Diagnostics - Antibodies as Diagnostic tools Flashcards
What are the different types of reporters
- Enzymes
- Fluorescent probes
- Radioisotopes
- Magnetic beads
How do enzyme reporters function
- Wash the antigens over with an antibody with the enzyme attached
- Add colourless substrate that changes colour when in the presence of antigen bound enzyme
How do magnetic bead reporters function
- Have an antibody that binds to the target
- Special column can be used that has a magnet to extract the cells that are bound to the magnetic beads
What are the three methods for manufacturing antibodies
- Antisera from immunised animals
- Monoclonal antibodies
- Genetically engineered antibodies (using recombinant DNA technology)
How are monoclonal antibodies produced
- Fuse a B lymphocyte that is producing the antibody of interest to a myeloma cell line
- This produces a hybridoma
- These cells can then produce the antibody of interest
Describe production of antibodies by antisera from animals
- Historically the antigen would be inserted into the animal and the antisera is removed once the animal produces an immune response
- This gives a limited amount of antibody
Describe production of antibodies using Recombinant DNA Technology
- Make a library of all possible V segments
- Display V segments on a coat protein or bacteriophage
- Then use this library of phages to screen plates with the mobilised antigen
- The phage with the correct specificity V segment will stick to the plate and the others can be washed off
What are the therapeutic uses of manufactured antibodies
- Prophylactic protection against microbial infection - give to people who cant make their own antibodies
- Removal of T-cells from bone marrow grafts - prevents graft-versus-host disease
- Block cytokine activity
- Anti-cancer therapy - many therapies based on monoclonal antibodies targeting overexpressed molecules in tumour cells
What are the diagnostic uses of manufactured antibodies
- Tissue typing
- Blood grouping serology
- Immunoassays (hormones, antibodies, antigens)
- Immunodiagnosis (infectious diseases, autoimmunity, allergy, malignancy)
What are the two cases in which somebody could have antibodies for HIV but not have HIV
- Maternal antibodies
- People who volunteer for clinical trailers
- If someone is very recently infected then they might not have had time to produce antibodies
Why do smaller immune complexes form and what is the problem with them
- The ratio of antigen to antibody determines the size of the immune complex
- The more antigens there are the larger they will be
- Large immune complexes can be cleared easily by the immune system but small immune complexes don’t efficiently activate complement
- This can cause glomerulonephritis
How do smaller immune complexes cause glomerulonephritis
- Large immune complexes will activate platelets and neutrophils
- Neutrophils will release mediators that affect the endothelial layer
- Smaller immune complexes can pass through the cell layer and basement membrane getting trapped in the subendothelial layer
- Once they stick to the surface they activate complement which attracts neutrophils
- This will then cause damage to the cell membrane and basement membrane, which will have a detrimental effect on kidney function
What is flow cytometry used for
- To determine lymphocyte subsets
- Antibodies against specific markers are used to identify different lymphocytes
How can serum electrophoresis be used to determine different types of infection
- In a healthy person there is a diffuse gamma globulin region
- In someone with an infection you will have more gamma globulin making the region darker
- This shows polyclonal expansion
- A single, sharp, clear band, then this indicates a monoclonal expansion of B cells
How are cell populations measured
- Label several different monoclonal antibodies with different coloured fluorescent dye
- Cells passed in a stream through a laser beam and the fluorescence is detected
- Each cell can then be categorised based on the basis of its fluorescence
