CCAT L9 Polyclonal Antibodies Flashcards
Lecture outline: Define the main steps involved in generating polyclonal antibodies, the problems involved at various stages and how they are overcome. Suggest why anti-peptide antibodies can be useful and how they are produced.
With a polyclonal response, how many antigens are needed to activate several B-lymphocytes?
One antigen will activate several B-lymphocytes.
This is because one antigen will have multiple epitopes, and antibodies with different paratopes will be produced against this one antigen.
Each antibody will be specific for one antigen epitope.
This increases the liklihood that antibodies will bind the antigen.
What are the two regions of an antibody?
How many chains are they composed of?
Antigen binding site (Fab)
Constant domain (Fc)
Four polypeptide chains (2 heavy, 2 light).
What is the antigen binding site on an antibody composed of?
~120 amino acids at tip. Two variable regions, VH or VL from the two chains.
In polyclonal antibody production, what must be considered when choosing immunogen?
Complexity/size/solubility of molecule.
Complex, large, insoluble molecules generate good response.
Purity - must be pure, purified via 2D-PAGE, size exclusion chromatography, affinity chromatography.
When designing immunogen for polyclonal antibody production, what is different when considering viruses or cancer epitopes?
For virus, heat inactivated virus can be used and injected to give polyclonal response.
For cancer, specific epitopes for targets must be used.
What can be done to improve polyclonal production?
Modification of antigen (to increase size and insolubility)
- denaturing - exposing more epitopes
- linkage to carrier proteins (BSA)
- cross link monomers into large complexes.
What must be done to peptide immunogens to make them immunogenic for poAb production?
Conjugated to carrier protein to make it immunogenic
When selecting a peptide to induce poAb production, what general characteristics should it have?
Which three amino acids present generates better antibody response?
At least 10 amino acids long.
C- or N-terminal sequences, more likely to be hydrophillic on outside of lipid bilayer and outside of cells in vivo.
Gutamic acid (present in epitopes)
Proline (gives shape)
Tyrosine (large, used to couple to carrier proteins)
Purity above 90%,
Peptide to carrier ratio 5:1
Choice of model is important in generating poAb response.
Which animal is generally used for moAb and which are used for poAb?
moAb usually mice.
poAb generally larger animals.
Depends upon amount of serum required (larger animals, more serum can be harvested).
Also must consider compatibility with immunogen source and model animal.
During immunisation of animal to produce poAbs, how is the optimal dose for immune response identified?
Several animals are immunised with a range of antigen concentrations to identify the correct level. Don’t want too high of a response. For peptides, dose that immunises 50% of animals given it is correct dose.
What is the purpose of the adjuvant during immunisation?
What drawbacks does this have?
When is an adjuvant not needed?
It acts as a ‘highlighter’ of antigen to the immune system, enhancing the immunogenicity of antigen.
Bad side effects can be seen, fever, inflammation from IL-2 production is stimulated, but this is a positive sign as it shows successful immunisation.
Using intact bacterial cells to immunise means adjuvant is not needed.
What are three basic mechanisms by which adjuvants work?
Enhance long term release of antigen - increasing exposure time to immune system.
Stimulates modulating immune cells.
Enhances macrophage phagocytosis.
What are the two main types of adjuvant?
How do their uses differ?
Freund’s Adjuvant (Complete and incomplete)
This is water in oil emulsion (the adjuvant) with heat killed mycobateria (if complete), comp used as primary immunisation.
Incomplete used for booster immunisations following initial antigen-CFA.
Aluminium salt adjuvants
These are used most frequently for human vaccine antigen delivery. Weaker adjuvant, so needs to be given with strongly immunogenic antigens.
Generates more mild inflammatory reactions.
Using Freunds adjuvants, how are antibodies generated from animals?
From the blood sample obtained during gen of poAbs, what is found, and what does each cell do?
Neutrophils confirm that injection is successful.
Plasma cells are cells of interest which take some time, why animals are rested for weeks.
How are antibodies confirmed to be in the blood sample from animal making poAbs?
Most cost-effective way is ELISA.
But IHC, IB, bioloigcal assays or cytotoxicity assays can be used.
Summary of the properties of monoclonal antibodies and polyclonal antibodies.
(answer is table)
