As a method for immobilizing a compound on a solvent-insoluble support for use as a ligand for specific adsorption, many investigations have been undertaken in the diverse field of immobilized enzymes, affinity chromatography, ion exchange chromatography, etc. and some methods are used on the industrial application.
As a representative method for immobilization known in the field of immobilized enzymes, there can be mentioned: (1) the method which comprises forming the imidocarbonate on the support by the cyanogen bromide activating method and, then, causing it to react with the amino group of the compound to serve as a ligand; (2) the so-called acid azide derivative method which comprises, sequentially, esterification of the carboxyl group on a support, hydrazide formation, conversion to the azide and substitution thereof with the amino group of the compound to serve as a ligand; (3) the so-called diazo method which comprises forming a diazonium salt on a support and letting it react with the amino group of the compound to serve as a ligand; (4) the so-called condensation reagent method which comprises condensing the amino or carboxyl group on a support with the carboxyl or amino group of the compound to serve as a ligand in the presence of a condensing agent; (5) the so-called alkylation method which comprises modifying a support with bromoacetyl or 4,6-dichloro-s-triazinyl and causing the resulting derivative to react with the amino group of the compound to serve as a ligand; and (6) the so-called matrix-crosslinking method which comprises crosslinking the amino group on a support and the amino group of the compound to serve as a ligand with glutaraldehyde followed by reduction (Atsuo Tanaka & Takuo Kawamoto: Modern Chemistry, 24 to 30, July 1992).
However, those methods have the drawbacks, namely
(a) when the compound to serve as a ligand contains an SH group, the efficiency of immobilization is relatively low; and PA1 (b) when the compound to serve as a ligand is an SH group-containing peptide or protein, in particular, the inherent activity of the compound (enzyme activity, binding activity, etc.) is sometimes sacrificed.
The inventors of the present invention discovered that when a peptide or protein to serve as a ligand contains an SH group, the above-mentioned phenomena (a) and (b) occur during its immobilization on a solvent-insoluble support. Similar phenomena are also observed with compounds other than peptides and proteins provided that they contain SH groups. There is no information in the available literature to the effect that the above phenomena occur when a compound to serve as a ligand is immobilized on an inert support.
USPTO Backgrounds
USPTO Backgrounds