title: Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats.
abstract: Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE, United States.
authors.forename: Erika L
authors.initials: EL
authors.lastname: Pfaunmiller
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE, United States.
authors.forename: Jeanethe A
authors.initials: JA
authors.lastname: Anguizola
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE, United States.
authors.forename: Mitchell L
authors.initials: ML
authors.lastname: Milanuk
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE, United States.
authors.forename: NaTasha
authors.initials: N
authors.lastname: Carter
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE, United States. Electronic address: dhage1@unl.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D012709:Serum Albumin
keywords: Affinity microcolumn; Chromatographic immunoassay; Competitive binding immunoassay; Human serum albumin; Protein G