L7-8 - Immunological Techniques

Question 1

What do immunoassays do?

Answer 1

Use the antibody-antigen reaction to quantify a substance

Question 2

What is another name for the antibody-antigen complex?

Answer 2

Immunocomplex

Question 3

What is a paratope and an epitope?

Answer 3

Paratope - the antigen binding site on an antibody
Epitope - the area of the antigen that the paratope binds

These are what gives antibodies their specificity

Question 4

What is the difference between monoclonal and polyclonal antibodies?

Answer 4

Polyclonal - heterogeneous antibody population with different paratopes for the same antigen

Monoclonal - homogeneous antibody population with the same paratopes (arise from the same parent cell)

Question 5

How are monoclonal antibodies produced in the lab?

Answer 5

Fuse B cells to myeloma cells to form a hybridomas

These have a long life and the potential to make almost infinite antibodies with the same paratope

Question 6

How are polyclonal antibodies produced in the lab?

Answer 6

Inject a host with antigen, wait for their immune system to produce many antibody secreting plasma cells that recognise different epitopes, and extract serum containing the antibodies

Question 7

What are the advantages of using monoclonal antibodies?

3

Answer 7

Epitope specific, low cross-reactivity and background noise

Question 8

What are the advantages of using polyclonal antibodies?

3

Answer 8

Inexpensive and quick to produce, can be obtained from numerous host species based on amount of antibodies needed

Question 9

What is specificity in terms of antibody characteristics?

Answer 9

Ability of an antibody to distinguish the immunogen from other antigens

Question 10

What is affinity in terms of antibody characteristics?

Answer 10

The strength of antibody binding to one antigen binding site

Question 11

What is avidity in terms of antibody characteristics?

Answer 11

The strength of antibody binding where there are multiple binding sites

Question 12

What types of labels can be used in immunoassays?

4

Answer 12

Enzymes, radioisotopes, chemiluminescent labels, fluorescent labels

Question 13

What is radial immunodiffusion (RID)?

Answer 13

Antibody is mixed into gel and sample is added to a well

Forms immunoprecipitate in rings, with ring size corresponding to the amount of antigen in the sample

Question 14

What are homogeneous assays?

What is the difference between turbidimetry and nephelometry

Answer 14

Insoluble immunocomplexes form in liquid media, and the absorption/scattering of light is proportional to the amount of antigen

Turbidimetry measures absorption
Nephelometry measures scatter

Question 15

What immunological techniques are qualitative or semi-quantitative?

Answer 15

• Histology, including immunohistochemistry and immunofluorescence
• Latex enhanced agglutination

Question 16

How is histology used in immunoassays?

Answer 16

Antibodies can identify molecules and their distribution in tissues

Visualised with immunohistochemistry or immunofluorescence

Question 17

What types of sections are used in histology?

4

Answer 17

Paraffin embedded, frozen, free floating, cytological specimens

Question 18

How does immunohistochemistry work?

Answer 18

Enzymes attached to antibodies against a molecule in tissue

Add substrate to elicit a colour change

Question 19

Give an example of an enzyme used in immunohistochemistry

Answer 19

Horseradish peroxidase (HRP)
Alkaline phosphatase (ALP)

Question 20

What is the difference between direct and indirect immunofluorescence?

Answer 20

Direct - primary antibody against antigen in tissue is fluorescently labelled

Indirect - secondary antibody against the primary antibody is labelled

Question 21

How does latex enhanced agglutination work?

Answer 21

Beads are coated with antibody/antigen

When sample is added, agglutination indicates a reaction has occurred