ELISA for Antibody Flashcards
Blotto
Unrelated protein from nonfat dried milk that nonspecifically blocks unbound sites
No antigen controls
Wells coated w PBS
for non-specific binding of the hybridoma supernatants or secondary reagents such as AP-GAM
Reactivity is only found in presence of coated antigen or coated antibody
Can determine if background is factor
Unrelated Antigen for Antigen Specificity Controls
Wells coated with unrelated antigen should not have reactivity
Used test antigen and an unrelated antigen for control
NMS
Normal Mouse Sera
Negative Control
From unimmunized mice –> should not have Ab that bind to target Ag
Should not bind to antigen-coated wells
PMS
Polyclonal Mouse Sera
Positive Control
Shows Ag binding was successful, Ab can bind Ag and secondary reagents are working
Shows nice were sufficiently immunized to obtain strong Ab response
Blanks
wells that give minimal colour signal in absence of Ag
Used to subtract out non-specific signal incl background
Substrate
2 buffer tablets and 2 p-NPP (p-nitrophenyl phosphate, disodium) tablets in ddH2O
Color changes when bound enzymes active it
AP-GAM
Often responsible for incr background
Alkaline Phosphatse goat anti-mouse IgG
2ndary Ab
EDTA
0.1M
Stopping sol’n
Polystyrene
Protein never comes off w/o chemicals
Etched and rough surface incr SA, max binding
Add areas of + and - charge to incr van der Waals
Very hydrophobic and small <20aa pep don’t bind
Must fill spaces b/w prot so non-sp don’t bind
Polyclonal vs Monoclonal
Polyclonal:
Cheaper, binds many epitopes, some non-sp binding
Monoclonal:
can’t use for primary and secondary, usually only for secondary, sometimes use mix of diff monoclonal
Antibody
For Ag detection
Must stick to prot with Fc region
W monoclonal, 75% of Fab sites available for binding
CTRL AG
Normal mouse (preimmune) serum
Control Plate
AG w PMS, NMS, PBS-T, AP-GAM(7-12 no 2ndary Ab, PBS instead)
CTRL AG w PMS, NMS, PBS-T, AP-GAM(7-12 no 2ndary Ab, PBS instead)
PBS/No AG w PMS, NMS, AP-GAM
Reading
OD 405nm
