Antibodies & diagnostics

Question 1

Give examples of what can be attached to antibodies for diagnostic purposes

Answer 1

• Enzymes: e.g. peroxidase, alkaline phosphatase
• Fluorescent probes: dyes, beads of different sizes
• Magnetic beads: e.g. purification of cell types
• Drugs: e.g. Kadcyla, anti-HER2 linked to emtansine

Question 2

List 3 ways antibodies can be manufactured

Answer 2

antisera from immunised animals (polyclonal) monoclonal antibodies “genetically engineered” antibodies

Question 3

How are monoclonal antibodies produced?

Answer 3

• immunise mouse
• take out spleen
  
  • source of cells producing HGPRT+ve antibodies but have limited cell division.
• These cells are fused with immortal HGPRT-ve myeloma cells to form hybridomas
• Culture in a medium selective for HGPRT+ve cells
• Clone cells by limiting dilution
• Harvest antibodies produced by cells.

Question 4

With examples list the therapeutic uses of antibodies

Answer 4

• Drugs - targeting drugs to certain cell types e.g. breast cancer Kadcyla - anti-HER2 linked to emtansine
• Prophylactic protection against microbal infection - synagis to protect from respiratory syncytial virus
• Removal of t-cells from bone marrow grafts prevent graft vs host disease (Anti-CD3)
• Block cytokine activity (anti-TNFα)

Question 5

list examples of types of substance that are identified diagnostically by antibodies

Answer 5

• Blood group serology
• Immunoassays
  
  • hormones
  • antibodies
  • antigens

Question 6

How are genetically modified antibodies produced?

Answer 6

Take the V section gene of an antibody and fuse it with a bacteriophage coat protein - displays V section on its surface

Create a library of these

Add the library to the antigen and wash unbound phage away

Expand the right phage population

Question 7

What is serum electrophoresis?

Answer 7

Electrophoresis of blood antibodies, shouldnt see thin dense bands - bands should be spread out more

Question 8

How can serum electrophoresis be used?

Answer 8

If a single band is very dense - indicates monoclonal expansion Could be B-cell malignancy Investigate for myeoma

Question 9

How does ELISA work?

Answer 9

Washed after adding antibody to remove any unbound antibody

If colour change detected = positive result

Question 10

With an example how does rapid testing work?

Answer 10

Pregnancy testing

Question 11

What is flow cytometry?

Answer 11

Antibodies to specific antigens labelled with florescent markers

Mixed with cells

Bind to cells with their antigen

Passed through machine

laser meaures size granulation and floresence.

Question 12

What kind of conditions is immunodiagnosis useful for?

Answer 12

• Infectious diseases
• Autoimmunity Allergy (IgE)
• Malignancy (myeloma)