13 - monoclonal antibodies Flashcards
inject 1 antigen into a mouse - what kind of response?
polyclonal
polyclonal response
complex mixture/pool of antibodies produced
many different B cells switched on
no discrimination between antigens
why do we get a polyclonal response
1 antigen carries many different epitopes
each antibody specific for a different epitope
what does the monoclonal response allow
recognition of just the 1 B cell producing the antibody of interest
no cross reaction with other epitopes
relevance of Kohler and Milstein 1975
hybridoma technology
production of monoclonal antibodiees
what does hybridoma technology allow
identification and culture of cells secreting identical antibodies with pre-defined specificity
method of hybridoma technology
immunise animal and allow immune response to build up
B cells become activated by specific antigen causing disease
B cells produce monoclonal antibodies
fuse splenocyte with myeloma cell
results in immortal hybridoma cell
splenocyte
antibody producing B cell from the spleen with a finite lifespani
myeloma cell
non antibody producing B cell with infinite life span
produces immortality in hybridoma cells
3 methods to immunise/inject the mouse
intrasplenically
into tail vein
into peritoneum
most common way to immunise mouse
inject antigen into peritoneum
antigen can enter bloodstream quickly to amount immune response
time course of immunisation of mouse
after a few days
- build up and activation of multivalent, low affinity IgM antibodies
after 2 weeks
- immunise again
- allow build up of immune response then drop off
after multiple boosts
- build up of bivalent, higher affinity IgG
method to test which cells are fully fused hybridoma cells
tail vein removed from mouse and serum from blood tested for immune response
if +ve, then booster injected
spleen then removed from mouse and cells fused with myeloma cells in vitro to produce hybridoma cells
isolation
- HAT medium: knocks out un-fused splenocytes and myeloma cells
- ELISA used as screening assay to identify well producing antibody of interest
- expand cell
hybrid cell selection using HAT medium
hybridoma cells have HGPRT from spleen and immortality from myeloma cells
therefore they are the only cells that will grow in the HAT medium
HAT medium =
Hypoxanthine-Aminopterin-Thymidine medium)
aminopterin
blocks main biosynthetic pathways for nucleic acids
prevents cells from making RNA/DNA
importance of adding Hypoxanthine and Thymidine
they can salvage the biosynthetic pathways for making nucleic acid
importance of HGPRT
only cells that have HGPRT can make RNA via the salvage pathway
use of ELISA
assay used by immunologists that allows identification of antibody of interest
method of ELISA
microtitre well with antibody attached
expose hybridoma solution to the well
add antibodies to bind to Fab region of antigen
add secondary antibodies to target Fc domain of primary antibody
colour reaction allows identification
example secondary antibody used in ELISA
rabbit-antimouse enzyme conjugate
phage display technology
takes antibody fragments and expresses them as fusion proteins on the outside of bacteriophage
method of phage display technology
take B cell source and make a combinatorial library
convert mRNA to cDNA
PCR –> amplifies V-gene families
reassemble V-genes into new combinations
phage then expresses new combinations on surface as antibody fragments
V-gene families
variable light and heavy genes of antibody
scFv
single chain variable fragment
a fusion protein of the variable regions of the heavy and light chains of immunoglobulins
where to bacteria express scFv
on their terminal coat protein
pII protein
allows Fab fragments to also be expressed on the phage as an antibody fragment
if antibody fragments are expressed along the length of the phage
then there is low affinity of antibody expression
isolating antibodies using phage technology
use ELISA on phage population
process called biopanning
limitations of biopanning
very expensive and fiddly
method of biopannign
expose antigen to phage population
wash off non-binding antigens
identify bound phages using reporter molecule
use UV to eliminate non-bound
elute/remove bound phages and increase population using E.Coli
3 methods of in vitro diagnositc assays using monoclonal antibodies
1 - ELISA
2 - Lateral-flow assay (LFA)
3 - Proximity Ligation Assay (PLA)
PLA - proximity ligation assay
use PCR from antigen instead of from nucleic acid
uses of monoclonal antibodies
- diagnostic pathology to identify cancerous cells
- in vitro diagnostic assays
- diagnostics to track pathogens
- affinity purification
- characterisation of antigens
recent surge in ability to use mouse monoclonal antibodies (mAbs) in vivo due to
phage display and hybridoma technology
problems with monoclonal antibody use
need to “humanise” monoclonal antibodies otherwise the immune system recognises them as foreign and the mouse antibdy will be destroyed quickly (HAMA reactions)
xenogenic
antibodies coming from another species
HAMA reaction
Human Anti-Mouse Antibody reaction
4 methods of humanisation
1 - produce human hybridomas
2 - create chimeric antibody
3 - CDR grafting
4 - make a transgenic mouse
limitations to the production of human hybridomas
low efficiency
ethical issues - source of human B cell pool?
production of chimeric antibodies
replace constant region of mouse antibody with humans
CDR grafting
replace CDR of human antibody with mouse CDR
advantages of transgenic mouse
fully humanised
disadvantages of transgenic mouse
expensive
transgenic mouse method
knock out all mechanisms to create mouse antibodies
replace with humans
only human antibodies expressed
Rituximab
chimeric antibody drug
