Download ESPRIT user manual 4.4.0-2

148
SPR Theory
•
Non-covalent binding of biotinylated ligand to a streptavidinimmobilized gold disk. Due to the severe biotin-streptavidin
interaction, it is possible to regenerate the surface without refractive
the non-covalent biotin-streptavidin bond. This method is suitable to
couple synthetic DNA molecules to the surface.
•
Immobilization of capturing antibodies. Capturing antibodies are used
when the activity of antibodies is reduced by the immobilization
procedure. Detection of antigens is achieved in three steps. Firstly,
immobilization of the capturing antibody (for example anti-Rabbit Anti
Mouse-Fc). Secondly, binding of the second antibody (a mouse
antibody) by the capturing antibody. Thirdly, specific binding of the
antigen by the second antibody. Regeneration of the surface will
usually break all non-covalent interactions.
These methods have been used to study biomolecular interactions
intensively. Examples of biomolecular interactions are mentioned here:
•
•
•
•
•
•
•
•
•
•
Peptide-antibody interaction18
Peptide-MHC interaction19
Protein-antibody interaction, epitope mapping20
Protein-DNA interaction21
Protein-polysaccharide interaction22
Protein-virus interaction23
Protein-cell interaction24
Protein-T cell receptor interaction25
Antibody-antibody interaction, capturing antibody26
DNA-DNA interaction27
The activated NHS-ester reacts with uncharged primary amino groups of
biomolecules. This means that the reaction rate is favoured by high pH
values of the buffer.
The reaction can only occur if the ligand is available for reaction, i.e. when it
is inside the dextran layer. This is achieved by pre-concentration of the
ligand. Positively charged biomolecules adsorb electrostatically to the
negatively charged dextran layer by pre-concentration. Consequently, the
ligand buffer should be lower than the isoelectric point (pI) of the ligand.
A compromise for pH values should be chosen for ligand solutions to fulfil the
pre-concentration condition and the reaction rate condition for ligands.