Antibodies

Question 1

What is the advantage of using antibodies as a reagent rather than as synthetic molecules?

Answer 1

• High specificity
• Unlimited range - possible avidity for any antigen, including novel synthetic molecules
• Synthetic peptides need to conjugated with large carrier molecules to be immunogenic

Question 2

How are polyclonal antisera produced?

Answer 2

1. Immunise - to activate B cells
   
   • Use adjuvant (aluminium salts, surfactants, mineral oils) - ensures slow Ag release, directs and enhances humoral stimulation.
   • BOOST several times, few weeks apart - increases Ab production, allows IgM to IgG class-switching, increasing affinity for the antigen.
2. 4-8 weeks - collect blood, clot, aliquot serum (test for activity). Rabbit/goat generates 100-200mg polyclonal Ab.
3. Store at -20 or 4 °C - stable for many years.

Question 3

How are monoclonal antibodies involved in cancer diagnosis?

Answer 3

• Tumour overproduction of the same monoclonal antibody can cause life threatening disease
• Useful in diagnosis of some B lymphocyte tumours

Question 4

How are monoclonal antibodies produced?

Answer 4

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Question 5

What are native monoclonal antibodies and how are they used?

Answer 5

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Question 6

How are antibodies made in plants? What are the pros and cons?

Answer 6

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Question 7

Explain how Western blotting is used to separate and detect specific proteins from a mixture.

Answer 7

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Question 8

Explain how immunoblotting is used to detect autoantibodies in Graves’ disease.

Answer 8

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Question 9

What are the advantages of monoclonal antibodies?

Answer 9

• Produced by isolating a single lymphocyte and growing it in vitro
• High specificity
• Low probability of cross-reactivity
• Inexhaustible supply of homogeneous reagent
• Commercial applications - e.g. ELISA