title: Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary.
abstract: For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.
authors.affiliation: Division of Advanced Chemistry, Fukushima Medical University, 1 Hikarigaoka, Fukushima, 960-1295, Japan.
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Shimura
authors.affiliation: Division of Advanced Chemistry, Fukushima Medical University, 1 Hikarigaoka, Fukushima, 960-1295, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Nagai
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D012822:Silicon Dioxide