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Use of Antibodies (ELISA (Enzyme-Linked Immunosorbent Assay) (Types of…
Use of Antibodies
ELISA (Enzyme-Linked Immunosorbent Assay)
Types of Assay
Detect Protein
Indirect Assay
utilize secondary antibody compared to Direct method
Direct Assay
Proteins bind to well
add antibody with conjugated enzyme
add substrate and quantify signal
many proteins dumped at the same time, target antigen might not bind
low sensitivity
Conjugated Assay
very slow. can attach enzyme to primary antibody but less signal
Well has primary antibody
binds antigen only
add 2nd primary body that binds antigen
secondary antibody with conjugated enzyme
Detect Antibody
use Indirect by coating the well with antigen
Quantify signal
standard curve of known concentration of analyze
Control
Positive
antigen with known concentration, and then make standard curve
Negative
no antigen added
Production of Antibodies
Animal production
Inject antigen into animal
Detect Antibody
first exposure and second exposure
another antigen injected for the first time -> initial antibody production is similar
B cells
organisms produce different B cells
each B cell produce 1 specific antigen
the one that binds to the antigen proliferate
Types of Antibodies
Polyclonal
Multiple B cell clones
Recognize multiple epitopes
Monoclonal
B-cell clone "Hybridoma"
B Cells can't survive in culture
fuse it with a mutant that need to be provided purine
select only the hybrid by cutting purine in culture
ELISA to test antibody
Immunoprecipitation
isolate specific proteins + binding partners
lyse cells to release antigen and its binding partners
mix cell lysate + antibody coated beads
Attach antibody to protein A, which tethered to beads
wash off others, and pallet target
release target by boiling or changing in pH, or increase in salt
Western Blotting
Identify protein antigen after SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Immunofluorescence microscopy
localize specific proteins
analyze with confocal microscope
secondary antibody has fluorescent dye
cell is fixed
primary antibodies enter and bind antigen