pre-IC4

Question 1

What is an immunoassay?

Answer 1

Assays that employ antibodies to detect and

quantify a specific analyte (usually a biomolecule)

Question 2

Key component in immunoassays

Answer 2

Antibodies against the biomolecule of interest; Polyclonal or Monoclonal antibodies may be used; usually raised in an animal

Question 3

What are the 2 means of detection of Ag-Ab binding in immunoassays?

Answer 3

1. Using a marker (radioimmunoassays/ enzyme immunoassays)

2. Agglutination/ haemagglutination

Question 4

What is agglutination, and how is this observed?

Answer 4

Clumping of red blood cells (bound to antibodies); visibly observed as cloudiness/ turbidity/ precipitation

Question 4

What is agglutination, and how is this observed?

Answer 4

Clumping of red blood cells (bound to antibodies); visibly observed as cloudiness/ turbidity/ precipitation

Question 5

Solid phase enzyme immunoassay: What does a low absorbance mean?

Answer 5

Greater amount of antigen present in sample, lesser enzyme-linked antigens bound to solid phase, lesser enzyme activity thus lesser product formed.

Question 6

What does ELISA stand for?

Answer 6

Enzyme-linked immunosorbent assay

Question 7

What can be found on the solid surface of ELISA?

Answer 7

Can be antibody/ antigen depending on analyte to be determined in sample

Question 8

What does indirect ELISA detect in a sample?

Answer 8

Indirect ELISA employs antigen to detect the presence of a specific antibody in the sample.

Question 9

What does sandwich ELISA detect in a sample?

Answer 9

Sandwich ELISA employs antibodies to detect the presence of a particular antigen in the sample.

Question 10

What happens in competitive ELISA?

Answer 10

Enzyme-linked antigen (AKA inhibitor antigens) competes with antigen in sample for antibody binding.

Question 11

Advantages of ELISA

Answer 11

1. Ease of use (usually only UV spectrophotometer needed)

2. Safe and specific (depending on specificity of Ab used)

Question 12

Disadvantages of ELISA

Answer 12

1. False positive (inadequate washing, use of polyclonal antibodies, inadequate blocking, cross reactivity of secondary Ab)
2. False negative (antibodies and conjugated enzymes are denatured)

Question 13

How does haemagglutination assay detect enveloped viruses in samples?

Answer 13

Viral particles interact with red blood cells through a viral surface glycoprotein called hemagglutinin. Presence of virus causes clumping of red blood cells (hemagglutination), forming a lattice instead of a nice full red dot.

Question 14

When does clumping (agglutination) occur?

Answer 14

It happens as long as long antigen and/or antibody is particulate in nature (i.e. semi-solid or solid by being conjugated to a solid particle).

Question 15

How can agglutination be measured quantitatively?

Answer 15

Agglutination is a visible change in turbidity, which can be detected by measuring the UV absorbance.

Question 16

How does pregnancy test direct/ passive agglutination work?

Answer 16

hCG present in urine binds to anti-hCG Ab on latex particles, showing cloudiness.

Question 17

What is passive agglutination?

Answer 17

Only works with particulate Ag. RBCs coated with a soluble Ag is used to test for Ab.

Question 18

What is haemagglutination inhibition?

Answer 18

Soluble Ag in sample inhibits agglutination of semi-solid Ag-coated RBCs by Ab.

Question 19

For haemagglutination inhibition, what would a positive test result show?

Answer 19

No agglutination as the Ag in sample successfully competed with other particulate for binding to Ab. Thus lower turbidity, and low absorbance.