Lecture 14 - Monoclonal antibodies

Question 1

What part of the antibody detects the antigen?

Answer 1

The hypervariable region

Question 2

What is the standard way that antibodies are made?

Answer 2

• Injecting small samples of antigens into an animal

- Antigens contain many different epitopes (Polyclonal)

Question 3

How were antitoxins made?

Answer 3

By injecting an animal with sub-lethal doses of toxins they make antibodies for it

Question 4

How were monoclonal antibodies made?

Answer 4

• Normal B-cells fused with myeloma cells

- Hybridoma formed with antibody producing genes and immortal properties of the tumour cells

Question 5

What are the advantages of Monoclonal antibodies? [5]

Answer 5

• Very high specificity
• Low cross-reactivity
• Unlimited supple
• Can be standardised
• Used as therapeutic agents

Question 6

List some of the immunological methods using monoclonal antiodies. [7]

Answer 6

• Affinity chromatography to purify molecules
• Enzyme Linked ImmunoSorbant Assay (ELISA)
• Haemagglutination
• Immunodetection on tissue samples
• Western Blotting
  Flow cytometry
• Magnetic cell separation

Question 7

What is affinity chromatography used for?

Answer 7

To purify antigen

Question 8

How does affinity chromatography work?

Answer 8

• Bind antibody to beads
• Add mixture then wash away unbound molecules
• Elute the bound molecule
• Purified antigen formed

Question 9

How can antibodies be tagged?

Answer 9

• Fluorescent markers

- Enzyme markers that change colour when substrate is added

Question 10

What is the role of an ELISA test?

Answer 10

To determine: viral diagnostics, levels of hormone/antibody etc

Question 11

How is a direct ELISA performed?

Answer 11

• Samples are added to wells that have different antigens in them
• Only the well that matches the antibody will show colour

Question 12

How is an indirect ELISA performed?

Answer 12

• Well is coated with antigen
• Specific antibody to be measured is added
• Enzyme-conjugated 2°Ab is added
• Substrate added and amount of 2°Ab (absorbance) measured by ELISA reader

Question 13

What does Agglutination determine?

Answer 13

If there is antigen present there will be clumping

Question 14

What does Haemoagglutination determine?

Answer 14

The blood type of the individual

Question 15

What can Immunohistochemistry and Immunofluorescence show?

Answer 15

Localisation of antigen (quantitative)

Question 16

How does Immunohistochemistry work?

Answer 16

• By combining an antibody with a peroxidase enzyme.

- The antibody is detected with a substrate

Question 17

How does Immunofluorescence work?

Answer 17

Tag the antibody with fluorescent molecule

Question 18

What is Western blotting used for?

Answer 18

Qualitative and Quantitative analysis of protein expression

Question 19

How does Flow cytometry work?

Answer 19

Uses lasers to detect fluorescent antibodies bound to cells

Question 20

When is Flow cytometry used?

Answer 20

To give scatter plots indicating the number of each cell type

Question 21

What kind of information does Flow cytometry give?

Answer 21

Quantitative and qualitative information

Question 22

What is the purpose of Magnetic Separation of cell populations?

Answer 22

• Used to obtain purified cell populations

- May be used clinically

Question 23

What household object used monoclonal antibodies?

Answer 23

Pregnancy tests