Lecture 13- Monoclonal antibodies

Question 1

Why are antibodies such specific reagents? and easily tagged?

Answer 1

They have hypervariable regions.

They are large proteins

Question 2

How do you “make” antibodies?

Answer 2

Inject small samples of antigens into an animal
combine antigen with an “adjuvant”
Give “boosts”, more rounds of injections to get loads of antibodies in the serum.
Then serum is enriched for specific antibodies against injected antigen

Question 3

Why do we say that antibodies in the serum are polyclonal?

Answer 3

antigens usually contain many different epitopes. so lots of different antibody affinities.
However you can purify them and use them in lots of different ways

Question 4

What are anti-toxins? how do they get made?

Answer 4

Injecting animal with sub lethal doses of toxins- make anti-toxins (antibodies)
First successful anti-toxin was to cobra venom, still have anti-venoms now

Question 5

What did Kohler and Milstein do? Breakthrough with monoclonal antibodies?

Answer 5

Kohler and Milstein. Technique of immortalising clones of cells that could make antibodies.
Fuse a normal B cell with a myeloma cell (tumour cell)
Hybridoma has the antibody producing genes from the B cell and the immortal properties from the tumour cell

Question 6

How do you get monoclonal antibodies in practise?

Answer 6

1. Mice are immunised with an antigen- a couple of times to enhance class switching and increase affinity
2. Spleens removed which contain the antigen-specific B cells
   splenocytes fused in vitro with a dividing myeolma cell line (which cant secret Ig)
3. Using polyethylene glycol Hybrid cells then put into plate with HAT medium.
4. Myeloma cells cant survive in HAT medium, spleen cells eventually die too. so only fused cells will survive.
5. Some wells will make antibodies against the original antigen. So screen for those and will have some immortal cell lines secreting monoclonal antibodies.

Question 7

Advantages of monoclonal antibodies?

Answer 7

• high sepcificity and low cross-reactivity
• standardised worldwide
• unlimited supply
• used in immunoassays, diagnosis, tissue typing, separation of cell types, clinical therapeutic agents

Question 8

What kind of immunological methods can we use with monoclonal antibodies?

Answer 8

• affinity chromatography to purify molecules
• ELISA
• western blotting
• flow cytometry

Question 9

Describe affinity chromatography

Answer 9

1. Antibody to antigen A is bound to beads
2. Add a mixture of molecules
3. Wash away unbound molecules
4. Elute specifically bound molecules

Question 10

Antibodies are colourless so how do we detect antibody/antigen interactions?

Answer 10

Antibodies can be tagged to reveal where they bind- with fluorescent markers e.g. FITC
or enzyme markers that change colour when a substrate is added e.g. peroxidase

Question 11

Describe direct ELISA process

Answer 11

1. Add anti-A antibody covalently linked to enzyme
2. Wash away unbound antibody
3. Enzyme makes coloured product from added colourless substrate
4. Measure absorbance of light by coloured product

Question 12

How do you increase sensitivity?

Answer 12

add an anti-antibody-> a secondary antibody. Secondary antibody can interact with many epitopes on the primary antibody, thus amplifying the signal

Question 13

Describe indirect ELISA

Answer 13

Same prinicpal as direct but secondary antibody.

1. Well is coated with antigen
2. Specific antibody to be measured is added
3. enzyme-conjugated secondary antibody is added
4. substrate added and amount of secondary antibody measured by ELISA reader

Question 14

Agglutination

Answer 14

Identify if antibody is present for an antigen.
Visualise clumping
Commonly used in blood typing and to diagnose if a patient has had an infection, will see clumping if they have

Question 15

Immunohistochemistry and immunofluorescence

Answer 15

-Shows localisation of antigen and can be quantitative
-Detection and localization of the presence or absence of specific antigens
- Can use it on many different tissues and specimens (even dna)
Enables you to see what’s there and where it is

Question 16

Western Blotting

Answer 16

• Qualitative and quantitative analysis of protein expression
• Can be used diagnostically
  1. Get protein of interest, 2. dissociate in solution, 3. SDS page,
  4. Transfer to nitrocellulose and overlay with antiserum
  5. Detect bound antibody with enzyme-linked anti-IgG

Question 17

Flow cytometry

Answer 17

On blood smear the white cells look similar but they have different cell-surface proteins.
-uses lasers to detect fluorescent antibodies bound to cells
get different scattering depending on how many granules and size of cell
-Get scatter plots

Question 18

Flow cytometry uses?

Answer 18

gives quantitative and qualitative information
levels of cell activation
cell viability
cytokines secreted
rare cell types
clinical use common

Question 19

Magnetic cell separation?

Answer 19

Often used in lab to purify cell populations. May be used clinically.

1. heterogenerous population of lymphocytes mixed with antibodies couples to paramagnetic particles or beads and poured over an iron wool mesh
2. when a magnetic field is applied the coupled cells stick tot he iron wool; unlabelled cells are washed out
3. the magnetic field is removed, releasing the coupled cells

Question 20

When are monoclonal antibodies used in the home?

Answer 20

Pregnancy tests