Generating antibodies for immune-based techniques in research and medicine

Question 1

What properties of antibodies make them useful for research and medicine?

Answer 1

-diverse (so many diff specificities!)
-highly specific with high affinity (very small Kd)
-stable domain structure, which can be genetically engineered
-multivalent (improves binding and enables crosslinking)
-have effector properties

Question 2

What labels can be added to antibodies?

Answer 2

-fluorescent labels (eg. for FACS)
-enzyme (eg. to produce coloured product, for ELISA or immunoblotting)
-radioisotope (eg. for imaging)
-gold particles (eg. for immuno-electron microscopy)
-sepharose (eg. for affinity purification, immunoprecipitation)

Question 3

What are linear epitopes?

Answer 3

non-conformational
ajacent in sequence

Question 3

What is immunogenicity?

Answer 3

an antigen’s ability to induce an immune response

Question 4

What are discontinuous epitopes?

Answer 4

conformational (depend on protein conf!)
non-adjacent in sequence
(diff points in seq, brought together by conformational folds, etc)

Question 5

What affects immunogenicity?

Answer 5

-how foreign the antigen is (the more different the antigen is, the greater the immunogenicity)
-molecular size (smaller antigens will be removed from the body quicker)
-chemical composition (charged or aromatic residues facilitate antibody binding better)
-ability to provoke T cell responses
-use of adjuvants

Question 6

What is polyclonal antisera?

Answer 6

antisera made up of a mixture of antibodies, specific to different epitopes
-from several B cells being stimulated

Question 7

What is monoclonal antisera?

Answer 7

antisera made up of a single epitope
-from a single B cell (cloned to produce lots!)

-can be fully mouse, chimeric, humanised or fully human

Question 8

Advantages of polyclonal antisera

Answer 8

-cheap production
-robust (some antibodies in mixture will bind to proteins even if partially unfolded/denatured -linear epitopes)

Question 9

Disadvantages of polyclonal antisera

Answer 9

-recognise multiple epitopes so not specific enough
-pure antigen needed for immunisation
-hard to standardised (varied composition!)

Question 10

Advantages of monoclonal antisera

Answer 10

-highly specific (only recognise 1 epitope)
-easy to standardise
-no requirement for pure antigen for immunisation

Question 11

Disadvantages of monoclonal antisera

Answer 11

-epitopes recognised are often conformational-sensitive
-expensive

Question 12

How can antibodies be engineered?

Answer 12

Antibody chimeras
-mouse V region spliced to human C regions

Humanised antibodies
-human framework region (less variable bits of V region) genes splices to mouse CDR genes

Question 13

How can fully human antibodies be generated?

Answer 13

-using transgenic mice to express human immunoglobulin genes
-making antibody gene libraries by using a vector to express B cell antibody V region genes in bacteria or bacteriophages
-isolate specific B cell from patient and amplify/clone expressed antibody V genes

Question 14

How can antibodies be improved for use in immunotherapy?

Answer 14

-effector sites (eg. hinge, specific residues) mapped onto antibodies to generate “designer antibodies”
-glycosylation (eg. removing fucose improves IgG interaction with NK cell Fc receptor)
-label antibody (with drug, prodrug, toxin or radionuclide)
-use antibody fragment (eg. Fab region)
-bi-specific antibodies (dual specificities to link functions, eg. link tumour binding to effector cell functions)