Antibodies Flashcards
Lecture 5
Describe an antibodies structure
- Constant Fab region (made up heavy chains)
- Variable Fc region (made up of heavy and light chains)
- Overall structure is made up of 4 protein chains but has a symmetrical structure.
- In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical
- Inter-chain and intra-chain disulphide bonds
- Hinge region in heavy chain
- Fab region frequently glycosylated
What functions do the Fab and Fc regions play?
Fc region binds to receptors on cells
Fab region = for antigen binding
Can also use experimentally - attach to an enzyme like peroxidase or alkaline phosphatase as a reporter or for fluorescent probes etc…
What is the disadvantage of using antibodies from a patient as a source of antibodies against an antigen?
The antibodies are polyclonal
What does polyclonal mean?
Antibodies that are secreted by different B cell lineages within the body
This means that they will all react to the same antigen, but different epitopes thus have different affinities etc…
Describe the hybridoma method of producing antibodies?
B cell producing antibody of interest
Fuse with myeloma cell (polyethylene glycol in medium)
These cells can divide indefinitely and produce lots of antibody
Describe how DNA recombinant technology can be used to produce antibodies using a phage library is the model?
- Isolate populations of genes encoding variable regions
- Recombine with a phage coat protein
- Have antigens fixed + wash over phages with the attached variable regions
- The phage that gets stuck to the desired antigen - well then you’ve isolated a protein that targets your target antigen
Easy extrapolation that adding some form of mutagenesis will allow you to develop a variable protein region with greater affinity
What therapeutic use do manufactured antibodies have? (4)
- Prophylaxis - give an antibody specific to some pathogen and the person will be immune to said pathogen (although obviously this is short lived - need cells for long-lived immunity
- Anti-cancer therapy - the classic example everybody talks about is Herceptin (anti-HER2) (HER2 aka neu aka EGFR2 whatever you call it plays a role in breast cancer development)
- Block cytokine activity (or other hormones really)
- Removal of T cells from bone-marrow grafts (stop development of GvHD)
What other uses do manufactured antibodies have?
ELISA, blood group serology, all sorts of immunoassays
What is a lateral flow assay?
The classic that most of us have seen if not used is the home-pregnancy test
Essentially have a series of capillary beds
Capillary flow drives … flow
The first capillary bed absorbs excess fluid
On the second bed you have antibodies which bind to an antigen you are targeting (hCG in pregnancy tests)
The antibody-antigen complexes migrate to the third bed where they cause some sort of colour change
Someone presents with an infection - they complain of vague aches and pains what immunological concern might you have?
Immune complexes. Immune complexes are more likely in a large scale immune response. If the antibody is in large excess to the antigen they form larger complexes.
What is the relationship between immune complexes and glomerulonephritis?
Large immune complexes activate platelets and neutrophils
The net effect is damage to the basement membrane which leads to a deterioration in kidney function (glomerulonephritis)
How can you use antibodies to identify different cell populations?
Have antibodies that target specific antigens on the cell surface. The antibodies have some sort of marker (fluorescent). Run flow cytometry. As cells run through can pick up the fluorescence and identify each cell type. You can have multiple antibodies to target different cells so then can can study a heterogenous cell population. Could extend this technology for cell counting too.
