Practicals Flashcards
1
Q
What is a monoclonal antibody? (2)
A
- Proteins made in laboratories that act like antibodies
- They are clones of just one antibody, and they bind to one antigen only.
2
Q
How do we produce mAbs using murine derived spleen cells? (3)
A
- Take lymphocytes from the spleen of an immunized mouse
- Lymphocytes are fused with myeloma cells to form hybridomas
- These hybridomas divide and produce millions of monoclonal antibodies specific to the original antigen
3
Q
What is a hybridoma and how do we screen for their presence? (3)
A
- A fusion product of B lymphocytes and myeloma cells
- They retain the properties of both classes of cell in that they secrete large amounts of antibody, proliferate rapidly and remain immortal
- Screening for hybridomas – Dot blot
4
Q
Dot blot technique (3)
A
- The supernatant can be quickly screened for antibody production by a simple dot blot technique where supernatants are applied to nitrocellulose paper
- Following washing and blocking, an enzyme conjugated antibody is added, this will bind to any mouse antibody on the nitrocellulose paper and can be visualised by addition of appropriate substrate
- The positive dots indicate that the cells in the wells from which the supernatant came from were hybridomas producing monoclonal antibodies to the original antigen
5
Q
What is CD20? (1)
A
- A 33-37 kDa non-glycosylated protein expressed on the surface of normal and malignant B lymphocytes
6
Q
Why might CD20 be a useful therapeutic target? (3)
A
- It is highly expressed, with approximately 100,000 CD20 molecules expressed on the surface of normal B-cells (with similarly high levels on most malignant cells), which facilitates efficient target opsonization and deletion
- Moreover, given the extracellular structure of the molecule, the available mAb binding epitopes are located close to the plasma membrane, a feature that has been reported to facilitate efficient binding and recruitment of effector mechanisms for deletion
- CD20 has no known ligand to interfere with mAb binding and does not exhibit extracellular post-translational modifications, reducing the variation in, and potential loss of, binding epitopes
7
Q
What cells are present during the fusion process? (3)
A
- Unfused myeloma cells that are deficient in an enzyme called HGPRT
- Unfused spleen cells
- Fused hybridoma cells
8
Q
How is HAT media selective? (6)
A
- Unfused spleen cells are easily selected against since they do not replicate in culture.
- Unfused myelomas can be selected against using media containing HAT.
- The aminopterin found in the medium blocks the de novo DNA nucleotide synthesis pathway.
- Typically when the de novo pathway is blocked, cells will then utilize the salvage pathway as an alternative means to replicate (only if hypoxanthine and thymidine are present).
- However, these myelomas are unable to do so since they are deficient in an enzyme called HGPRT, which is required for the salvage pathway. Hence, myelomas are unable to replicate in culture.
- Only hybridomas survive. Hybridomas inherit a functioning HGPRT enzyme from the spleen cells, so even though the de novo pathway is blocked, they can still use the salvage pathway to replicate.
9
Q
When might we use mAbs? (4)
A
- As probes to identify materials in labs or for use in home-testing kits like those for pregnancy or ovulation
- To type tissue and blood for use in transplants
- For diagnosis
- For disease treatment
10
Q
What conditions are mAbs used for? (1)
A
- Cancer, organ transplant rejection, inflammatory and autoimmune disorders (inc. allergies), infections, osteoporosis, eye conditions, migraines, high cholesterol, and nervous system disorders
11
Q
What are the mechanisms of action of mAbs? (7)
A
- Impeding tumour cell survival cascades
- Inhibiting tumour growth by interfering with tumour angiogenesis
- Eluding programmed cell death
- Evading immune checkpoints
- Direct tumour cell killing: mAb binding to an enzyme can lead to neutralisation and apoptosis
- Immune-mediated tumour cell killing: Phagocytosis, complement activation, antibody-dependent cellular cytotoxicity (ADCC), inhibition of T-cell inhibitory receptors
- Vascular and stromal cell ablation: Vasculature receptor antagonism or ligand trapping, stromal cell inhibition, delivery of toxin to stromal cells or delivery of toxin to vasculature
12
Q
Why might we humanise mAbs? (2)
A
- Humanised mAbs are a combination of a small part of mouse mAb and a human antibody
- The mouse part binds to the target antigen, the human part makes it less likely to be destroyed by the body’s immune system
