Epitope Mapping Flashcards
1
Q
What is a linear epitope? (2)
A
- A straight line of amino acid residues in a particular region of a protein, which are nearby in the sequence as well as structure
- Epitope where a stretch of continuous amino acids is sufficient for binding, referred to as a continuous epitope
2
Q
When might a linear epitope be preferred to use? (2)
A
- Applications in which the protein target is wholly or partially denatured during the sample preparation prior to the immunoassay
- such as in Western blot (WB), immunohistochemistry (IHC) or immunofluorescence-based confocal microscopy.
3
Q
What is a confirmational epitope? (1)
A
- Epitope where key amino acid residues are brought together by protein folding
4
Q
When might a confirmational epitope be preferred to use? (2)
A
- for applications involving protein targets in their native state
- such as therapeutic applications or flow cytometry
5
Q
What is a discontinuous epitope? (1)
A
- consists of non-adjacent parts of the protein sequence that form a specific 3D conformation in a protein tertiary structure
6
Q
What are the methods used to map epitopes? (6)
A
- X-ray Crystallography
- Cryo-EM
- Array-based oligopeptide scanning
- Site-directed mutagenesis mapping
- High-throughput shotgun mutagenesis epitope mapping
- Cross-linking coupled mass spectrometry
7
Q
Why might you develop mAb approaches to diseases including Ebola/Sars COV2 viral infection? (5)
A
- SARS-CoV-2 binds to the ACE2 receptor
- T-helper cell binds to viral peptide on antigen presenting cell (which has ingested the virus)
- Helper T-cells stimulate B-cells to make antibodies and help killer cells develop. Killer T-cells or cytotoxic T-cells directly kill cells that have already been infected by a foreign invader.
- B-cells make anti-SARS-CoV-2 antibodies, cytotoxic T cell kills any SARS-CoV-2 infected cells
- Thus infected cells are destroyed, antibodies are produced, and memory B cells and T cells created
8
Q
Why is epitope mapping important? (3)
A
- Epitope mapping is instrumental in therapeutic antibody development
- Plays a key role in functional understanding and selection of antibodies, IP protection and medical body approval
- Shows strongest reactions, slightly weaker responses found within a few aa of the core area
9
Q
Steps in epitope mapping (5)
A
- Select protein target
- Generate overlapping peptides
- Test sample in ELISA-like assay
- Record a binding profile
- Deduce the epitope
10
Q
Cryo-EM (3)
A
- Involves flash-freezing solutions of proteins or other biomolecules and then bombarding them with electrons to produce microscope images of individual molecules.
- These are used to reconstruct the 3D shape, or structure, of the molecule
- Doesn’t require protein crystals, but the technique languished because it tended to produce low-resolution structures — some scientists called it blobology
11
Q
How does oligopeptide scanning work? (5)
A
- Amino acid sections of antigen are cloned and produced
- Get recombinant proteins of different small sections of entire antigen, peptides overlap by one or more nucleotides
- Put on solid surface to allow antibodies to bind, antibodies will only bind to where the epitope is
- Signal readout (luminescence, fluorescence)
- From then you can map where on the antigen the epitope is
12
Q
What are “CLIPS” and why are they sometimes used in epitope mapping? (3)
A
- CLIPS – Chemical Linkage of Peptides onto Scaffolds
- Technology to map epitopes contained within looped, beta sheet or helical structures with constrained peptides. This means we can characterize such epitopes with high accuracy
- CLIPS discontinuous epitope mapping also enables detection of discontinuous epitopes involving dimeric or multimeric protein complexes, simply by introducing screening of one sequence in one loop against the sequence of a second protein in the second loop
13
Q
What is the therapeutic purpose of a neutralising mAb? (1)
A
- bind to specific sites on the spike protein of the SARS-CoV-2 virus particle, blocking its entry into cells and therefore inhibiting its replication
