Method for immobilization of (an) affinity reagent(s) on a hydrophobic solid phase
The invention relates to a method of immobilizing an affinity reagent or affinity reagents on a hydrophobic solid phase which can be used in biological assays for analyte detection. The invention relates more particularly to a method of immobilizing an affinity reagent on a hydrophobic solid phase functionalized by a carboxyl group, said method comprising a step for activation of said solid phase and a step for coupling of the affinity reagent to said solid phase, characterized in that the step for activation of said solid phase uses a combination of a carbodiimide and a phosphate buffer in the presence of a co-activator and in an acid medium, and in that the coupling step is performed in a basic medium. The invention further relates to the reactive complexes obtained by this method and to their use in immunoassay kits, hybridization kits or enzymatic assay kits.
1. Method of immobilizing an affinity reagent on a hydrophobic solid phase functionalized by a carboxyl group, said method comprising a step for activation of said solid phase and a step for coupling of the affinity reagent to said solid phase, wherein the step for activation of said solid phase uses a combination of a carbodiimide and a phosphate buffer in the presence of a co-activator and in an acid medium with a pH ranging from about 4 to about 6.5, and the coupling step is performed in a basic medium with a pH ranging from about 7.2 to about 10.5.
2. Method according to claim 1 , wherein the carbodiimide used is a compound selected from the group comprising CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide).
3. Method according to claim 1 wherein the co-activator used is a compound selected from the group comprising s-NHS (sulfo-N-hydroxysuccinimide), HOBt (1-hydroxybenzotriazole) and N-hydroxysuccinimide.
4. Method according to claim 1 wherein the carbodiimide used is CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) and the co-activator used is s-NHS (sulfo-N-hydroxysuccinimide).
5. Method according to claim 1 , wherein the carbodiimide is used in an amount of 20 to 50 molar equivalents per COOH group.
6. Method according to claim 1 , wherein the co-activator is used in an amount of 3 to 10 molar equivalents per COOH group.
7. Method according to claim 1 , wherein the step for activation of the solid phase uses a combination of 20 to 50 molar equivalents per COOH group of CMC, in 30–200 mM phosphate buffer, in the presence of 3 to 10 molar equivalents per COOH group of sulfo-N-hydroxysuccinimide co-activator, in an acid medium with a pH ranging from about 4 to about 6.5, and in that the coupling is performed in a basic medium with a pH ranging from about 7.2 to out 10.5.
8. Method according to claim 1 , wherein the step for activation of the solid phase uses a combination of 30 molar equivalents per COOH group of CMC, in 50 mM KH 2 P 4 phosphate buffer, in the presence of 5 molar equivalents per COOH group of sulfo-N-hydroxysuccinimide co-activator, at pH 6, and the coupling is performed in a medium containing one volume of a buffer of pH 8.5 and one volume of said phosphate buffer.
9. Method according to claim 1 , wherein the affinity reagent contains an amine group.
10. Method according to claim 9 , wherein the affinity reagent is selected from the group comprising proteins, peptides, immunoglobulins, antigens, haptens, antibodies, enzymes, enzyme substrates, oligonucleotides and polynucleotides.
11. Reactive solid complex obtained by a method according to claim 1 , said reactive solid complex selected from the group consisting of solid phase/antigen complexes, solid phase/protein complexes, solid phase/peptide complexes, solid phase/hapten complexes, solid phase/antibody complexes, solid phase/immunoglobulin complexes, solid phase/oligonucleotide complexes, solid phase/polynucleotide complexes, solid phase/enzyme complexes and solid phase/enzyme substrate complexes.