Antibodies Flashcards
Is the normal immune response mono/polyclonal?
- Polyclonal
- Antibodies are a mixture of Igs to different epitopes (vary in hypervarible region)
- normal immune response involves several monoclonal lymphocytes
Why do we need a polyclonal response?
Target antigens have several different epitopes, promotes a range of diverse, specific antibody binding sites
Why are antibodies good reagents?
- high specificity
- possible avidity for virtually any antigen
- better than alternatives (peptides have to be conjugated with large “carrier” molecules to be immunogenic)
What 4 things do we have to think about when producing polyclonal antisera?
- Cost (e.g. animal housing facilities)
- Amount of antigen available (goat will require a lot more than a mouse)
- Volume of sera required (mouse will provide 1-2ml of blood)
- Phylogenetic relationship between antigen from origin and host
How do we produce polyclonal antisera?
- We immunise the animal to activate the B cells
- Use an adjuvant that is a slow Ag release and direct stimulation of immune system
- Then do boosters several times, a few weeks apart - increases antibody production and allows class switching to IgG
What are B cell tumours?
Derive from uncontrolled growth of a single cell
Advantages of monoclonal antibodies
- high specificity
- low possibility of cross reactivity with unrelated Ag
- an unlimited supply
How do we produce monoclonal antibodies
- immunise animal of choice
- harvest B cells
- take HPRT sensitive tumour culture cells and immortalise them using Sendai virus
- Fuse the B cells and the tumour cells and then treat with a drug
- unfused B cells will die as they are not immortal
- unfused tumour cells will die from drug
- fused cells which are both B and tumour cells will immortal and resistant
- plate out individual fused hybridomas and grow
- each is derived from a single B lymphocyte, so the secreted antibody is monoclonal
How do we test if hybridomas are making the antibody we want?
Use an ELISA test
What adaptions can we do to native monoclonals?
- Can re-engineer DNA coding heavy and light V region, which can be fused to active molecules to target activity
- Can combine antigen binding site engineered in mouse with Ig constant regions from humans - increases half life and decreases adverse effects
What is biopharming?
- plants can be transgenically altered to express antibodies
What are advantages of plantibodies over mammalian cell lines?
- more efficient expression and cheaper purification
- allow complex Ab folding
- stable if stored in seeds
- can be post-translationally processed improving activity
- oral delivery
What plants have been used to produce antibodies so far?
- Tobacco plant - IgG against plant pathogens (resistance), HIV, cancer and rabies (therapy), hepB (purification)
- Glycine max (soya bean) - IgG against Herpes (therapy)
- Alfalfa - IgG diagnostic antibody
What is immunoblotting/ Western blotting?
Separates and detects specific proteins from a protein mixture
How is Western blotting carried out?
- Denature by treating with agent (e.g. SDS)
- Fractionate by length of peptides using SDS-page (electrophoresis)
- Stain proteins to visualise
- transfer onto a robust membrane
- hybridise to specific labelled antibodies
What can we use immunoblotting for?
- determining size of antigen - detecting proteins that are secreted by ebola infected cells.
- use known quantity in control well and compare intensity of band
- screen samples for antibody presence
What do we use immunocapture for?
- pregnancy test - have a control line and test line. Put sample on end, liquid flows along the paper. Antibodies spread along with it. If there is beta-HCG, the antibodies will take them up and give a positive line. A second line isnt specific for Beta-HCG, but is specific for antibody. One line, the test has worked, two lines, it is positive.
- Rapid ELISA - the same sort of thing but using wells. If when you have put an indicator enzyme in there is a colour, then it is positive for the antibody.
What can we use immunoprecipitation for?
- detect proteins of interest in a sample
- purify native proteins of interest
- characterise molecular weight of proteins
- study structure of proteins or antibody/antigen complexes
- detect specific antibodies in a sample
What is immunoprecipitation?
A technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. Can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.
Give an example of a disease that can be detected using immunoprecipitation
Grave’s Disease
What is affinity chromatography?
- purifying a soluble molecule from a mixture, requires specific and reversible binding
How do we do affinity chromatography?
- couple a ligand (antigen antibody) to a solid matrix in a chromatography column
- pass sample through column allowing bindign and discard the rest of the mixture
- elute off bound molecule
What is immunomagnetic separation?
- Specific antibodu coupled to magnetic beads
- mix sample and beads and bind
- apply magnet
- wash out supernatant
- re-suspend purified bead
- elute off beads if necessary
