Monoclonal antibodies Flashcards
antibodies can be used to
exploit immune mechanisms we use naturally
antibodies which have been refined for specificity and affinity can be used to
track diseases in vitro and in vivo
specific antibodies can be used to also
turn immune systems on to kill cancer cell
most antigens have
many epitopes
- animals injected with a single antigen will produce a complex mixture of antibodies- each made by a different clone of cells
polyclonal response
when body becomes exposed to an antigen, a mixture of antibodies will be produced due to the fact antigens have many epitopes
- does not discriminate between different antigens
to discriminate between different antibodies
antibodies need to be identified that bind to epitopes specific to individual antigens
monoclonal anitbodies
bind to epitopes specific to individual antigens
monoclonal antibodies will not
cross react with conserved antigens- due to the antigen being focused on being specific to that antigen
need to find a B cell
producing antibodies specific to antigen-monoclonal
two ways of creating monoclonal antibodies
Hybridisation
Phage Display
hybridisation is brought about using
hybridoma technology
hydrbidoma technology allows
the identification and culture of cells secreting identical antibodies with a pre-defined specificity
Hybridoma simple-
A single clone of cells secreting a single
antibody is made by fusing a B cell (Ig+ splenocyte
with finite lifespan) with a myeloma cell (cancerous
Ig- B cell with infinite lifespan)
B ells activated within
mouses immunised with unique antigen- to create an immune response- with hyper mutations- class switching to produce highly specific monoclonal antibodies
B cell is a
Ig+ spelnocyte with finite lifespan
Myeloma cell
cancerous Ig- B cell with infinite lifespan
basic principle of hybridoma
fuse a mortal cell with immortal cell 9which can live outside the body)to produce an immortal B cell hypridoma- producing monoclonal antibodies
Hybridisation process;
1) inject mice with antigen of interest (directly into spleen)
2) antigen goes into blood stream and activates B cells
3) within the first few days: activates low affinity multivalent IgM
4) as you carry on immunising (i.e. primary infection, secondary injection, tertiary injection, multiple boosts), the immune response builds up by genetic switching- making antibodies more and more specific to the antigen- increasing affinity
5) then you cut the mouse’s tai and the serum of the blood that comes out can be tested for response to target antigen
4) if response is large enough- the mouse will be euthanised and spleen removed
5) B cells isolated
6) fuse spleen cells (splenocyte) with myeloma in vitro
7) hybrid cell
spleen is
a secondary lymphoid organ
in the mix produced after B cells and myeloma are fused
there will also be many unfused myeloma and splenocytes in the the mix
there will also be many unfused myeloma and splenocytes in the the mix- how do we get around this
Isolation of hybrid cells using HAD
Isolation of hybrid cells using HAD medium
-kocks out splenocytes and B cells which havens been fused- therefore only left with hybrids
what is used to isolate hybrid cells using HAD medium
20, 96 well culture plates
- each could have a hybrid cells which is producing the correct monoclonal antibody
what is used to identify if a well contains the hybrid cell producing correct monoclonal ntibody
EISA
if you want to produce larger quantities of antibodies
hybridisation is carried out within a rabbit or goat
unfused myeloma cells dies in
HAT mediaum
unfused B cell
dies naturally
hybridoma cells are
immortal and survive in HAT
mechanism of hybrid cell election in HAT medium
within HAT medium there is a component called Aminopterin (blocks the main biosynthetic pathways for nucleic acids- stops cell producing RNA and DNA).
- Within the cells there are two salvage pathways- which rely on two enemies (hypoxanthine and thymidine kinases)- which slaavege the nucleic acid biosynthesis
- both cells types have thymidine kinase (so can make DNA)
- only cells with HGPRT can make RNA
spleen B cell is HGPRT
positive
Myeloma cell is HGPRT
negative
- therefore not able to use the slavge pathway for RNA synthesis
hybridoma cells
has the HGPRT from the spleen cell (RNA synthesis) and immortality from myeloma cells- so able to grow in HAT medium
- providing there is an outside supply of H and T
What is used to then isolate the hybridoma in the wells which have survived
Plate-trapped-antigen- enzyme- Linked immunosorbent assay
Plate-trapped-antigen- enzyme- Linked immunosorbent assay
PTA-ELISA
PTA-ELISA process allows identification of antibody of interest
1) on wall of the well you have your Ag of interest
2) mAb specific to Ag will bind to the epitome via Fab domain
3) then rabbit anti-mouse enzyme conjugate added which binds to the Fc region of mAb
4) this antibody is bound to an enzyme- which causes colour change when substrate is added
rabbit anti-mouse enzyme
a second antibody- raised in mice, using mouse antibodies which have been injected into rabbits which ave made antibodies agains the mouse antibody)
If colour change occurs in PTA-ELISA
monoclonal antibody is present
which technique allows you to get away from using a live host to produce monoclonal antibodies
phage display
phage display
taking a phage which infects bacteria and getting them to display antibody fragments on their surface
phage display is a techniue
in which peptides and proteins (antibody fragments) are expressed as a fusion with a coat protein or bacteriophage
- fuse proteins are displayed not eh surface of VIRION- while the DNA encoding fusion resides within the virion
example of a virus which is used to express antibodies
M13
M13
ssDNA genome- integarre DNA inro genome of host cell to express antibodies
making antibody combinatorial libraries using phage display
1) B cell source (immunised animal)
2) production of mRNA
3) production off cDNA from mRNA
4) PCR to amplify each of the V-gene families (VL?VH)
5) assembles the VH and VL genes ar random
6) clone as scFv onto PIII phage
scFV
VL and VH domains of an immunoglobulin linked by a peptide spacer
scF are expressed
on surfaces of phages
up to ….. ca be pressed on a phage
5 copies
fusion pteoteins form between
scFV and PII minor coat proteins to allow expression of scFV on surface of the phage
expression of Fab fragments on filamentous phage
Fab fragments of antibodies can be expressed on the surface of phage as fusion proteins, with PIII
expression of Fab garments on filamentous phage
Fab fragments can also be expressed on the surface of phage as fusion proteins with pVII- multiple copies expressed along the length of the phage particle
- useful for isolation of low affinity antibodies
if you want to epees slow affinity antibodies
multiple copies can be expressed along the length of the phage particle- rather than on the PII
isolating antibodies using phage technology
take a population of phage and do ELISA this allows you to pull out the phage producing the antibody of interest
phage selection
1) need to eliminate phages not producing antibody of interest
2) to get rid of all phages not of interest big UV
3) these phages are then eluted and used to infect E.coli
4) e.coli used to bulk u population of phage and is lysed
phage display and selection means
never have to back to immunization- have combinatorial library
