title: Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.
abstract: Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti-insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol-reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab-gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral-polymer coated fused silica capillary (50 μm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser-induced fluorescence under focusing conditions. The insulin-AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first-order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.
authors.affiliation: Laboratory of Chemistry, School of Medicine, Fukushima Medical University, Fukushima, Fukushima, Japan.
authors.forename: Kiyohito
authors.initials: K
authors.lastname: Shimura
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kasai
mesh_terms: D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007328:Insulin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007525:Isoelectric Focusing; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007328:Insulin; D011994:Recombinant Proteins
keywords: Affinity capillary electrophoresis; Capillary IEF; Immunochemical detection; Insulin; Recombinant antibody