Practical 5b: ELISA Flashcards
What does ELISA stand for?
Enzyme-Linked Immunosorbent Assay
What is an ELISA?
A highly sensitive assay that uses antibodies to quantify molecules of interest in a solution
In an ELISA, what is used to measure the molecule of interest?
Antibodies (which are conjugated to some reporter molecule)
Give three examples of reporter molecules
An enzyme that will act on a substrate and give a coloured product
A fluorescent molecule
A chemiluminescent label
How many wells are usually used in an ELISA?
96 well plates
List the four different types of ELISA
Direct
Indirect
Sandwich
Competitive
How is a direct ELISA carried out and give a use?
(2)
Antigen is coated on the plate and detected using a conjugated antibody
e.g. screening for an antibody that binds well to a specific antigen
How is indirect ELISA carried out and give a use?
(5)
Antigen is coated on the plate
Patient sample is added
Conjugated anti-human antibody
If there is a patient antibody in sample the conjugated anti-human antibody will bind to it
e.g. detection of a patient antibody against a protein from an infectious agent like the spike protein of SARS-CoV-2
How is a sandwich ELISA carried out and give a use
(4)
A capture antibody is added to the plate
The sample antigen competes for binding
Antigen is detected by a conjugated antibody
e.g. detection of a cytokine like IL-4 in solution
How is a competitive ELISA carried out and give a use?
(4)
A known amount of target antigen is added
The sample antigen competes for binding
Lower results mean higher concentration of molecule of interest in the sample
e.g. detection of an antigen found in very low concentration
What must be done after every step of an ELISA?
The plates must be washed after every step
In an indirect ELISA, what is the colour proportional to?
Proportional to how much antibody is present
