5: Immunology

Question 1

What are reporters?

Answer 1

Molecules that you can attach onto the Fc part of antibodies without changing the antigen binding site

Question 2

Examples of reporters?

Answer 2

Enzymes: Peroxidase, alkaline phosphatase

Fluorescent probes: dyes, beads

Radioisotopes

Magnetic beads - good for separating cell types using magnet

Can also add drugs to target

Question 3

4 sources of antibodies?

Answer 3

1. Produced by patient
2. Anti-sera from immunised animals (polyclonal)
3. Monoclonal antibodies
4. Genetically engineered antibodies (recombinant DNA)

Question 4

How do you generate monoclonal antibodies?

Answer 4

1. Take B lymphocytes from spleen which is producing antibody of interest
2. FUSE IT with myeloma cells (tumour of B cells)
3. This gives you HYBRIDOMAS - cells that can produce antibody of interest, plus it can divide indefinitely so lots of antibody can be produced
4. Clone by limiting dilution
5. Harvest monoclonal antibodies

Question 5

How do you make antibodies using recombinant DNA technology?

Answer 5

1. Make a library of all possible V (variable region) segments by isolating population of genes encoding for them
2. Fuse the V segments with bacteriophage coat protein
3. Clone random population of V regions, which gives rise to a mixture of bacteriophages
4. Select bacteriophage with desired V regions (i.e. binds to antigens on an agar plate, doesn’t get washed off) and clone

Question 6

What are the uses of manufactures antibodies?

Answer 6

• Prophylactic protection against microbial infection (vaccine)
• Anti-cancer (antiHER2 antibodies for breast cancer)
• Removal of T-cells from bone marrow grafts
○ Prevent GvHD (graft versus host disease)
• Block cytokine activity (e.g. anti-TNFa for treating RhA)

Question 7

Explain the nomenclature for monoclonal antibodies

Answer 7

• omab = MOUSE monoclonal
• imab = Chimeric (mouse + human) e.g. Infliximab
• umab = HUMAN

Question 8

What is ELISA?

Answer 8

Enzyme Linked Immunosorbent Assay

1. Add antigen A into well
2. Add anti-A antibodies
3. Wash away unbound antibody
4. Antibody has enzyme reporter. If antibody (hence the antigen) is present, enzyme will break down colourless substrate into COLOURED PRODUCT

Amount of colour is proportional to amount of antibody

Question 9

What is lateral flow assay?

Answer 9

Similar to ELISA in principle
Uses gold nanoparticles
Gives RAPID result

Question 10

What are 2 cases where someone would have HIV antibodies but NOT HIV?

Answer 10

1. Maternal antibodies

2. People who volunteer for clinical trials

Question 11

If someone has HIV antibodies what does it mean?

Answer 11

They CANT have been infected recently as HIV antibodies take time to be produced

Question 12

What are immune complexes and what do they cause?

Answer 12

Antibody-antigen complexes
Cause:
- Inflammation/complement activation
- Serum sickness
- Immune complex glomerulo