Week 5: Antibodies Flashcards
What is the purpose of antibodies?
Mammals produce antibodies - bind to targets antigens (foreign agents - often biomolecules of virus, bacteria, parasite or cancer cell, each binds target tightly and specifically)
Describe antibody structural framework
All have common structural framework. Variation in their amino acid sequence generates antibody diversity - occurs in part that contacts target.
Describe the overall structure of an antibody
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).
What are the 5 classes of immunoglobulins?
5 Classes of Immunoglobulins - IgM, IgG, IgA, IgE and IgD. Cover IgG only.
What is a tandem repeat and what does this mean for antibodies?
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.
What is an Ig fold? (see images)
Ig fold is a beta-sandwich made of two stacked beta sheets. Residues exposed to interior are hydrophobic, exterior - polar hydrophilic.
Describe antigen-antibody interactions
Are surface driven - bind to antigen binding site. Bottom non-variable heavy chain is interaction site with other effectors of immune response
How are antibodies studied?
Are fragmented into two Fab fragments (carrying variable region) and one Fc fragment containing the bottom of heavy chains.
What is a CDR?
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
How do CDRs bind to antigen?
Non-covalent interactions.
What are epitopes?
Antigenic determinants Antigen can have multiple -multiple antibodies can bind - polyclonal antibodies.
Describe process from antigen -> macrophage
Antibodies bind to antigenic determinants and link with other antibodies which are bound to other antigens - then macrophage consumes this collection
How are antibodies used in disease treatment?
Antibodies as drug-targeting agents in disease treatment - ie. targeted chemo/radiotherapy - any drugs ending in ‘ab’ are antibody-based.
Describe a cytotoxic antibody? (see pictures)
Targets cancer cells - contains linkers that join the antibodies and the cytotoxic drug - destroys the cancer cells.
Describe how synthetic polyclonal antibodies work
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.
What are 3 applications of antibody technology?
- quantify these molecules within biological samples. 2. Detect their locations ie. fluorescent staining. 3. Detect protein-protein interactions within cells.
