title: Development of microcolumn-based one-site immunometric assays for protein biomarkers.
abstract: One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1mm i.d. ×5mm and contained 8-9nmol of immobilized HSA. These microcolumns were used from 0.10 to 1.0mL/min and gave results within 35s to 2.8min of sample injection. Limits of detection down to 0.10-0.28ng/mL (1.5-4.2pM) or 25-30pg/mL (0.38-0.45pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ±0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Erika L
authors.initials: EL
authors.lastname: Pfaunmiller
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Jeanethe A
authors.initials: JA
authors.lastname: Anguizola
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Mitchell L
authors.initials: ML
authors.lastname: Milanuk
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Efthimia
authors.initials: E
authors.lastname: Papastavros
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: NaTasha
authors.initials: N
authors.lastname: Carter
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Matsuda
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA.
authors.forename: Xiwei
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588 USA. Electronic address: dhage1@unl.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D000906:Antibodies / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000032:analysis*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D012709:Serum Albumin / Q000032:analysis
chemical_list: D000906:Antibodies; D015415:Biomarkers; D005456:Fluorescent Dyes; D012709:Serum Albumin
keywords: Affinity microcolumn; Chromatographic immunoassay; Human serum albumin; Near-infrared fluorescence; One-site immunometric assay; Protein biomarker