Antibody Basics

Question 1

Why are carbohydrates essential for Ab function?

Answer 1

Studies have shown that N-linked oligosaccharides on the Fc region are key for assembly and function of IgD (respiratory defence) and IgA1 (mucous membrane). Discovered using site directed mutagenesis.

Question 2

What is somatic recombination?

Answer 2

Assembly of the Ab domains. Occurs in the bone marrow. Process of converting the progenitor lymphoid cells to immature B cells. Involved VDJ recombination.
First Heavy chain recombination (produces IgM)
Then Light chain recombination (produces immature B cells)
Involves the use of Recombination Signal Sequence (RSS) and Rag 1/2

Question 3

What is clonal selection?

Answer 3

Process of selecting B cells that only bind to non-self antigen with high affinity. Involved somatic hypermutation, clonal deletion, and class switching and maturation. Occurs in secondary lymphoid tissues. Final product is a B cell with high affinity for non-self antigen.

Question 4

How do we generate polyclonal Abs?

Answer 4

From Antisera (purify using chromatography from soluble proteins and growth factors). Non specific - contains multiple antibodies for the same epitope.Run out.

Question 5

How do we generate monoclonal Abs?

Answer 5

Fusion of B cells with Ig negative Myeloma cells using PEG. Select for hybidomas using selective media. Select for specific Abs using Elisa.

Question 6

What were the key findings that suggest why we can use Abs as tools?

Answer 6

1. Variable region can function without constant region (Papain, Pepsin)
2. Hypervariable regions (HV1, HV2, HV3) govern antigen binding on the variable region