Antibodies as Diagnostic Tools

Question 1

What can you attach to antibodies?

Answer 1

Enzymes – e.g. peroxidase, alkaline phosphatase
- Antigens washed over antibodies with enzyme, colourless substrate is then added which turns colour

Fluorescent probes – e.g. dyes and beads of various size

Magnetic beads – e.g. purification of cell types
- Magnet-antibody attaches to receptor and then run sample over a magnet and only linked-cells bind

Drugs – e.g. Kadcyla, anti-HER2 linked to emtansine

Question 2

What are the to categories of antibodies?

Answer 2

Produced by patient – for autoimmune disease OR defence against infection

Produced artificially – antisera from immunised animals (polyclonal – have many specificities), monoclonal antibodies and genetically engineered antibodies

Question 3

How are monoclonal antibodies produced?

Answer 3

Take normal B lymphocytes from the spleen which have limited cell division and fuse them with myeloma cells that are immortal to create hybridomas

The hybridomas then can produce antibodies indefinitely

Question 4

How are antibodies produced using recombinant DNA technology?

Answer 4

1. Isolate all the V-segments of antibodies you need
2. Display all the V-segments on a protein or bacteriophage so all bacteriophages display different specificities
3. Use the phage library to screen plates for the antigen and the complimentary phage will stick to the plate whilst others will be washed off

Question 5

What are some uses of manufactures antibodies?

Answer 5

Therapeutic:
- Prophylactic against microbial infection – e.g. Synagis (anti-RSV)
> Suffix “-umab” – human – e.g. Synagis (anti-RSV)
- Anti-cancer therapy – e.g. anti-HER2
- Removal of T-cells (bone marrow transplant) – e.g. anti-CD3
> Suffix “-omab” – mouse monoclonal – e.g. anti-CD3
- Block cytokine activity – e.g. anti-TNF-alpha
> Suffix “imab” – chimeric/partly humanised – e.g. anti-TNF-alpha

Diagnostic:
- Blood group serology
- Immunoassays – hormones, antibodies, antigens
Immunodiagnosis – infectious disease, autoimmunity, allergy (IgE), malignancy (myeloma)

Question 6

What is ELISA?

Answer 6

Enzyme Linked Immuno-Sorbent Assay

Two samples are used to coat the walls of some wells

Anti-A antibody is covalently linked to a reporter (enzyme) that when it binds (whilst any non-binding antibody is washed away), a colourless substrate is added and turns colour if the antibody is still present in the well after washing

Absorbance of the light can then be measured

Question 7

What happens in a lateral flow assay?

Answer 7

Pre-made antibodies bonded to gold nanoparticles have an analyte passed over them

If the antibodies do successfully bind to the analyte (+ve result), the antibodies will bind to the positive strip and it will show up

E.G. hCG protein in pregnancy test

Question 8

What can large and small immune complexes activate?

Answer 8

Large immune complexes:

• recognised more easily by the immune system and cleared quickly
• Activate platelets and neutrophils that release mediators.

Small immune complexes – get trapped in sub-endothelial layer
- Can activate complement when stuck to the surface and so attract neutrophils that can damage kidney function

Question 9

What would you look at to determine immunodefficiency?

Answer 9

Serum immunoglobulin levels:
IgG, IgM, IgA and IgG1, 2, 3, 4 - via serum electrophoresis/ELISA/Nephelometry.

Specific antibodies:
protein antigens (tetanus) and polysaccharide antigens (pneumococcus) - via ELISA

Lymphocyte subsets:
CD3, 4, 8, 19, NK cells - via Flow Cytometry (you can use specific antibodies against these receptors)
- CD3+ All T-Cells.
- CD4+ T-helper Cells
- CD8+ Cytotoxic T-Cells.
- CD19+ B-Cells.
- CD56+ NK-Cells

Question 10

How can electrophoresis show immunodeficiency?

Answer 10

In a healthy person, smear at top is gamma globulin region

*It is diffuse due to a varying range of antibodies

In someone with an active immune response, this region becomes DARKER due to more gamma globulins

If you see a SINGLE SHARP BAND, this indicates a monoclonal expansion of b-cells – e.g. myeloma?

Question 11

What happens in flow cytometry?

Answer 11

To measure cell populations, you attach different monoclonal antibodies with fluorescent dyes attached to them to each cell type

The cells are then passed into a stream through a laser and the dyes are detected and each cell is categorised based on its fluorescence

The results are then shown on a flow cytometry graph

• *Lymphocyte subsets :
• CD3+ All T-Cells.
• CD4+ T-helper Cells
• CD8+ Cytotoxic T-Cells.
• CD19+ B-Cells.
• CD56+ NK-Cells

Question 12

Describe the method of treatment of HIV

Answer 12

Antibody HIV test ->Low CD4, high viral load -> commence ART (1st line) -> monitor CD4/viral load -> CD4 down or viral load up -> ART (2nd line) increase

This increases life expectancy

• When CD4 gets very low, the patient will be prone to opportunistic disease and viral load will shoot up
• At LOW CD4, you get MAC infections (mycobacterium avium complex) – normal people don’t get this