Enzyme-linked immunosorbent assay (ELISA)

Question 1

What is an ELISA test?

Answer 1

• ELISA = an immunological assay used to measure antibodies, antigens, proteins and glycoproteins in biological samples
• a plate-based assay generally carried out in 96 well plates
• allowing multiple samples to be measured in a single experiment

Question 2

What are the 4 main steps in ELISA?

Answer 2

• Coating (with either antigen or antibody)
• Blocking (typically with the addition of bovine serum albumin (BSA)
• Detection
• Final read

Question 3

What is direct ELISA?

Answer 3

• The antigen is immobilised to the surface of plate and detected with an antibody specific for the antigen. The antibody is directly conjugated to HRP

Question 4

What is indirect ELISA?

Answer 4

• uses a 2 step process for detection
• a primary antibody specific for the antigen binds to the target and a labelled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection

Question 5

What is capture assay sandwich ELISA?

Answer 5

• sandwich begins with a capture antibody coated onto the wells of the plate
• antigens are sandwiched between 2 layers of antibodies (capture and detection antibodies)
• highly sensitive

Question 6

What are some ELISA detection strategies (chromogenic and chemiluminescent)?

Answer 6

• ELISA assays = usually chromogenic using a reaction that converts the substrate (TMB solution etc) into a coloured product which can be measured using a plate reader
• Chemiiluminescent ELISA - uses the peroxidase conjugated detection antibodies in a chemiiluminescent reaction
• a luminol based enhancer solution is added along with a substrate and the signal is read using a luminometer