Preparation and use of a reactive solid support surface

Abstract

A method of preparing a protein-resistant reactive solid support surface is disclosed. The method comprises the steps of providing a solid support having a hydrogel coating with a plurality of binding elements, coupling a protein resistant compound to the hydrogel via a first fraction of the binding elements, and coupling at least one binding agent to the hydrogel via a second fraction of the binding elements, whereby the protein resistant compound and the at least one binding agent are co-immobilized to the hydrogel. Also the use of the reactive surface in analysis, such as immunogenicity assays, is disclosed.

1 . A method of preparing a reactive solid support surface, which method comprises the steps of: 
 a) providing a solid support having a hydrogel coating with a plurality of binding elements;    b) coupling a protein resistant compound to the hydrogel via a first fraction of the binding elements; and    c) coupling at least one binding agent to the hydrogel via a second fraction of the binding elements;    whereby the protein resistant compound and the at least one binding agent are co-immobilized to the hydrogel.    
     
     
         2 . The method according to  claim 1 , wherein step c) is performed after step b).  
     
     
         3 . The method according to  claim 2 , wherein the second fraction of the binding elements are comprised by those remaining on the hydrogel after coupling of the protein resistant compound via the first fraction of the binding elements.  
     
     
         4 . The method according to  claim 1 , wherein the binding elements of the hydrogel comprise functional groups, and the protein resistant compound and the at least one binding agent are covalently coupled to the hydrogel via the functional groups.  
     
     
         5 . The method according to  claim 4 , wherein the protein resistant compound and the at least one binding agent are coupled to the same kind of functional group of the hydrogel.  
     
     
         6 . The method according to  claim 4 , wherein the functional groups are selected from hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, vinyl and thiol.  
     
     
         7 . The method according to  claim 4 , wherein in at least one of steps b) and c) of  claim 1  functional groups are activated by an activating agent prior to the coupling.  
     
     
         8 . The method according to  claim 7 , wherein the activated functional groups are selected from reactive ester, hydrazide, thiol, maleimide and reactive disulphide-containing derivative.  
     
     
         9 . The method according to  claim 4 , wherein the protein resistant compound and the binding agent each contain a functional group independently selected from amine, thiol and a reactive disulphide-containing derivative.  
     
     
         10 . The method according to  claim 1 , wherein the binding elements of the hydrogel comprise one member of a specific binding pair and the protein resistant compound contains the other member of the specific binding pair.  
     
     
         11 . The method according to  claim 1 , wherein the protein resistant compound is a hydrophilic polymer.  
     
     
         12 . The method according to  claim 1 , wherein the protein resistant compound comprises poly(ethylene glycol) or a derivative thereof.  
     
     
         13 . The method according to  claim 1 , wherein the hydrogel is a polysaccharide.  
     
     
         14 . The method according to  claim 13 , wherein the polysaccharide is selected from agarose, dextran, carrageenan, alginic acid, starch, cellulose, and derivatives thereof.  
     
     
         15 . The method according to  claim 13 , wherein the polysaccharide is dextran or a derivative thereof.  
     
     
         16 . The method according to  claim 13 , wherein the polysaccharide is cellulose or a derivative thereof.  
     
     
         17 . The method according to  claim 1 , wherein the binding agent is a ligand capable of specifically binding to an analyte.  
     
     
         18 . The method according to  claim 1 , wherein the binding agent is a capture agent capable of binding to an analyte-specific ligand.  
     
     
         19 . The method according to  claim 1 , wherein the amount of protein resistant compound that is coupled in step b) of  claim 1  is not less than about 2 ng/mm 2 .  
     
     
         20 . The method according to  claim 1 , wherein the amount of binding agent that is coupled in step c) of  claim 1  is not less than about 9 ng/mm 2 .  
     
     
         21 . The method according to  claim 1 , wherein the solid support surface to which the hydrogel is attached comprises a metal layer.  
     
     
         22 . The method according to  claim 21 , wherein the hydrogel is attached to the metal layer via an ordered monolayer of alkyl chains.  
     
     
         23 . The method according to  claim 1 , wherein the hydrogel coating on the solid support comprises an array of defined discrete areas.  
     
     
         24 . The method according to  claim 1 , wherein the solid support surface comprises a sensor surface.  
     
     
         25 . Use of a reactive solid support surface prepared according to the method of  claim 1  for analysis of at least one analyte in a sample.  
     
     
         26 . The use according to  claim 25 , wherein at least one of (i) analyte concentration and (ii) a kinetic parameter for at least one interaction of analyte with ligand is determined.  
     
     
         27 . The use according to  claim 25 , wherein the sample is selected from blood serum, blood plasma and whole blood.  
     
     
         28 . The use according to  claim 25 , wherein the analyte is a protein.  
     
     
         29 . The use according to  claim 25 , wherein the analysis comprises detection based on mass sensing.  
     
     
         30 . The use according to  claim 25 , wherein the analysis comprises an immunogenicity study.  
     
     
         31 . A method of determining if an immune response against an antigen has occurred in a mammal, which method comprises determining the presence of antibodies against the antigen in a blood sample by a surface sensitive detection technique using a sensor surface with a hydrogel coating which has co-immobilized thereon an antibody specific binding agent and a protein resistant compound.  
     
     
         32 . A protein resistant solid support surface for coupling of binding agents, comprising a hydrogel layer containing a plurality of binding elements, wherein a selected fraction of the binding elements are coupled to a protein resistant compound, and the remaining binding elements are free for coupling of at least one binding agent to the solid support.