ELISA

Question 1

What is an ELISA and what steps are the three core concepts?

Answer 1

An ELISA is an assay used to detect and quantify the concentration of a particular molecule

1. Immobilising the antigen (attach the ELISA plate)
2. Probing for antigen
3. Detecting product (enzyme substrate and reaction produces a signal

Question 2

Why are wells washed?

Answer 2

To remove unbound material, wells are washed in each ELISA step

Phosphate buffered saline with a non ionic detergent Tween 20

3-5 washes

Insufficient washing can result in a high background signal while excessive washing can decrease the sensitivity of the assay by watching away the antibody or antigen

The non ionic detergent is added to wash buffers to reduce non specific binding

Question 3

Blocking buffer

Answer 3

A blocking buffer is applied to each well

A blocking buffer is a mixture of proteins with no affinity for the target protein or ELISA reagents, these proteins bind passively to areas of the well base not occupied by an antibody

They prevent binding non specifically to the plate

Reduces background signal and increases sensitivity of the assay

Question 4

Steps of a sandwich ELISA

Answer 4

Capture antibody

Blocking buffer

Sample

Primary antibody

Secondary antibody with enzyme

Enzyme substrate

Question 5

What controls are used in an ELISA

Answer 5

Standards include known concentration of the analyte, created using serial dilution of a stock solution. By plotting absorbency v conc a standard curve is reduced and used to calculate the concentration of the analyte

Positive control - sample which expresses protein of interest

Negative control - sample that does not express protein if interest