Monoclonal Antibodies

Question 1

Examples of uses?

Answer 1

Blood/pregnancy tests
Checkpoint therapy (?)
TNF therapy (?)
Plasma therapy for covid19

Question 2

Why are antibodies good for detecting things?

Answer 2

Specific hypervariable regions

Large - easily tagged

Question 3

Making antibodies?

Answer 3

Mimic infections e.g. small amount of antigen injected
Combine with adjuvant for immune system boosting
Repeat this over about 10 weeks to increase levels
This produces polyclonal antibodies (due to differing epitopes on antigen) so are purified for use

Question 4

What is convalescent plasma?

Answer 4

Blood plasma from recovered patients (e.g. ebola, covid19) used in convalescents or purified for hyperimmune globulin for post-exposure prophylacis

Question 5

Deriving monoclonal antibodies?

Answer 5

Fusing normal B cells with a myeloma (tumour) cell = hybridoma
This immortalises the antibody-producing cells
Immunised repeatedly for Ab class switching and increased affinity
Spleen cells extracted

Splenocytes fused with dividing myeloma cell line using PEG. Cell line must have purine enzyme deficiency so cannot secrete Ig
Plate cells on HAT medium - myeloma cells will die, only hybridomas will survive

Question 6

Advantages of monoclonal antibodies?

Answer 6

Low cross-reactivity
Unlimited supply
Worldwide standardisation
Highly specific
Many uses

Question 7

Disadvantages of monoclonal antibodies?

Answer 7

May trigger immune response
May not access site of need
Could cause off target effects

Question 8

Overcoming disadvantages of monoclonals?

Answer 8

Bioengineering - insect systems allow bypassing of immune system to avoid autoimmune attacks because surface structures don’t contain sugars that flag cells as foreign
Only Fab fragment produced
Improved delivery to target regions

Question 9

Applications (methodological) of monoclonal antibodies?

Answer 9

Affinity chromatography (purification)
Magnetic separation of cell populations (immuno-isolation, purification)
Agglutination (blood typing)
Detect antibody:antigen interactions (direct and indirect ELISA, ELISpot)
Immunohistochemistry and immunofluorescene (antigen localisation, gene expression)
Western blotting (protein expression)
Flow cytometry (differentiate between morphologically identical cells)

Question 10

What is checkpoint inhibitor therapy?

Answer 10

Used in cancer treatment - activation of T cells by inhibiting checkpoint systems e.g. with CTLA-4 and PD-1
Tumour cells activate checkpoints so suppress immune response - reversing this will kill the tumour cells

Question 11

Practical uses for monoclonals?

Answer 11

Pregnancy tests, lateral flows
Diagnostics
Treatments e.g. TNF, checkpoint therapy, anti-serum

Question 12

Plots obtained from flow cytometry?

Answer 12

Scatter plots - more granular cells = more sideways scattering
Larger cells = blocks light so less forward scatter
Can show if cells possess a certain molecules (e.g. if they are MHCII positive)