ELISA Analysis

Question 1

What is the real name of ELISA Analysis?

Answer 1

Enzyme
Linked
Immunotherapy
Sorbent
Assay

Question 2

What is the ELISA analysis used for?

Answer 2

antibody counting (test vaccines)

Question 3

What are the pros of the ELISA Analysis?

Answer 3

• most accurate method for investigation
• conjugated antibodies used for signal (attached to ENZYMES (not nanoparticles!)
• allows for the testing of multiple samples
• allows to quantify the amount of substance searched for (eg. quantify number of antibodies in a given sample)
• easy result investigation, either positive or negative.

Question 4

What are the cons of the ELISA Analysis?

Answer 4

• complicated method (all steps done manually) (vs. strip in chromatography method does it itself)

Question 5

What is the conjugated particle in ELISA Analysis?

Answer 5

enzyme

Question 6

Explain how the ELISA Analysis test works.

Answer 6

• 8 wells in a strip which is coated in antigen/antibody (if searching for antibody, then wells are coated in antigens)
• step 1: insertion of the liquid sample (usually serum)
• antibody-antigen complex formed –> immobilized antigens keep the antibodies stuck to the well
• step 2: washing step, antibodies are immobilized, but must remove anything else present within the sample
• step 3: addition of the conjugated solution: conjugated antibodies (in ELIZA marked by an enzyme eg. horse radish peroxidase)
• antibodies are specific against antibodies (conjugated antibodies interact with antibodies immobilized on the surface of the antigen) (would be the same if looking for antibody counting antigen not antibody!)
• step 4: washing step, remove residual, unnatached conjugated antibodies (complex remains)
• step 5: addition of substrate
• enzyme works on specific substrates (on the conjugated antibody enzyme (eg. horseradish peroxidase)
• clear, but when attached to enzyme, structure is changed –> colour is seen
• if colour seen = positive test result!
• intensity of colour correlates with the amount of antibodies in the sample
• use spectrometer to assess colour intensity (set a specific wavelength) –> assesses optical density
• optical density–> numerical value of colour intensity
• use formula to use optical density correlation with antibody number

Question 7

How does the ELISA Analysis work when testing for the antigen?

Answer 7

• antibody is immobilized on the well bottom
• antibody interacts with the antigen (added from solution)
• conjugated antibody interacts with the antigen –> SANDWITCH
• (either antibody or antigen is immobilized)