ELISA

Question 1

What does ELISA stand for and what is it

Answer 1

• Enzyme-linked immunosorbent assay

- A technique used to detect (assay) specific molecules in samples

Question 2

What are the 3 ways in which ELISA is used in veterinary medicine

Answer 2

• In disease detection
• In detection of illegal drugs
• Detection of hormones

Question 3

What is indirect ELISA used for

Answer 3

• used to detect/measure a specific antibody in a sample

Question 4

How does an indirect ELISA work

Answer 4

1. Antigen bound to plastic well
2. Well is coated with blocking reagent to prevent non-specific binding and washed to remove excess reagent
3. Test sample is added and any antiodies in that sample that react against antigen will bind. Wash to remove excess
4. Secondary antibody with attached enzyme added. Wash to remove excess
5. Substrate is added
6. Substrate converted by enzyme if antibody is present

Question 5

What is direct ELISA used for

Answer 5

used to detect / measure the concentration of specific antigens in a sample

Question 6

What are the 2 types of direct ELISA

Answer 6

• Sandwich ELISA

- Competitive ELISA

Question 7

How does a sandwich ELISA work

Answer 7

1. Wells coated with capture antibody
2. Well is coated with blocking reagent to prevent non-specific binding and washed to remove excess reagent
3. Test sample is added and any antigen can bind to capture antibody. Wash to remove excess
4. Addition of primary antibody results in sandwich formation. Wash to remove excess
5. Add secondary antibody with attached enzyme. Wash to remove excess
6. Substrate is added and converted by enzyme if antigen present

Question 8

When is competitive ELISA used instead of sandwich ELISA

Answer 8

If the antigen is very small and has only one epitope (binding site for antibody)

Question 9

What does a positive control in ELISA allow for

Answer 9

Confidence in the diagnostic test results, confirms that the negative results are accurate and assists in any protocol adjustments or optimizations that may be necessary

Question 10

What does a negative control in ELISA show

Answer 10

If any solutions of the assay are contaminated adding validity to any positive results

Question 11

In a sandwich ELISA what would happen if the blocking step was ommitted

Answer 11

All wells including the negative control wells, would show uniform over development

Question 12

In a sandwich ELISA what would happen if there was no washing steps after the detection antibody was added to the wells

Answer 12

All wells, including the negative control wells would show uniform over development