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? Give examples of each.
- Enzymes: e.g. peroxidase, alkaline phosphatase
- Fluorescent probes: dyes, beads of different sizes
- Magnetic beads: e.g. purification of cell types
- Drugs: e.g. Kadcyla, anti-HER2 linked to emtansine
Why are antibodies used in diagnostic tests?
Their unique specificity for their target antigens
What is the name of antibodies which are raised against other immunoglobulins?
anti-antibodies
Where do the antibodies that are used come from?
Produced by the patient
- in autoimmune disease
- for defence against infection
Manufactured antibodies
- antisera from immunised animals (polyclonal)
- 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
Describe the production of antibodies using recombinant DNA technology.
- You make a library of all the possible V segments
- You display these V segments on a coat protein or bacteriophage so each bacteriophage is displaying different specificity V segments
- Then you use this library of phages to screen plates that have the antigen mobilised on it
- The phage with the correct specificity V segment will stick to the plate and the others can be washed off
What are manufactured antibodies used for therapeutically?
- Prophylactic protection against microbial infection e.g. IVIG, synagis (anti-RSV)
- Anti-cancer therapy e.g. anti-HER2
- Removal of T-cells from bone marrow grafts e.g. Anti-CD3
- Block cytokine activity e.g. anti-TNA-alpha
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
What are manufactured antibodies used diagnostically?
Blood group serology
Immunoassays
- hormones
- antibodies
- antigens
Immunodiagnosis
- Infectious diseases
- Autoimmunity
- Allergy (IgE)
- Malignancy (myeloma)
What does ELISA mean?
Enzyme Linked ImmunoSorbent Assay
How does ELISA work?
- There are two samples containing antigen which have been used to coat two wells
- The wells will be covered with an antibody against the antigen that we’re trying to measure
- If the antigen is present, the antibody will stick and the reporter is an enzyme
- You then add a colourless substrate and if the antibody is present it will remain in the wells and so will breakdown the colourless substrate to produce a coloured product
What is a lateral flow assay and how does it work?
The most common example of lateral flow assay is a pregnancy test.
If an antigen is present in the analyte it will keep moving down the strip with the antibodies (atached to gold nanoparticles) and then will attach to another anotibody. An anti-IgG antibody is present in the control line to catch any remaining unbound antibodies.
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?
Larger immune complexes are recognised by immune system and cleared more easily but can activate platelets and neutrophils freely
Smaller immune complexes don’t efficiently activate complement, it will only activate complement when it is bound to a surface
What are the consequences of immune complexes in the body?
- Inflammation / complement activation
- Serum sickness (immune complexes in circulation)
- Immune complex glomerulonephritis
- Immune complex deposition at other sites
- Skin
- Joints
- Lungs
What is a particular problem related to immune complexes? Describe this.
Glomerulonephritis
- Large immune complexes will activate platelets and neutrophils
- Neutrophils will release mediators that affect the endothelial layer
- Smaller immune complexes will eventually pass through the cell layer and basement membrane and get trapped in the subendothelial layer
- Once they are stuck to a surface, they will activate complement
- Following complement activation, neutrophils will be attracted to the small immune complexes
- This will lead to the cell membrane and basement membrane becoming damaged which will eventually lead to a detrimental effect on kidney function
What is the difference between bands in a monoclonal, polyclonal expansion and healthy person in serum electrophoresis smears?
- 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, there’s a lot more gamma globulin so smudge will be much darker
- In this case there is also a smear showing polyclonal expansion - very sharp, dark band. A bone marrow aspiration would show abnormal B cells and plasma cells in the bone marrow.
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 tests can be done to test for serum immunoglobulin levels?
Serum electrophoresis
ELISA
Nephelometry
Subtypes are: IgG, IgM, IgG1, IgG2, IgG3, IgG4
What test uses specific antibodies for finding specific antigens/antibodies?
ELISA
e. g. protein antigens in Tetanus and haemophilia
e. g. polysaccharide antigens in pneumococcus
Which test is used to look at the different lymphocyte subsets?
Flow cytometry
CD3/CD4/CD8/CD19/NK cells
List the differnet lymphocyte subsets.
–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
What can be measured to define the extent of immune damage and to predict the short term outlook for HIV patients?
CD4 cell count
What infection do HIV patients get when CD4 count is extremely low?
- When you get an extremely low CD4 count you get MAC infections (mycobacterium avium complex)
- This is an environmental mycobacterium that is everywhere and normal people can deal with it easily
Describe how patients who test positive for antibodies to HIV are managed to increase life expectancy.
