Antibodies as diagnostic tools Flashcards
What can you attach to the ends of antibodies?
Enzymes e.g. peroxidase
Fluorescent probes e.g. dyes
Magnetic beads e.g. purification of cell types
Drugs e.g. Kadcyla, anti-HER2
What is the basis for many diagnostic tests that involve antibodies?
Unique specificity of antibodies to antigens
What are the two types of antibodies?
Produced by the patient
Manufactured - antisera from immunised animals, monoclonal
How are monoclonal antibodies generated?
mouse challenged with antigen
Spleen cell removed that produce the Ab - limited cell division so fused with myeloma cells - forming Hybridomas
Then culture in HAT medium and select for positive cells
Then harvest the monoclonal antibodies
what does -omab, -imab and -umab mean?
- omab = derived from mice
- imab = recombinant
- umab = human only
How are recombinant antibodies made?
library of V-segments and library of bacteriophages
Fusion protein
What are the THERAPEUTIC uses of manufactured antibodies?
prophylactic against microbial infection e.g. IVIG, synagis
Anti-cancer therapy
Removal of T cells from bone marrow grafts e.g. anti-CD3
Block cytokine activity e.g. anti-TNFa
What are the DIAGNOSTIC uses of manufactured antibodies?
blood group serology
immunoassays
immunodiagnosis: infectious disease, autoimmune, allergy, malignancy
What is the ELISA test?
Enzyme linked immuno-sorbent assay
Antibodies linked to an enzyme are added to a well that could contain the target antigen
If present, bind so cannot be washed away
Uncoloured substrate added, the enzyme on the antibody converts it to a coloured substrate
Light absorbance can be measured via mass spectrometer
What is rapid testing?
put on a sample
capillary action along the strip moves the sample alaong
antibodies conjugated to gold particle bind to antigen
Test line - visible if positive test
Control line - anti-G antibodies - visible line to show it’s working
What is the problem with immune complexes?
result in inflammation and complement activation
How is immunodeficiency determined?
serum immunoglobulin levels - using ELISA, electrophoresis, nephelometry
Specific antibodies - using ELISA - protein antigens, polysaccharide antigens
Lymphocytes subsets (Flow cytometry) - CD3, CD4…
What does serum electrophoresis show?
Thick band of gamma globulin - active immune response
sharp thin band of gamma globulin - monoclonal expansion of B cells - myeloma
What is flow cytometry?
Specific marker (CD) used against various lymphocytes
CD3+ for T cells
CD56+ for NK cells
monoclonal antibody fluorescently labelled
pass through laser beam in a stream and fluorescence is detected - each cell can then be characterised based on fluorescence
