Epitope mapping Flashcards
Using examples explain the difference between linear and conformational epitopes
Epitopes are molecular structures on an antigen that make direct contact with the antibody, made of amino acids or protein sequences. They can be linear consisting of amino acid sequences which can be directly identified by antibodies or conformational which require specific 3d folding to be recognised by the antibody or receptor
Linear - CD20 epitopes which Rituximab and Ofatumumab monoclonal antibodies act against.
Confirmational - HIV contains confirmation epitopes as the glycoproteins gp41 and gp120 require specific calibration
Describe approaches to map epitopes using X-ray crystallography.
Initiatlly a pure crystal is obtained of the epitope using crystallization, this is then flash frozen preserving crystals.
the crystal is exposed to a beam of x-rays which interact with the electron density of the crystal lattice causing a refraction pattern detected by a specialised camera. By analysing the diffraction pattern and electron density overlayed the structure of the crystal the positions of the atoms are obtained and the epitope position can be obtained.
Describe approaches to map epitopes using cryogenic EM.
In this approach, purified protein samples are flash-frozen and imaged using an electron microscope. The resulting images are processed to generate a three-dimensional reconstruction of the protein structure. To map epitopes,
Describe the principle to map epitopes using peptide scans
peptide library is designed to cover the entire sequence of the protein of interest. Typically, short peptides with a length of 10-20 amino acids are used.
The peptides in the library are designed to overlap with neighboring peptides by a fixed number of amino acids.
The peptide library is tested for binding to the target antibodies or receptors using an appropriate binding assay, such as enzyme-linked immunosorbent assay (ELISA).
The peptides that show binding to the antibodies or receptors are identified as potential epitopes by m measuring signal intensity.
Explain the principle of fine mapping in epitope mapping
Fine mapping aims to narrow down the epitope region to a smaller segment and pinpoint the key residues responsible for binding through the substitution of singular amino acid residues.
Alanine scanning involves systematically substituting each amino acid within the epitope region with alanine (or sometimes glycine) while keeping the rest of the protein sequence intact. The mutated peptides are then tested for their ability to bind to the antibodies or receptors.
Explain how you would use Array-based oligo-peptide scanning to do epitope mapping.
Design a library of short peptides that covers the entire protein sequence or a specific region of interest. The peptides are typically around 10-20 amino acids in length.
Synthesize the peptides directly on the surface of an array, such as a glass slide or a microplate.
ncubate the peptide array with the target antibodies or receptors of interest. The antibodies or receptors can be labeled with a fluorescent or enzyme tag for detection.
detection methods such as fluorescence imaging, enzyme-linked detection, or mass spectrometry to identify the bound antibodies or receptors.
This can then be followed by fine mapping techniques for validation.
Explain how you would use Array-based oligo-peptide scanning to do epitope mapping.
Design a library of short peptides that covers the entire protein sequence or a specific region of interest. The peptides are typically around 10-20 amino acids in length.
Synthesize the peptides directly on the surface of an array, such as a glass slide or a microplate.
ncubate the peptide array with the target antibodies or receptors of interest. The antibodies or receptors can be labeled with a fluorescent or enzyme tag for detection.
detection methods such as fluorescence imaging, enzyme-linked detection, or mass spectrometry to identify the bound antibodies or receptors.
This can then be followed by fine mapping techniques for validation.
What is the difference between continuous and discontinuous epitopes?
Continous or linear epitopes consist of amino acid sequences directly next to each other however discontinuous require 3d configuration / folding
Describe The principle of chemiluminescence-based epitope mapping
The target antigen, which contains the epitope of interest, is immobilized onto a solid support, such as a membrane or a microplate. The immobilization can be achieved through various methods, such as covalent binding or passive adsorption.
A specific antibody that recognizes and binds to the antigen is incubated with the immobilized antigen. The antibody is usually conjugated with an enzyme, such as horseradish peroxidase (HRP), that can catalyze a chemiluminescent reaction.
A chemiluminescent substrate specific to the enzyme conjugated to the antibody is added to the system. The substrate typically consists of a luminol derivative that, upon enzymatic reaction, produces light.
he enzyme conjugated to the antibody catalyzes the oxidation of the chemiluminescent substrate, resulting in the generation of excited intermediates. As these intermediates decay to the ground state, they emit photons of light, producing a detectable chemiluminescent signal.
What was the objective of creating B cells hybridomas in the practical?
We want to produce immortal and high replicating B cells to be antibody factories
What was the principle of the B cell hybridomas test explaining the use of HAT medium.
We need immortal and high replication B cells to produce Mab which B cells cannot do so to achieve this we:
Immunised rats with CD20 B cells, these cells are then fused with myeloma cells to create hybridomas.
These cells are then Cultured in HAT medium- (hypoxanthine-aminopterin-thymidine ) Aminopterin present in HAT media blocks the power of cells to synthesize nucleotides by the de novo synthesis pathway.
Hypoxanthine and deoxythymidine allow cells with functional hypoxanthine-guanine phosphoribosyltransferase (HGPRT) genes to survive through salvage pathways (hybridomas) . Due to a limited life span, unfused B cells perish within a few days. Unfused malignant neoplastic cells die as a result of the lack of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene.
Finally harvest Mab
What is CD20 and what is its role in B cells.
Cultural differentiation 20 CD20 is a membrane-embedded surface molecule which plays a role in the development and differentiation of B cells into plasma cells
CD20 is seen more in mature b cells.
Explain the principle of CLIPS - chemical linkage of peptides onto scaffolding.
The addition of chemicals such as cl- or br- to forum bonds and folds within AA to aid in epitope mapping of confirmation structures
What Mab are clinically trailed for Covid ?
COV 1/2 - Strovimab
REGEN COV 2 - Casirivimab & imdevimab
How does Mab effect bacterial infections?
Neutralising toxins and antibodies
