Week 5: Antibodies

Question 1

What is the purpose of antibodies?

Answer 1

Mammals produce antibodies - bind to targets antigens (foreign agents - often biomolecules of virus, bacteria, parasite or cancer cell, each binds target tightly and specifically)

Question 2

Describe antibody structural framework

Answer 2

All have common structural framework. Variation in their amino acid sequence generates antibody diversity - occurs in part that contacts target.

Question 3

Describe the overall structure of an antibody

Answer 3

Two heavy chains (blue) - MW 53,000 Da each, H

Two light chains (white) - MW 23,000 Da each, L

Chains held together by disulfide bonds. Heavy chains are modified by addition of carbohydrates (CHO)

Each chain has constant domains (identical in amino acid sequence in all class members). Each chain also has variable domain (lets diff antibodies bind diff targets).

Question 4

What are the 5 classes of immunoglobulins?

Answer 4

5 Classes of Immunoglobulins - IgM, IgG, IgA, IgE and IgD. Cover IgG only.

Question 5

What is a tandem repeat and what does this mean for antibodies?

Answer 5

Tandem repeat - contiguous arrangement of multiple copies of repeating structural unit in linear sequence. The antibody structure is based on tandem repeats of the immunoglobulin (Ig) fold - 4 in each heavy chain, 2 in each light chain.

Question 6

What is an Ig fold? (see images)

Answer 6

Ig fold is a beta-sandwich made of two stacked beta sheets. Residues exposed to interior are hydrophobic, exterior - polar hydrophilic.

Question 7

Describe antigen-antibody interactions

Answer 7

Are surface driven - bind to antigen binding site. Bottom non-variable heavy chain is interaction site with other effectors of immune response

Question 8

How are antibodies studied?

Answer 8

Are fragmented into two Fab fragments (carrying variable region) and one Fc fragment containing the bottom of heavy chains.

Question 9

What is a CDR?

Answer 9

Complementary Determining Regions (CDRs) determine specificity - are complementary in charge and shape to their antigen. Match shape to antigen - complementary. Fab -> has constant region, variable region with antigen binding site

Question 10

How do CDRs bind to antigen?

Answer 10

Non-covalent interactions.

Question 11

What are epitopes?

Answer 11

Antigenic determinants Antigen can have multiple -multiple antibodies can bind - polyclonal antibodies.

Question 12

Describe process from antigen -> macrophage

Answer 12

Antibodies bind to antigenic determinants and link with other antibodies which are bound to other antigens - then macrophage consumes this collection

Question 13

How are antibodies used in disease treatment?

Answer 13

Antibodies as drug-targeting agents in disease treatment - ie. targeted chemo/radiotherapy - any drugs ending in ‘ab’ are antibody-based.

Question 14

Describe a cytotoxic antibody? (see pictures)

Answer 14

Targets cancer cells - contains linkers that join the antibodies and the cytotoxic drug - destroys the cancer cells.

Question 15

Describe how synthetic polyclonal antibodies work

Answer 15

Companies prepare polyclonal antibodies - inject host with antigen, host begins to raise antibodies against it, repeat antigen injection several weeks later to boost antibody production. After 2-3 months, draw blood from host, centrifuge to obtain serum, isolate antibodies from serum by affinity chromatography using antigen linked to column matrix. Keep host alive so further antibody can be obtained later.

Question 16

What are 3 applications of antibody technology?

Answer 16

1. quantify these molecules within biological samples. 2. Detect their locations ie. fluorescent staining. 3. Detect protein-protein interactions within cells.