Antibodies Flashcards
What 4 things may be attached to the constant region of antibodies?
Enzymes e.g. Peroxidase
Fluorescent probes e.g. dyes
Magnetic beads e.g. purification of cell types
Drugs e.g. Kadcyla
What is the basis that allows antibodies to be used in many diagnostic tests?
The specificity of antibodies for their target antigens
How can recombinant DNA technology be used to produce antibodies?
- Isolate all the V-segments of antibodies required
- Display all the V-segments on a protein or bacteriophage so all bacteriophages display different specificities
- Use the phage library to screen plates for the antigen; the complimentary phage will stick to the plate whilst others will be washed off
List 5 therapeutic uses of manufactured antibodies
Prophylactic protection against microbial infection e.g. when deficient
Anti-cancer therapy
Removal of T-cells from bone marrow grafts (so they don’t attack host)
Block cytokine activity e.g. as treatment of RA
Anti-calcitonin gene-related peptide for migraine
List 3 diagnostic uses of antibodies
Blood group serology
Quantitative immunoassays (levels of hormones, antibodies, antigens)
Immunodiagnosis (infectious diseases, AI, allergy (IgE), malignancy)
Describe the process of an ELISA assay
Antigen is coated onto wells
Antibody conjugated with enzyme is added + binds to target antigen
Substrate is added + signal produced by enzyme-substrate reaction is measured e.g. absorbance of light
How may antibodies be produced for diagnostic purposed?
Antibodies can be raised against almost any antigen
Can attach molecules to the constant Fc region without affecting specificity of the antibody
What are the 2 categories of antibodies?
Produced by patient: AI disease OR defence against infection
Produced artificially: antisera from immunised animals (polyclonal; have many specificities), monoclonal antibodies + genetically engineered antibodies
How are monoclonal antibodies produced?
Fuse normal B lymphocytes from the spleen (can produce antibodies but have limited cell division) with myeloma cells (no antibody production but immortal) to create hybridomas
Hybridomas can produce antibodies indefinitely
What is meant by the suffixes -umab, -omab and -imab when describing source of monoclonal antibodies?
umab: Human
omab: Mouse
imab: Chimeric/ partly humanised
What happens in a lateral flow assay?
Pre-made antibodies bonded to gold nanoparticles have an analyte passed over them
If antibodies successfully bind to the analyte (+ve result), the antibodies will bind to the positive strip + it will show up
E.G. hCG protein in pregnancy test
Describe the difference between large and small immune complexes
Large immune complexes:
Recognised more easily by immune system + cleared quickly
Activate platelets + neutrophils that release mediators.
Small immune complexes:
Get trapped in sub-endothelial layer
Can activate complement when stuck to the surface + so attract neutrophils that can damage kidney function
What would you look at to determine immunodefficiency?
Serum immunoglobulin levels- via serum electrophoresis/ ELISA
IgG, IgM, IgA + IgG1, 2, 3, 4
Specific antibodies - via ELISA
Protein antigens (tetanus)
Polysaccharide antigens (pneumococcus)
Lymphocyte subsets - via Flow Cytometry
CD3, 4, 8, 19, NK cells (can use specific antibodies against these receptors)
How can electrophoresis show immunodeficiency?
Healthy person: smear at top is gamma globulin region- diffuse due to a varying range of antibodies
Active immune response: this region becomes DARKER due to more gamma globulins
SINGLE SHARP BAND: indicates a monoclonal expansion of B-cells, indicates myeloma
What markers are recognised in flow cytometry to distinguish between lymphocyte subpopulations?
CD3+ ALL T cells CD4+ T helper cells CD8+ Cytotoxic T cells CD19+ B cells CD56+ NK cells
