Polyclonal and monoclonal antibodies Flashcards
How do B cells generate antibodies? (3)
Somatic rearrangement
Affinity maturation
Class switching
Applications of antibodies (5)
Physiological role- fighting disease
Research - detecting “invisible substances”, blocking function, etc
Diagnosis- detecting disease markers, blood types, pregnancy hormones
Therapeutics- anti-venom, passive vaccination, immunotherapy
Industrial uses- abzymes, detectors
What are polyclonal antibodies? (2)
Those produced by many different B cells responding to the same antigen.
Polyclonal preparations contain a mixture of antibodies targeting different parts of the protein.
What are monoclonal antibodies? (2)
Produced by a population of identical clonal - B cells
They are homogeneous and all recognise the same epitope
Describe the process of polyclonal antibodies produced? (6)
Immunisation of animal
Normally immunised with purified protein plus an adjuvant
Adjuvant needed to elicit local immune response
Recruitment of cells, slow release of antigens, also activate APCs via pattern recognition receptors- release
of cytokines, T cell help needed for effective antibody production CD40-CD40L interaction etc)
A small hapten requires a carrier protein to elicit an immune response
Whole sera (liquid fraction of clotted blood) is collected and purified
Why are multiple boosts required? (2)
Multiple boosts generates higher titre, higher affinity antibodies
Advantages of polyclonal antibodies (3)
Technically easy to obtain
Antibodies are against numerous epitopes
Allows more effective crossing-linking/neutralisation also higher chance of cross-reactivity)
Give example of use of polyclonal antibodies (3)
Examples- passive protect against rabies, neutralisation of snake venom, Anti-D
What is the Haemolytic Disease of the Newborn? (3)
Rh- mother is sensitised to Rh+ foetus during first pregnancy/birth
Production of IgG against Rh by memory
B cells during second pregnancy destroys foetus (IgG is transfered across placenta)
Treatment for Haemolytic Disease of the Newborn (1)
Therapeutic anti-Rh Ab before birth prevents memory response
Disadvantages of polyclonal antibodies (4)
Polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen. (can not easily manipulate via recombinant means)
Each antisera preparation differs in specificity, average affinity, crossreactive specificies, etc.
Not optimised for application
Supply is limited.
How are monoclonal antibodies generated? (3)
Immunisation of animal and inject with adjuvant and antigen
Get poly-sera
Harvest the spleen for B cells
B cells produce antibodies
Immortalise B cells by fusing with cancer cells to prevent B cells from dying
Continued production of antibodies - hybridoma - mixture of B cells and cancer cells
Separate B cells from cancer cells by growing cancer cells in a selected media
Uses of monoclonal antibodies
Research
Diagnostic/prognostic indicators
Therapeutic antibodies
What are ELISAs? (3)
Allows for specific, sensitive and quantitative detection of soluble molecules within a
complex mixture. eg blood, saliva, tissue culture growth media (in both research and diagnostics)
Multiplex ELISAs can be performed (ie measuring multiple analytes and the same time)
What is Western blot analysis (2)
Allows specific detection from complex mixtures (eg cell lysate)
Not particular quantitative, but provides size information
What is flow cytometry? (4)
Depends on specificity of antibody
Defines individual cells from a mixture of cells- allows analysis of multiple antigens simultaneously on millions of cells.
Sensitive, quantitative, versatile
What is immunohistochemistry/Immunofluorescence (2)
Specific antibody linked to enzyme or fluorophore
Used to look at individual cells and localisation of cells
Look at components of cells
Therapeutic uses of monoclonal antibodies (3)
Rheumatoid arthritis, Crohn’s disease - Anti-TNF alpha (Infliximab), anti-CD11a (Efalizumab)
B-cell leukaemia anti-CD20 (Rituximab)
Breast cancer- anti-HER2/neu receptor (Herceptin)
What are the problems with monoclonal antibody therapy (3)
Repeat doses are often immunogenic, resulting in neutralising anti-antibody antibodies
Side-effects mediated via Fc region, eg complement activation, FcR activation, ADCC
scFv have different pr biological half lives, greater tissue penetrance (important for tumours/delivery to the CNS).
What is radioisotope arming method? (1)
DNA damage, can penetrate solid tumour
What is cytotoxic drug arming method? (1)
Cytotoxicity. Drug must be internalized
What is toxin arming method? (1)
Cytotoxicity. Toxin must be internalized
What is Antibody dimer arming method? (1)
More effective engagement of physiological killing mechanisms
What is enzyme arming method? (1)
Activation of prodrug
What is Bispecific antibody arming method? (1)
Engagement of cytotoxic T cells or natural killer cells
Advantages of monoclonal antibodies (5)
Consistent
Limitless supply of specific reagent
More easily tested for cross-reactivity
Can optimise for application
Can be manipulated via recombinant technologies
Disadvantages of monoclonal antibodies (2)
technically more difficult and time consuming, less likely to neutralise/cross react
Problem with mouse antibodies (2)
They’re mouse:
immunogenic, need to house and kill mice, once antibody is generated one needs
to clone the DNA sequences before manipulation.
Can’t generate antibodies against very homologous (similar) proteins
Solution for mouse antibodies problem (2)
In vitro generation, phage display technology
Why are antibodies good at eliminating pathogens? (4)
Specificity, high affinity, stability and their
effector Fc region provides an ideal mechanism
for binding and eliminating antigens
Polyclonal antibodies can be used to treat which disease? (1)
Haemolytic disease of newborn
Cloning hybridoma (5)
Spleen cells and cancer cells mixed
Polyethylene glycol to allow fusion
Separate out hybridomas and place at limiting dilution
Allow clones to expand
Expand positive well and test for production of antibody of desired specificity
Which tests are used for research? (3)
Tissue staining
Western blot
ELISA
Which tests are diagnostic/prognostic indicators? (3)
Immunohistochemistry
Flow cytometry
Confocal microscopy
What are the therapeutic antibodies? (2)
Toxin
Abzymes
How to work out the concentration of an unknown protein with ELISA? (4)
Standard amount of protein
Generate a standard curve
Plot a graph
Annulytes and read off the graph
Describe Western Blot Analysis (11)
SDS page gel - separate proteins based on size
Stain
Only works with pure proteins
Use specificity of antibodies
Separate proteins using gel electrophoresis
Transfer
Probe with antibody
Visualisation
See proteins of certain sizes
See isoforms of proteins
See degrading proteins
Can be fluorescence
Lateral flow tests
Test zone has an antibody against a specific hormone
Phage display (5)
Rearranged variable-region gene segments are randomly combined through the use
of recombinant molecular biology techniques to yield a large phage ‘library’ of
antibodies/scFvs
Bacteriophage library is used to infected E. Coli which display the “antibody”
fragment on the surfaces of the generated phage
Binding phage are then isolated and used to re-infect E. Coli
Totally human-derived antibody fragment with known sequence, ready for manipulation
Alternatives for antibodies (2)
Antibodies are composed of Ig domains
VLR genes from jawless vertebrates
consist of LRRs
