Lecture 12: Uses of monoclonal antibodies Flashcards
Why are antibodies good biological tools?
Secreted in high amounts from differentiated B cells/plasma cells
Bind almost all biological materials
Binding well characterised and often high affinity (SHM)
What were the early approaches with purifying antibodies?
Purified antigen antibodies found in plasma
What is serum?
The plasma once blood clot removed
From immunised person/animal known as antiserum
Antiserum does not contain cells or clotting proteins
What will antiserum contains?
Many different antibodies secreted by different B cells and ma bind same antigen (different parts of pathogen)
How do antibodies bind different parts of the antigen?
As they are epitopes
How can different antibodies be purified from other serum proteins?
Using gel filtration chromatography (based on molecular weight)
Or
Affinity chromatography (binds to bead which filters out the other proteins)
What are the imitations of antisera?
The individual antibodies are separated out
Another individual has to be immunised however the antibodies will not be the same
What was the method devised by George Kohler, Cesar Milstein and Niles Jerne?
Fusing of mouse myeloma cells with mouse plasma cells making antibodies of know specificity to generate hybridomas
Why are myelomas useful in antibody purification?
They produce large amounts of homogenous antibodies
How are monoclonal antibodies produced?
Immunise organism (with specific antigen)
Harvest spleen
Spleen produces antibody and myeloma cells are produced
Cells mixed and fused
Transfered to HAT medium
Select hybrid making specific antibody
Clone
How can antibodies be detected once separated?
Using labels once bound to antigen which should not affect antibody/antigen binding
Why do methods using antibodies use secondary antibodies?
To detect primary antibody as it increases sensitivity
How are secondary antibodies produced?
Collect mouse sera
Contains mouse antibodies
Purify mouse antibodies and immunise rat
collect rat anti-mouse antiserum
Purify rat anti-mouse polyclonal antibodies
What are uses of antibodies in research?
Purifying biological molecules from a mixture (affinity chromatography)
Identifying the location of a protein within a cell using immunofluorescence microscopy
What are the uses of antibodies in diagnostics?
Western blot:
HIV dissociate in SDS (lyse virus)
SDS-PAGE (separate proteins based on molecular weight)
Transfer proteins to membrane and incubate with monoclonal antibody
Detect bound antibody with enzyme-linked anti-IgG
ELISA:
Linear and non-linear epitopes
Sensitive for specific biological material in wide range of complex samples
Quantitate amount of antigen present
Flow cytometry:
Characterisation based on light scattering properties
Natural or induced by pre-incubation of cells with antibodies labelled with dyes
Rapid and measures multiple parameters from each individual cell
