Antibodies as diagnostic tools Flashcards
Which part of antibody is constant?
Fc
What can you do because the Fc part is constant?
Attach various things to this constant part without affecting the binding ability of the antibody to the antigen
Which part of the antibody is variable?
Fab- antigen binding part
What sort of things are reporters?
Enzymes- peroxidase, alkaline phosphatase etc
Fluorescent probes- dyes, beads of different sizes
Radioisotopes
Magnetic beads
Why are antibodies used in diagnostic tests?
Their unique specificity for their target antigens
Where do the antibodies that are used come from?
Patient- In autoimmune disease Defence against infection Manufactured- Antisera from immunised animals Monoclonal antibodies Genetically engineered antibodies
How do you generate monoclonal antibodies?
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
What are manufactured antibodies used for therapeutically?
Prophylactic protection against microbial infection
Anti-cancer therapy
Removal of T-cells from bone marrow grafts
Block cytokine activity
How do manufactured antibodies act as anti-cancer therapy?
Monoclonal antibodies target molecules that are over-expressed on certain types of tumours
Why is removal of T cells from bone marrow grafts important?
T cells cause graft versus host disease in transplants
For the nomenclature of therapeutic monoclonal antibodies, what does the suffix -omab mean?
It is mouse monoclonal e.g. muronomab (anti CD3, transplant immunosuppression)
What does the suffix -imab mean?
Chimeric or partly humanised e.g. infliximab (remicade) (Anti TNFalpha)
What does the suffix -umab mean?
Human origin e.g. palivizumab (anti-RSV)
Which ones are ideal that are used therapeutically?
-umab
What are manufactured antibodies used diagnostically?
Tissue typing
Blood group serology
Immunoassays- hormones, antibodies and antigens
Immunodiagnosis- infectious diseases, autoimmunity, allergy and malignancy
What does ELISA mean?
Enzyme
Linked
ImmunoSorbent
Assay
How does ELISA work?
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
What is an immune complex?
Antibody bound to antigen
What governs the size of an immune complex?
Ratio of antigen to antibody- excess of antigen to antibodies leads to smaller complexes
What is the difference in response to larger or smaller immune complexes?
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
What is a particular problem related to immune complexes?
Glomerulonephritis
What is the difference between someone developing an acute response and healthy person in terms of serum electrophoresis?
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)
What does a very sharp single band in serum electrophoresis indicate?
Monoclonal expansion of B cells e.g. myeloma
How can you measure different cell populations simultaneously?
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
What is the natural progression of HIV in someone that hasn’t had treatment in terms of CD4 T cell count and viral load?
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
What are MAC infections and what are there relation to HIV?
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
What are the targets of lymphocytes that is used in flow cytometry?
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
