Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-06
2001-07-17
Low, Christopher S. F. (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S051000, C525S054200
Reexamination Certificate
active
06262256
ABSTRACT:
The present invention relates to a method of modifying the binding properties of the surface of a solid phase on which nucleophilic groups are present, a method of immobilizing a chemical compound to the surface of a solid phase, and the solid phase modified by the method in the form of an article, and the use thereof in solid phase reactions.
THE TECHNICAL FIELD
Immobilization of various chemical compounds to surfaces of solid phases, such as glass and plastic materials or other solid materials, is used in a variety of chemical and biochemical techniques in which the chemical compound is bound to the surface of the solid phase, in order to be able at this locality to form part of solid phase reactions with other chemical compounds. Such solid phase reactions occur e.g. within chromatography, solid phase assays, including biosensors and solid phase peptide and oligonucleotide synthesis, and they are also used in bioreactors. Further, solid phase reactions occur e.g. in chemical surface treatment of biological surfaces, such as teeth, as a step in glueing items thereon.
Methods of immobilizing chemical compounds to solid phases are multifarious and depend on the object, but within the fields of application mentioned below the present invention constitutes a clear improvement over the existing techniques, since e.g. the durability of immobilized proteins is improved, the functionality is retained, and it is possible to recognize/immobilize peptides.
DESCRIPTION OF THE KNOWN ARE
Use of solid phase assay technique, including Enzyme Linked Immunosorbent Assays (ELISA) (Engvall E. and Perlmann P. “Enzyme-linked immunosorbent assay, ELISA. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigene-coated tubes”, J. Immunol, 109: 129-35, 1972) and solid phase radioimmunoassays (RIA), has during recent years been growing strongly for concentration determination of both antigenes and anti-bodies. Furthermore solid phase assays and the appertaining radioactive or non-radioactive detection systems are used in gene probe based hybridization assays and in biosensors.
By conventional ELISA or solid phase-RIA-technique antigenes/antibodies are most often immobilized to the solid phase by passive adsorption. Certain proteins, peptides, polysaccharides, haptenes and oligo-/poly-nucleotides can, however, not be immobilized/detected in this way, and other compounds are denatured and destroyed in the adsorption process (Kurki P. and Virtanen I. “The detection of human antibodies against cytoskeletal components”. J. Immunol. Methods, 67: 209-23, 1984).
Further, the orientation of the antigene after immobilization to the solid phase may in certain cases be such that some parts are sterically hindered against interaction with e.g. the antibody. Finally, there may in several cases be a number of serious problems of unspecific (and undesired) binding of the succeeding (maybe enzyme- or isotope-labeled) compounds in the ELISA-or RIA-methods.
Some of the problems can be overcome by methods where the micro-titre-plate is chemically modified with functional groups, whereafter non-adsorbing molecules can be bound covalently by conventional cross-linking technology (cf. e.g. Neurath, AR and Strick, N “Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates. J. Virol. Methods. 3: 155-65, 1981, and e.g. GlueTech Aps “A method for covalent fixation of molecules on a solid phase”, WO89/05329).
Often, however, this does not solve the problems of denaturing and consequently deterioration of the quality of the immobilized oligo-/polypeptides or oligo-/poly-nucleotides, since the hydrophobic solid surface is still present. Furthermore, the technique requires quite some chemical expertise on the part of the performer, since use must be made of biofunctional cross-linking reagents. This is also so if the problem is sought solved by non-adsorbing molecules, such as e.g. peptides, being conjugated to larger proteins before the latter subsequently are adsorbed to the solid phase. In this case it is further necessary to use relatively large peptide quantities as compared with the present method.
Covalent immobilization of proteins, such as e.g. anti-bodies, is well-known in connection with affinity chromatography, where the molecules at issue are coupled to a solid phase consisting of a cross-linked, insoluble, activated, hydrophilic polymer, such as e.g. agarose or dextran (Pharmacia “Affinity chromatography. Principles & Methods”, 1986). Such surfaces are capable of efficiently and covalently binding large quantities of molecules without the biological function or structure thereof being affected. Additionally, the numerous hydrophilic groups (e.g. ether and alcohol groups) in the said polymers cause the surface to be hydrophilic, whereby succeeding unintended adsorption of e.g. protein is avoided. In other words, the surface has “non-binder” properties, except as regards the covalent coupling of the protein, which takes place via its functional groups (e.g. amino groups). Cross-linked dextran and agarose thus have a number of expedient binding properties which as such would be well-suited in solid phase techniques, but the disadvantages of these materials are firstly that they cannot be shaped as e.g. micro-titre-plates, and secondly that they are totally opaque, which makes it impossible to use the material in assays in which optical detection methods are employed.
Published international patent application No. WO91/09877 discloses a method of covalent binding of a protein to a hydrophilic surface consisting of a non-ionic polymer exhibiting protein-reactive groups, by which a low spontaneous adsorption of undesired products is simultaneously obtained. According to the invention the non-ionic polymer which is bound to a carrier in a manner known per se must in order that the protein subsequently can be coupled effectively to the surface, also exhibit a “cloud-point” which lies at least 5° above the temperature at which the hydrophilic surface is to immobilize the protein. Hereby it is purportedly achieved that the non-ionic immobilized polymer at an elevated temperature becomes more willing to establish the binding to the protein. Binding to anionic surfaces is specifically described.
Published international patent application WO90/06954 describes monoclonal antibodies which recognize poly-sulphated polysaccharides. Coating of activated polyvinyl chloride-immunoassay plates with poly-L-lysine to which dextran sulphate is bound by an ionic binding is specifically described.
Both synthetic oligonucleotides and peptides are today typically prepared by solid phase chemical synthesis on so-called resins (cf. e.g. E. Atherton and R. C. Sheppard “Solid-phase peptide synthesis: A practical approach”, IRL Press). Frequently used resins for peptide synthesis consist of diatomaceous earth grafted with polyacrylamide, but they may also consist of cross-linked polystyrene (Merrifield's resin) (see e.g. E. Atherton and R. C. Sheppard “The solid phase in solid phase peptide synthesis”, in “Perspectives in Peptide Chemistry”, Eds. E. Eberle, R. Geiger, and T. Wieland, 1981, p. 101). It has, however, turned out to be an advantage synthesis-wise that the resin has hydrophilic properties like e.g. the PEG-resin which consists of a copolymer of polystyrene and polyethylene glycol (PEG) (see e.g. Jiang, Ying; Liang, Xun; Chen, Weizhu; He, Binglin “Synthesis of polystyrene-supported polyethylene glycol and study of its property in peptide synthesis”, Huaxue Xuebao, 45(11), 1112-18).
Technically, peptide synthesis can be performed on amino group modified, insoluble, cross-linked agarose or dextran, since such surfaces will also be hydrophilic. For mechanical reasons these materials are, however, not suited for this purpose, since they are compressible and fragile. Besides, the flow and swelling properties in organic solvents, such as DMF, are poor. Consequently, it would be expedient to have a technique which combines the advantages of the existi
Elsner Henrik
Mouritsen Søren
Jacobson & Holman PLLC
Low Christopher S. F.
Lukton D.
Mouritsen & Elsner A/S
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