Antibody Technologies Flashcards
What are monoclonal antibody
Ab from the same B cell for the same ag are all the same so are monoclonal (b cells are clonally distributed via allele exclusion)
Why are ab easy to purify
They are released from plasma cells in high amounts
What do ab bind to with high affinity
Can have ability to bind to any biological molecule = good for research or diagnosis
What is the fluid phase of blood called when it is centrifuged to remove blood cells
Plasma (sits on top)
What is removed from plasma (after centrifugatjon of blood) to form serum
Blood clotting agents
What is serum from a person who’s been immunised with an ag called
Anti sera
What does antiserum have
Many soluble proteins and also many types of antibody for the same ag
How can anti sera be polyclonal if exposed to only 1 ag
Many B cells react to same ag, produce diff antibodies which bind diff epitopes on the ag
How are ab separated from other soluble proteins in anti sera
Gel filtration chromatography (size exclusion)
Or
Affinity chromatography
How does gel filtration chromatography separate ab from anti sera proteins
Ab are larger in mw so will elute from column later
What is added to column in affinity chromatography to separate ab from anti sera
The ag the person was first immunised with
What is anti sera called bc many types of ab
Polyclonal anti sera
What is the issue with polyclonal anti sera (reason monoclonal ab produced)
You would want to separate diff types of ab for the ag but can’t separate them bc same mw and also same affinity for ag
Which B cell tumours were discovered to grow forever and can’t release of monoclonal ab (further used to produce ma)
Multiple myelomas
What was taken from immunised mice and fused with multiple myelomas to produce hybridoma cells
B cells which produce ab for the ag the mouse was immunised with
Which ab would be made from the myeloma and immunised B cell mix
The antibodies specific for the ag immunised by mouse, the myelomas used don’t produce any ab but have ability to grow forever and produce them
How are myeloma cells killed if they survive after trying to produce hybridomas
They are transferred to media which kills cells which don’t have a specific enzyme producing dna, myelomas don’t have the enzyme
Why won’t B cells be present if they haven’t fused with myeloma to form hybridoma
They die in tissue culture
How is the ab selected in ma production
The best ab will be tested through binding tests to the ag
How are MA when produced for research detected
Via primary or secondary antibodies which are labelled via Fluorochrome or enzyme substrate reaction
Why is using secondary antibody immunofluorescence better
Produces a stronger signal because chances of ab binding to are higher
What mustn’t labelling affect
Binding ability of ab to the samples
How are secondary antibodies produced which bind primary MAs
Antibodies produced from immunised mouse sera are purified from sera eg via GF chromatography
Transferred into a rat for example which is immunised with the antibodies
The rat then produced antibodies for the antibodies injected
The rat antibodies are purified from its anti sera = secondary ab
How can ab be used in research to purify / seperate molecules
Affinity chromatography
If ma are made which are specific to the molecule of interest they are bound to column and separate the molecules
How does macs work (magnetic bead isolation of molecules)
Magnetic beads with antibodies allow ab to only bind to molecules if a magnet is present
The molecules elute when magnet is removed
How does immunoprecipation for proteins work using ab
Proteins are radiolabelled due to Met aa,
Cells lyse and release these proteins
Ab are added and then removed , the protein bound is run on sds gel And visualise under light
How does immunofluorescence work to find if cells have ag
Using microscopy you bind ab to a cover slip and the ab has fluorochrome on it
The light will show where ab has bound on the cells or if it has. = cell has ag
How is western blot used for diagnostics or to detect level or protein (similar to immunoprecipitation)
Sds done first
Anti sera then added and ab will detect the protein of interest via binding on a membrane
Ab usually seen by light due to enzyme (in immuoprecipitation the protein is radiolabelled and run on gel after )
Why is Elisa used
To detect proteins with a low sample size
What are the 3 types of Elisa
Direct (using primary ab)
Indirect (using secondary ab too)
Sandwich (binds capture ab then sample added then a secondary antibody used)
What are the labels used for Elisa (same as western blot)
Enzymes Substrate colour change
Which technique is used to characterise cells from eg a blood sample
Flow cytometry
What does the laser measure when cells move through column in flow cytometry
The patterns of light scattering
What does forward scatter mean
The scatter which depends on the size of a cell
Eg macrophages are large so have a large forward scatter to others
What does side scatter measure in cells
Their granularity levels (low in lymphocytes but high in eg neutrophils)
What type of graph separates the cells depending on the ss and fs results
A dot plot
What is the term used to describe the focus on one type of cell in flow cytometry after the initial Fs and ss analysis
Gating using a computer eg gate lymphocytes from macrophages and neutrophils
(Low fs and ss)
What is added after the first gating to fully characterise cells
Antibodies with fluorochrome
Antibodies will be specific for cell type specific molecules eg cd19 ab binding = it’s a B cell
Cd3 antibody binds both t and B cell
Is just one ab used
No , different ab used with diff fluorochrome colours so fluoresce at diff wavelengths of light to detect diff cell types from diff laser detectors
So could use two at a time in first gate eg both cd3 and cd19 antibodies added
Another gate is done after ab binding. How
You gate positive cells ie cells which ab has bound to
Eg cd3 antibody will have positive B cells only in the second gate
What is the issue with using diff ab fluorochrome at a time
Fluorochromes have cross over at some wavelengths. This can give false positive or negative results for cells
What is the solution to cross over of fluorochromes
Compensation -
A control is done where cells only bind 1 ab at a time which should be picked up by 1 laser detector. The levels of defection by detector 2 are deleted because this is false
(The control is done with the other detector fluorochrome too)
Why can’t discontinuous / conformation epitomes be used in immunoprecipitation or western blot
Because the proteins are denatured on the sds page removing secondary structures so ab won’t be able to bind on gel
