Hydrocoating: a new method for coupling biomolecules to solid phases.
Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique--hydrocoating--for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays.
Gregorius
K
K
M & E, Lersø Parkallé, Copenhagen, Denmark.
Mouritsen
S
S
Elsner
H I
HI
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D003432:Cross-Linking Reagents; D003911:Dextrans / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D008024:Ligands; D051379:Mice; D008832:Microchemistry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D015717:Peptide T / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*
D003432:Cross-Linking Reagents; D003911:Dextrans; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D015717:Peptide T; C066759:hen egg lysozyme peptide (46-61); D001710:Biotin; D009113:Muramidase