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Immobilization of antibodies as a versatile tool in hybridized capillary electrophoresis
1Department of Analytical Chemistry and Toxicology, University Center for Pharmacy, Groningen, Netherlands.
Hybridization of capillary electrophoresis (CE) and immunoassays (IA) can theoretically lead to highly sensitive and selective assays. Immobilization of antibodies in the capillaries employed for CE can be achieved either by adsorption to the capillary wall, which was coated prior to use in order to improve the adsorption, or by covalent binding to modified capillaries. For the evaluation of the concept, a fluoroimmunoassay for the herbicide atrazine was used. Antibodies were immobilized by adsorption, and the specificity of the binding of the labeled ligand was confirmed by saturation and competition experiments. For this particular assay the use of a C8-modified capillary was shown to be preferable over C18- and mercaptodimethylsilane-modified capillaries. The first part of the C8 capillary wall was partially covered by antibodies and the remainder was covered by adsorbed bovine serum albumin to eliminate non-specific binding of the labeled ligand. In the present approach the antibody-bound fraction of the labelled ligand was quantitated, which means that after removal of the free fraction of the labeled ligand from the capillary, the binding of the labeled ligand and the analyte to the antibodies, should be broken. By changing the chemical environment such as pH, salts and organic solvents, this dissociation process can be facilitated. Addition of 25% methanol to the assay buffer increased the dissociation rate by 50% without inactivation or mobilization of the antibodies. On the other hand, these chemical tools should not interfere with the requirements for CE and fluorescence detection. Moreover, the methanol caused stacking of fluorescein-labeled atrazine (FA) in the sample plug by a factor of 30, which was very advantageous for the quantitation of FA. The results of this study imply that combination of antibodies and fluorescent labels with CE opens the way to multi-analyte immunoassays and forms a valuable tool for the selective preconcentration of analytes originating from complex biological matrices.
Hybridization of capillary electrophoresis (CE) and immunoassays (IA) can theoretically lead to highly sensitive and selective assays. Immobilization of antibodies in the capillaries employed for CE can be achieved either by adsorption to the capillary wall, which was coated prior to use in order to improve the adsorption, or by covalent binding to modified capillaries. For the evaluation of the concept, a fluoroimmunoassay for the herbicide atrazine was used. Antibodies were immobilized by adsorption, and the specificity of the binding of the labeled ligand was confirmed by saturation and competition experiments. For this particular assay the use of a C8-modified capillary was shown to be preferable over C18- and mercaptodimethylsilane-modified capillaries. The first part of the C8 capillary wall was partially covered by antibodies and the remainder was covered by adsorbed bovine serum albumin to eliminate non-specific binding of the labeled ligand. In the present approach the antibody-bound fraction of the labelled ligand was quantitated, which means that after removal of the free fraction of the labeled ligand from the capillary, the binding of the labeled ligand and the analyte to the antibodies, should be broken. By changing the chemical environment such as pH, salts and organic solvents, this dissociation process can be facilitated. Addition of 25% methanol to the assay buffer increased the dissociation rate by 50% without inactivation or mobilization of the antibodies. On the other hand, these chemical tools should not interfere with the requirements for CE and fluorescence detection. Moreover, the methanol caused stacking of fluorescein-labeled atrazine (FA) in the sample plug by a factor of 30, which was very advantageous for the quantitation of FA. The results of this study imply that combination of antibodies and fluorescent labels with CE opens the way to multi-analyte immunoassays and forms a valuable tool for the selective preconcentration of analytes originating from complex biological matrices.