Introduction to the preparation and use of aminated surfaces for immunological assays

Direction for use

Several recipes are routinely used for the coupling of biological molecules to amino groups. Specific directions for userequire the knowledge of the intended application. As general guideline, the interaction between the amino group on thesurface and the functional group of the molecule to be bound is based on covalent binding mediated by homo andheterofunctional crosslinkers.

In particular, Ethyldiethylaminopropylcarbodiimide (EDC), with or without the addition of N-hydroxysuccinimide, is apowerful 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 numberof maleimido groups containing crosslinkers as Succinimidyl 4-(N-maleimidomethyl)cycloexane-1-carboxylate (SMCC)for reacting with the amino group.

schematic chemical and physical configuration of biomat NH2 surface

example of reaction scheme:
an arbitrary NHS estherified compound ( R ) covalently combines with biomat NH2 surface through NHS splitting off

Hereunder are some examples of coupling agents to be used for covalent coating of the biomat NH2 surface with reactivegroups

A. Disuccinimidyl suberate (DSS).
This symmetric (homobifunctional) linker is capable of linking compounds containing secondary or primary aminogroups, and can thus be used for covalent immobilisation of peptides, proteins, glycoproteins, lipoproteins

Reaction A

B. Sulfosuccinimidyl maleimidomethyl cyclohexane carboxylate (SMCC).
This heterobifunctional linker is capable of linking compounds with SH-containing compounds.It can be usedespecially for covalent immobilisation of Fab-SH-antibody fragments or terminally cysteinized antigenic peptides,thereby exposing the active ends of these compounds to the liquid phase.

Reaction B

C. N-Hydroxysulfosuccinimide (Sulfo-NHS) or N-Hydroxysuccinimide (NHS) combined with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide Hydrochloride(EDC).
The EDC linker combined with sulfo-NHS is capable of linking small peptides (M.W. around 1000) via their carboxylgroup to the NH-activated strips surfaces.

Reaction C

  1. peptide
  2. Sulfo NHS (or NHS)
  3. EDC
  4. intermediate active compound resulting from the reaction
  5. biomat NH2 surface
  6. peptide covalently immobilised on biomat NH2 surface
Codes and suppliers of linkers
Product Supplier Code
Sulfo-SMCC PIERCE 22322
Sulfo-NHS PIERCE 24510