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 (1st, 2nd) / 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
Contain antibody against pneumococcal polysaccharide on bacteria
Polysaccharide solution added
Insoluble antibody-antigen complex formed
Precipitation occurs
Polyclonal Antibody
Inject an animal with specific antigen
Antibodies are produced against different epitopes.
Repeat antigen inoculation to boost antibody production
Harvest serum from blood
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 2nd antibody (attach enzyme)
Washes
Color development
Ex: HIV test
Sandwich ELISA for Antigen Detection
(Enzyme-Linked Immunosorbent Assay)
Capture 1st antibody in microplate
Washes
Block unbound sites in wells
Washes
Add antigen serum to wells
Washes
Add labeled 2nd antibody specific to antigen of interest (with substrate)
Washes
Color development
Ex: Pregnancy test
Enzyme-Mediated Detection
Horseradish Peroxidase Enzyme
DAB Substrate
DAB + H202 -------HRP-------> DAB ppt + H20

AP Enzyme
BCIP &NBT Substrate