14 - Immunology Based Techniques in Research & Medicine - Partridge Flashcards
Generally, how do Abs protect against infectious material?
label pathogens and infectious material for ELIMINATION
Why can Abs used in therapeutics? Give 5 reasons
- diverse. > 10^9 specificities
- robust and stable domain structure which facilitates structural engineering. member of the Ig gene superfamily
- effector properties. can exploit the natural functions of Abs eg in activating complement, binding to Fc receptros on surface of phagocytes and NK cells
- specific and high affinity. Kd = 10^-8 > -9 M
- multivalent with high avidity allowing cross-linking
What is antisera?
serum containing high levels of Abs specific to certain antigen
Draw a graph of [Ab] against time that highlights how antisera is produced
311 -14 word
When Abs are purified, they can be labelled with certain tags name the 5 types they can be labelled with and how these labels can then be identified
- gold particles; immuno-electron microscopy
- enzyme; adding a S -> coloured product, ELISA, immunohistology
- fluorescence eg fluorescin; FACS (fluorescence activated cell sorting), immunofluorescence microscopy
- radioisotope; radioimmunoassay imaging of eg tumours
- sepharose; Abs bound to stationary matrix. immunoprecipitation or affinity purification
Name an Ab that Partridge created and name its target
LP9 Ab targetting the CD63 TM protein
How are polyclonal Abs generated? Draw a diagram to help
antigen with many varying epitopes used to raise many Abs against it
different subsets of B cells bind to varying epitopes on it and produce varying Abs
polyclonal antisera is generated w/ a mix of different Abs
What are the advantages of polyclonal antisera?
- cheap
- robust (can recognise partially unfolded/denatured antigen because likely at least some Abs will recognise the 1ry sequence)
- form immune complexes well because there are high numbers of them that can cross Link
What are the disadvantages with polyclonal antisera?
- purified antigen is needed to immunise. do not want to get Abs specific to unwanted product ->. contamination
- poly-specific. if trying to distinguish between v conserved antigens then wont be useful because many different Ab types
- difficult to standardise. at certain times, the no.s & types of different Abs present in serum will differ
What are the 3 main uses of polyclonal antisera?
- 2ndry Ab used for immunoassays. instead of having to label many different monoclonal Abs, can raise Abs against this original Ab (eg IgG). these 2ndry ones can be labelled with ease
- can identify relationships between molecules eg Immunoglobulins. eg Abs will recognise different isotypes and allotypes
- gene product identification. eg couldn’t isolate protein from dystrophin gene. knew sequence -> protein sequence -> peptide. Abs raised against this theoretical protein sequence to purify it (human genome project was helped using this method)
Describe the type of specificity of monoclonal Abs and where they are derived from
single specificity
derived from single B cell type
describe how monoclonal Abs are produced
- mouse immunised with antigen
- B cells isolated from organisms (contains both specific B cells and other Ab-producing B cells)
- fused to myeloma cells (using PEG - polyethylene glycol) to give hybridoma cells
- hybridoma cells (fused cells) selected for by growing on drug-containing medium. unfused cells selected against
- hybridoma cells producing correct Ab selected for and isolated
- clone selected myeloma cells and purify Ab
Who won the Nobel prize for this technique in 1984?
Kohler and Milstein
What are the advantages of monoclonal Abs?
- highly specific
- pure antigen not needed to immunise. this is because myeloma cells grow indefinitely in tissue culture therefore produce large amounts of the same Ab
- can be standardised
What are the disadvantages of monoclonal Abs?
- does not form immune complexes as well as polyclonal antisera
- expensive
- conformation sensitive. will only work if protein has correct conformation
How can monoclonal Abs be used in diagnostics?
- detect/quantitate diagnostically important molecules eg Down’s syndrome, fertility/pregnancy testing
- serotyping pathogens (of closely related strains). therefore can be used to assign certain antibiotics
How are mAbs used in defining cell surface molecules?
Give an example
(1st reason on pp)
- CD (cluster of differentiation) classification system of human leucocytes (>300 CD numbers now)
eg CD3; defining cell types
CD8/4; defining subsets
defining stages of differentiation
eg LP9 identifying CD63 - used to clone this gene - member of the tetraspanin superfamily
List the 3 other ways in which mAbs can define cell surface molecules
- biochemical analysis/purification - eg immunoprecipitation, SDS-PAGE (purifying proteins)
- functional studies - binding to surface structure may activate/inhibit cell functions. may mimic the natural ligand
- gene cloning - identifying gene products, screen expression libraries
Why are Abs described as magic bullets?
Abs can be used to target cell surface structures found only on cancer cells
Name the man who won the first Nobel prize for developing serum therapy - passive immunisation
Emil von Behring
What diseases was serum therapy effective against and describe how it works
- tetanus and diphtheria
- serum from immunised animals was an effective treatment against these diseases
- serum effective because of the Abs it contained
What were some of the downsides of serum therapy?
people developed immune response to horse serum
serum sickness
Name a rodent-based Ab that can be used to induce an immune response in humans
HAMA; human anti mouse antibody
Describe the 2 types of antibody engineering and some of their disadvantages
ANTIBODY CHIMERAS;
- mouse V region combined with human C region
- mouse v regions were still immunogenic
“HUMANISED” ABs;
- human framework regions and mouse CDRs (CDR grafting)
- relies on the immunoglobulin structure being stable therefore can be modified
- may lose affinity/specificity - sometimes framework regions can be damaged
- also v time consuming
Describe how we obtain Abs from gene libraries
- isolate mRNA from Ab producing cells eg lymphoid tissue, blood, bone marrow
- amplify Fab fragment by PCR -> cDNA
- clone and express the cDNA using a vector into bacteria/phage -> Ab library
- in bacteria, Ab expressed as soluble proteins. in phage expressed as surface proteins on filamentous phage particles
- screen Ab phage display library against solid phase antigen (panning)
Draw a diagram and explain how we obtain fully human Abs from a gene library through selection by phage display
- 311 - 14 word
- obtaining the Fab fragment (fully human) we can fuse to another human C region
Describe how Greg Winter won the Nobel prize for Chemistry in 2018
- synthetic/synthetic human Ab gene library
- used human V regions and randomised CDRs giving > 10^9 specificities
- CDRs are continually mutated to give rise to more specific Abs against a particular target
Name and describe 2 further strategies for generating human Abs
1) SCID MICE reconstituted w/ human leucocytes
- because they have no adaptive immune system, can tolerate human lymphocytes.
- can be immunised to generate fully human Abs
- but repertoire is limited to that of the human cells used
2) TRANSGENIC mice expressing human immunoglobulin genes.
- these introduced using yeast artificial chromosome (YAC)
- mouse Ab genes replaced w/ human Ab genes (xenomouse)
- mice can be immunised to generate human Abs through conventional monoclonal or phage display techniques
