Diagnostics 5 - Antibodies as Diagnostic tools

Question 1

When using antibodies as diagnostic tools, where are the things attached and what things are attached?

Answer 1

Attached to Ab constant region - Ab binding unaffected

Things that are attached:

1. Enzymes (e.g. peroxides, alkaline phosphatase)
2. Fluorescent probes
3. Magnetic beads - magnet pulls away Abs conjugated with other beads
4. Drugs - e.g. Kadcyla (anti-HER2 Ab linked to emtansine)

Question 2

What are anti-antibodies

Answer 2

Immunoglobulins from other species

Question 3

Antibodies can come from the patient or can be manufactured. Give examples of each

Answer 3

Patient - autoimmune disease, or defence against infection

Manufactured antibodies - antisera from immunised animals, Monoclonal antibodies, genetically engineered Abs

Question 4

How are monoclonal antibodies produced

Answer 4

1. Produce a hybridoma by fusing B cells with Myeloma cells. Hybridomas are immortal, have enzyme and produce desired antibodies
2. Certain medium used to selectively grow hybridoma cells with enzyme
3. Clone by limiting dilution - put single cells in wells and screen clones for those that produce antibody

Question 5

How can Ab recombinant tech be used to produce Abs

Answer 5

1. Isolate population of genes encoding Ab variable regions
2. Construct fusion protein of V region using bacteriophage coat protein
3. Clone random population of V regions - gives mixture of bacteriophages
4. Select phage with desired variable regions by specific binding to antigen

Question 6

Name therapeutic uses of manufactured antibodies

Answer 6

1. Prophylactic protection against microbial infection - e.g. IV-IG, synagis
2. Anti-cancer therapy (e.g. anti-HER2)
3. Removal of T-cells from bone marrow grafts (e.g. anti-cd3)
4. Block cytokine activity (e.g. anti-TNF-a antibody)

Question 7

What are diagnostic uses of antibodies

Answer 7

1. Determine blood groups
2. Immunoassays - measure hormones, antibodies, antigens
3. Immunodiagnosis:
   - Infectious diseases
   - Autoimmunity
   - Allergy (look for IgE against agent)
   - Malignancy (myeloma)

Question 8

In ELISA, how is the amount of antigen detected

Answer 8

Strength of the colour indicates amount of antigen

Question 9

Describe Rapid testing

Answer 9

1. Strip containing Abs (conjugated to gold particles)
2. Sample/antigens added –> move down by capillary flow –> Ab binds antigen –> complex moves down and binds 2nd Ab on test line (control line further down to show that the sample has moved all the way down)

Rapid testing used for rapid diagnosis (Eg infections)

Question 10

Nomenclature of therapeutic Monoclonal Ab?

Answer 10

• omab = mouse monoclonal (e.g. muronomab - anti-CD3)
• imab = chimeric or partly immunised (e.g. infliximab - anti TNFa)
• umab - fully human (e.g. palivizumab - anti-RSV)