Antibodies And Molecular Probes Flashcards
What are molecular probes?
Molecules (usually proteins) that bind very specifically to another target molecule (protein or non-protein)
Why are molecular probes important?
Because they enable us to locate, track, purify or disable target molecules
What is an antibody?
An adaptive immune system protein that very specifically binds to target molecules (antigens) and has the ability to discriminate between self and non-self
What is a Fab fragment?
A fragment antigen binding
What is an epitope?
A small part of the antigen that the antibody binds to (2-8 amino acids/ 2-8 sugar residues)
Elicits the immune reaction
What is a paratope?
The part of the Fab fragment that binds to the epitope
Describe monoclonal antibodies
A population of antibodies that bind to the same epitope and as such are identical
Give some advantages and disadvantages of monoclonal antibodies
+ very specific
+ never ending supply
- expensive and time consuming to make
How are monoclonal antibodies made?
Immunisation by injection of antigen of interest
Antibody producing B cells are isolated from the spleen
B cells are fused with immortal cancerous myeloma cells that have been grown in a cell culture and their cell membrane disrupted to fuse with B cells through either electroporation, virus or PEG
Hybridomas secrete antibodies in media
Hybridomas are screened for antibody production and antibodies can be frozen and stored indefinitely under the right conditions
Single monoclonal antibodies are produced
Describe polyclonal antibodies and give some advantages and disadvantages
A mixed population of antibodies that all bind to the same antigen but different epitopes thus are not identical
+ cheap and easy to make
+ high overall avidity
- limited supply
- lack of specificity
How are polyclonal antibodies made?
Target molecule is injected into an animal which produces antibodies as part of its natural immune response
How are molecules localised using mAbs and give some examples?
Fluorescent tags such as FITC (cheap, stable, works well and binds away from the Fab fragment) or GFP which are connected with a chemical linker
Tubulin visualised using anti-tubulin antibodies
P&G develop enzymes for cleaning and use antibodies to determine what molecules are on fabrics
Cell wall specific antibodies used to visualise carbohydrates
Anti-xylem antibodies to visualise xylem vessels
How are molecules quantified using antibodies?
Marker molecules such as enzymes are used and when in contact with substrate change colour (more molecule = more enzyme = more colour)
Western blots form an antibody: protein complex that has a coloured product
ELISA tests have a fluorescent plate reader that reads the signal
How do pregnancy tests work?
hCG is present in urine only during pregnancy
Urine is drawn across the membrane from the left absorbent pad to the right
Anti-hCG antibodies bind to the hCG in urine on the conjugation pad
Anti anti-hCG antibodies on the test line bind to the anti-hCG antibodies and coloured substrate on the first antibody and an enzyme on the second forms a coloured line on the test line if hCG is present in the urine
Different anti-hCG antibodies are attached to the control line and bind regardless of hCG binding due to an excess of the first antibodies as a means of showing the test worked
Describe the use of antibodies in therapies
Immunomodulation is a change in the body’s immune system and can be regulated with antibodies
E.g. mAb binding to excess molecules and mop them up
Infliximab for Crohn’s disease that inhibits TNF-alpha
Only works for a certain amount of time as not humanised
