Antibodies as diagnostic tools

Question 1

Which part of antibody is constant?

Answer 1

Fc

Question 2

What can you do because the Fc part is constant?

Answer 2

Attach various things to this constant part without affecting the binding ability of the antibody to the antigen

Question 3

Which part of the antibody is variable?

Answer 3

Fab- antigen binding part

Question 4

What sort of things are reporters?

Answer 4

Enzymes- peroxidase, alkaline phosphatase etc
Fluorescent probes- dyes, beads of different sizes
Radioisotopes
Magnetic beads

Question 5

Why are antibodies used in diagnostic tests?

Answer 5

Their unique specificity for their target antigens

Question 6

Where do the antibodies that are used come from?

Answer 6

Patient-
In autoimmune disease
Defence against infection
Manufactured-
Antisera from immunised animals
Monoclonal antibodies
Genetically engineered antibodies

Question 7

How do you generate monoclonal antibodies?

Answer 7

You take a normal B lymphocyte which produces the antibody of interest and you fuse it with a myeloma cell line which gives you a hybridoma
These cells have the ability to produce the antibody of interest, furthermore, as it is fused with a tumour cell it can divide indefinitely

Question 8

What are manufactured antibodies used for therapeutically?

Answer 8

Prophylactic protection against microbial infection
Anti-cancer therapy
Removal of T-cells from bone marrow grafts
Block cytokine activity

Question 9

How do manufactured antibodies act as anti-cancer therapy?

Answer 9

Monoclonal antibodies target molecules that are over-expressed on certain types of tumours

Question 10

Why is removal of T cells from bone marrow grafts important?

Answer 10

T cells cause graft versus host disease in transplants

Question 11

For the nomenclature of therapeutic monoclonal antibodies, what does the suffix -omab mean?

Answer 11

It is mouse monoclonal e.g. muronomab (anti CD3, transplant immunosuppression)

Question 12

What does the suffix -imab mean?

Answer 12

Chimeric or partly humanised e.g. infliximab (remicade) (Anti TNFalpha)

Question 13

What does the suffix -umab mean?

Answer 13

Human origin e.g. palivizumab (anti-RSV)

Question 14

Which ones are ideal that are used therapeutically?

Answer 14

-umab

Question 15

What are manufactured antibodies used diagnostically?

Answer 15

Tissue typing
Blood group serology
Immunoassays- hormones, antibodies and antigens
Immunodiagnosis- infectious diseases, autoimmunity, allergy and malignancy

Question 16

What does ELISA mean?

Answer 16

Enzyme
Linked
ImmunoSorbent
Assay

Question 17

How does ELISA work?

Answer 17

Antigen (that you want to identify) is used to coat well
Specific antibody binds to antigen - excess washed away
Enzyme linked antibody binds to specific antibody
Substrate is added and converted into coloured product and, the rate of colour formation is proportional to the amount of specific antibody

Question 18

What is an immune complex?

Answer 18

Antibody bound to antigen

Question 19

What governs the size of an immune complex?

Answer 19

Ratio of antigen to antibody- excess of antigen to antibodies leads to smaller complexes

Question 20

What is the difference in response to larger or smaller immune complexes?

Answer 20

E.g. in glomerulonephritis
Larger immune complexes are too large so their are stuck at the endothelial cells. They are recognised by immune system and cleared more easily. However, they can also activate platelets and neutrophils freely causing tissue damage.
Smaller immune complexes can pass through endothelial cells freely and they don’t efficiently activate complement, it will only activate complement when it is bound to a surface e.g. the basement membrane under the endothelial surface

Question 21

What is a particular problem related to immune complexes?

Answer 21

Glomerulonephritis

Question 22

What is the difference between someone developing an acute response and healthy person in terms of serum electrophoresis?

Answer 22

At top of healthy person, there is a diffuse smear which is the gamma globulin region- diffuse because many different antibodies with different charges
If someone is developing an active immune response, a lot of gamma globulins produced so the area will be much darker (note: difference from monoclonal expansion of B cells)

Question 23

What does a very sharp single band in serum electrophoresis indicate?

Answer 23

Monoclonal expansion of B cells e.g. myeloma

Question 24

How can you measure different cell populations simultaneously?

Answer 24

Have several different monoclonal antibodies and label each with a different coloured fluorescent dye
Add the mixture of antibodies to the cell mixture
Then pass the cells in a stream through the laser beam and detect fluorescent so each cell can be categorised based on fluorescence

Question 25

What is the natural progression of HIV in someone that hasn’t had treatment in terms of CD4 T cell count and viral load?

Answer 25

Primary infection- CD4 will initially go down and then it will go up again after a few weeks
The viral will remain controlled by immune system for some time (clinical latency) but CD4 will keep going down
When CD4 gets very low, patient will show signs of opportunistic infection and viral load will go up

Question 26

What are MAC infections and what are there relation to HIV?

Answer 26

Mycobacterium avium complex- environmental mycobacterium that is everywhere and normal people can deal with it easily but it’s an opportunistic infection that develops when you have a low CD4 count

Question 27

What are the targets of lymphocytes that is used in flow cytometry?

Answer 27

Lymphocyte subpopulations
–CD3+   T cells – pan T cell marker
–CD4+   T cells – T helper/cells
–CD8+   T cells – cytotoxic T cells
–CD19+   B cells
–CD56+    Natural Killer (NK) Cells