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
