ELISA Flashcards
What is an ELISA?
Detection/quantification of a defined antigen or antibody of defined specificity
Where are ELISAs done?
In 96 well plates
What is the principle behind an ELISA?
- Relies on antibody-antigen binding and detection with an enzyme
- Enzyme catalyses a colour change and can be quantified
- Extend of colour change shows how much antibody-antigen is present
What is the Sandwich ELISA?
Antigen is trapped ebtween capture and detection antibodies
What are the advantages of Sandwich ELISa?
- High sensitive
- High specificity
What is the disadvantage of Sandwich ELISA?
Antibody optimisation can be difficult
What is a biotinylated antibody?
Blood proteins that bind to biotin via biotinylation
What is the enzyme is binded to by streptavidin after biotin binds to streptavidin?
HRP (Horse radish peroxidase)
What is the purpose of the coating buffer in ELISA?
Stick the first antibody to the plastic
What is the purpose of blocking buffer?
Contains BSA, blocks any unbound plastic
What is the purpose of Washing buffer?
Contains a detergent to block and open out molecules
What is the purpose of Subtrate buffer?
To maximise enzyme activity/colour change
What is BSA?
Bovine serum albumin
What is Tween-20?
Detergent
What is the purpose of the block solution?
Prevent antibodies or proteins in the sample binding non-specifically to the plastic of the plate
What is Steric hindrance?
An antibody unable to bind because another antibody is present
How is standard curve is done?
Diluting from the stock
What is the limit of detection??
If a sample of Optical Density is below 0.3 and less than 1pg/ml so it is undetectable
