Lecture 8 - Monoclonal Antibodies

Question 1

Can more than one antibody type recognise a single epitope?

Answer 1

Yes

Different parts of the epitope can be bound by different antibodies

Question 2

Generating B cell hybridomas
1)
2)
3)
4)
5)

Answer 2

1) Inoculate mouse with antigen of interest
2) Remove mouse spleen, lymph nodes, isolate B cells from there
3) Mix B cells with myeloma cells in a polyethylene glycol (PEG) fusion
4) Isolate hybridomas
5) Isolate hybridomas expressing antibody of interest

Question 3

Hybridoma isolation
1)
2)
3)
4)

Answer 3

1) B cells have a limited lifespan, myeloma cells don’t
2) B cells express HGPRT, which confers resistance to HAT. Myeloma cells don’t, and are therefore sensitive to HAT
3) HAT kills myelomas, B cells eventually die because of limited lifespan
4) Cells left over will be hybridomas

Question 4

Function of polyethylene glycol

Answer 4

Makes cell membranes able to fuse together

Used to fuse B cells and myeloma cells

Question 5

Selecting hybridoma clones
1)
2)
3)

Answer 5

1) Make a microarray with antigen of interest in wells
2) Limiting dilution of hybridoma suspension
3) Hybridomas of interest will be retained at high dilutions

Question 6

Do antigen mixtures have to be very pure for making hybridomas?

Answer 6

No.

Selection for antibody of interest occurs at end of process

Question 7

To make polyclonal antibodies against an antigen, is a very pure sample needed?

Answer 7

Yes

Question 8

Cross reactivity of serum

Answer 8

Reasonably good

Polyclonal response to antigen, so several different epitopes will have antibodies made against them

Question 9

Cross reactivity of monoclonal antibodies

Answer 9

Poor
Very specific, single target
Many antibodies against human leukocyte antigens are active against primate leukocyte antigens too

Question 10

What does ‘CD’ in ‘CD antigens’ stand for?

Answer 10

Cluster of differentiation

Based on which antibodies will bind to target receptor

Question 11

Uses for monoclonal antibodies
1)
2)
3)
4)

Answer 11

1) Serotyping
2) Isolating biological molecules
3) Identifying cells
4) Therapeutics

Question 12

How can MAbs be used to isolate biological molecules?

Answer 12

Immobilise MAbs on surface, run mixture over them.

Molecule of interest will bind to MAbs

Question 13

Anti-TNFa/TNFaR antibody

Answer 13

Remicade and humira

Question 14

Anti IgE/IgeR antibody

Answer 14

Xolair

Question 15

Possible issue with humanising mouse antibodies

Answer 15

Can alter specificity