (AP)
Substrate Abbrev. Color
Bromochloroindolylphosphate
Nitro Blue Tetrazolium
BCIP (AP substrate)
NBT (Enhance color)
Purple

Horseradish Peroxidase Enzyme
DAB Substrate
DAB + H
2
0
2
HRP > DAB ppt + H
2
0

AP Enzyme
BCIP &NBT Substrate