Lecture 8 - mAbs Flashcards
Why is the antibody response polyclonal?
Compare this w/ mAbs
When one is challenged with an antigen, the Ab response is polyclonal, because there are multiple antigens as well as multiple epitopes within the antigen present.
Multiple specificities of Ab will be induced.
On the other hand, mAbs are monoclonal, in that they have only one specificity, and come from a population of a single clone of B cell.
Describe the steps involved in generating mAb
- Mouse/rat injected with an antigen
- Fusion of myeloma cell + immune mice B cells
- Treatment w/ PEG + HAT
→ Hybridomas - Screening of hybridomas for desired specificity
- Dilution out in wells to one cell per well
- Screening for the Ab that we are looking for
What is a myeloma cell?
What features of these cells make them desirable for mAb production?
- ‘Professional’ Ab secretor
* Immortality
What is PEG?
What does it do?
- Polyethylene glycol
* Allows membrane of cells to fuse to form hybridomas
What is HGPRT?
This is a gene that allows tolerance of HAT
• Myeloma cells are sensitive to HAT → die
• B cells have the gene and are tolerant of HAT
What is HAT?
A drug that kills of the myeloma cells and not the B cells
What is CDR?
Complementarity determining region
One epitope, one CDR?
No
One epitope can be recognised by multiple CDRs
Why are sera more cross-reactive than mAbs?
• Sera contain Abs with many specificities
• mAbs are very specific for one epitope
NB some mAbs show some cross-reactivity, as a mAb can bind several similar epitopes
What does CD stand for?
What is it used for?
Cluster of differentiation
• Cell subsets express specific surface molecules
• These are used to classify the cells
What are the uses of mAbs?
- Serotyping
- Identification of cells
- Purification of biological molecules
- Therapeutics
What are some important mAb therapeutics
Rheumatoid arthritis:
• Humira / Remicade; Anti-TNF
Allergy
• Xolair; Anti-IgE/Fc(epsilon)R
B cell Malignancies
• Rituximab; Anti-CD20
Colon cancer
• Cituximab; Anti-EGFR
What are the draw backs of mouse derived mAbs?
How is this combatted?
The mouse parts of the Ig are rejected by humans
- In vitro CDR grafting
- Transgenic mice (have complete Ig gene loci)
Describe how molecules were classified by CD molecules
- Mice inject with human immune cells (CTCs, helper T cells, neutrophils etc.)
- Mice made antibody against surface molecules of the cells
- These molecules were named CD+number
Explain ‘Different mAbs can recognise the same antigen’.
An antigen can contain several epitopes; for example:
• HA has five different antigenic determinants.
Thus, different mAbs will recognise this one antigen (but the individual mAbs are recognising different epitopes)
