Surfaces with primary amino groups covalently bound are dedicated to promote the covalent immobilisation of compounds containing reactive moieties such as amino, carboxyl or thiol groups via well-known homoheterobifunctional linkers, e.g. N-Hydroxysuccinimide (NHS) or Succinimidyl 4-(N-maleidomethyl) cyclohexane-1-carboxylate (SMCC).
This kind of immobilisation can overcome some of the limitations connected with physical adsorption of the molecules to the surfaces such us:
- immobilisation of molecules which are bound weakly or not at all by physical adsorption, namely small peptides (M.W. 1000-5000 Da) drugs, toxins or hormones
- oriented immobilisation of molecules in order to secure the integrity and accessibility of their specific sites avoiding the risk of inhibition of these sites by casual physical adsorption for such molecules as Fab-SH-antibody fragments, streptavidin, polysaccharides or nucleic acids (single strand or double strand)
- increased storage stability compared with that of physical adsorption because of the reduced risk of spontaneous desorption
Introduction to the preparation and use of aminated surfaces for immunological assays
Several recipes are routinely used for the coupling of biological molecules to amino groups. Specific directions for use require the knowledge of the intended application. As general guideline, the interaction between the amino group on the surface and the functional group of the molecule to be bound is based on covalent binding mediated by homo and heterofunctional crosslinkers.
In particular, Ethyldiethylaminopropylcarbodiimide (EDC), with or without the addition of N-hydroxysuccinimide, is a powerful coupling agent of the carboxylic group of the molecule with the amino group of the surface.
If the biomolecule to be bound contains ε amino groups of lysine , the simplest method is coupling via Glutaraldehyde, with the formation of a stable amine linkage by reduction with Sodium Cyanoborohydride.
Other crosslinkers for this purpose are Dimethylpimelidate and Dissucinimidyl suberate.
Biomolecules containing thiolic groups, as Fab-SH or peptides with cystein at terminal end , can exploit the large number of maleimido groups containing crosslinkers as Succinimidyl 4-(N-maleimidomethyl)cycloexane-1-carboxylate (SMCC) for reacting with the amino group.
General directions for the use of surfaces with amino and carboxylic groups
The aminated surfaces offer the possibility to
• covalently immobilize small molecules which only bind weakly or not at all by physical adsorption
• orientate the immobilization of molecules in a defined way on the solid phase
Hereunder are some examples of application that can be used as guidelines to enable users to develop their own bio-specific assays.
- Coupling of NHS-activated compounds
- Coupling hapten or peptide, having a carboxylic group
1. DNA binding
2. Peptide Binding
The surface is modified with primary amino groups.
Volume of treatment: 200 µl/well.
Microplates show a CV% less than 5 when NHS-biotin is bound to the aminated surface in an ELISA format, using Streptavidin-HRP as detector and TMB as substrate.
Storage and Stability
The microplates are stored at room temperature in sealed PE bags and are stable until the expiration date printed on the label.