Diagnostics: ELISA

Question 1

Define antibody

Answer 1

Protein produced by the body in response to an invading substance

Question 2

Define antigen

Answer 2

Substance which the body is trying to attack by mounting an immune response

Question 3

Define ‘immuno’

Answer 3

An immune response that causes the body to generate antibodies

Question 4

Define ‘assay’

Answer 4

Refers to a test

Question 5

Define immunosassay

Answer 5

A test using antibody-antigen complexes as a means of generating a measurable result

Question 6

Define immuno-complex

Answer 6

An antibody-antigen complex

Question 7

Define enzyme immuno-tests

Answer 7

Any technique or method of analysis using enzymes to show antigen-antibody reactions

Question 8

Define test sensitivity

Answer 8

The proportion of subjects with disease who will have a positive result= true positives

Question 9

Define test specificity

Answer 9

The proportion of subjects without disease who will have a negative result= true negatives

Question 10

What are the 2 types of enzyme immunoassay?

Answer 10

• Homogenous
• Heterogeneous

Question 11

What are the characteristics of homogenous enzyme immunoassays?

Answer 11

• Enzyme inactivated when bound
• No washing stage
• Small quantities
• Easy to use
• Expensive
• Low sensitivity

Question 12

What are the characteristics of heterogenous enzyme immunoassays?

Answer 12

• Antibody & Antigen specific
• Structure and characteristics measured
• ELISA

Question 13

What are the characteristics of Enzyme-Linked Immunosorbent Assay (ELISA)

Answer 13

• Quantitative method showing antigen-antibody reactions
• Colour changes
• High level of specificity of antigens for antibody
• Can measure substances in very low concentrations

Question 14

What are the stages of an ELISA test?

Answer 14

1. Microplates with several wells to test different titres/dilutions
2. The antigen is bound to the solid phase
3. Antibodies bind to the antigen layer
4. Labelled antiglobulin binds to the antibody
5. Solution containing enzyme substrate added
6. Results read on a spectrophotometer

Question 15

How are ELISA results interpretted?

Answer 15

• Each well in microplate is a sample
• Colour intensity proportional to bound enzyme linked antiglobulin
• Antiglobulin proportionate to antibody
• Antibody proportionate to antigen number
• Colour depends on kit used

Question 16

What are the 4 types of ELISA?

Answer 16

• Direct
• Indirect
• Competitive
• Sandwich

Question 17

What are the advantages of direct ELISA?

Answer 17

• Short protocol: saves time and reagents
• No corss- reactivity from secondary antibody

Question 18

What are disadvantages of direct ELISA?

Answer 18

• Potential high background: all proteins in the sample bind to the surface
• No signal amplification
• Low flexibility: the primary antibody must be conjugated

Question 19

What are the advantages of indirect ELISA?

Answer 19

• Signal amplification: several secondary antibodies will bind to the primary antibody
• High flexibility: same antibody has different purposes

Question 20

What are disadvantages of indirect ELISA?

Answer 20

• Long protocol compared to direct
• Potential cross-reactivity from secondary antibody

Question 21

What are advantages of sandwich ELISA?

Answer 21

• High specificity: involves 2 antibodies detecting epitopes on the same antigen
• Suitable for complex samples
• High flexibility and sensitivity: both direct and indirect methods can be used

Question 22

What are disadvantages of sandwich ELISA?

Answer 22

• Demanding design: finding 2 antibodies against the same target that recognises different epitomes and work well together is hard

Question 23

What are advantages of competitive ELISA?

Answer 23

• Depends on base ELISA selected
• Suitable for small antigens
• No sample processing required

Question 24

What are disadvantages of competitive ELISA?

Answer 24

• Depends on base ELISA selected