title: Generation of human neutrophil gelatinase-associated lipocalin monoclonal antibodies for use in ARCHITECT® assay.
abstract: Development of a robust immunoassay requires the selection of monoclonal antibodies with desired properties. These properties generally include kinetics parameters such as on-rate and off-rate (i.e., binding affinity), and, often times, the ability to form a sandwich with the analyte of interest. We sought to obtain antibodies suitable for development of an immunoassay capable of detecting human neutrophil gelatinase-associated lipocalin (NGAL), a glycosylated lipocalin of 25 kDa expressed in kidney tubules in response to injury that has been shown to be a urinary biomarker capable of diagnosing acute kidney injury. We immunized CAF1/J and RBF/DnJ mouse strains with recombinant NGAL, and a robust immune response, as measured by serum antibody titer, was observed among all CAF1/J mice. Antibodies secreted from mouse B cell-myeloma hybridomas were screened by enzyme immunoassay (EIA) and by surface plasmon resonance using a method we termed hybrid supernatant kinetic screening. Approximately 300 hybrid clones were evaluated by this technique to identify antibodies with the kinetic binding parameters meeting criteria required for further assay development (i.e., rapid association and slow dissociation). This data, along with epitope grouping, cell growth, cell viability, and antibody secretion, were used to identify antibodies for testing in the ARCHITECT assay.
authors.affiliation: Biomolecular Research, Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6018, USA. David.hawksworth@abbott.com
authors.forename: David
authors.initials: D
authors.lastname: Hawksworth
authors.forename: Bailin
authors.initials: B
authors.lastname: Tu
authors.forename: Bryan
authors.initials: B
authors.lastname: Tieman
authors.forename: Susan
authors.initials: S
authors.lastname: Brophy
authors.forename: Joan
authors.initials: J
authors.lastname: Tyner
authors.forename: A Scott
authors.initials: AS
authors.lastname: Muerhoff
authors.forename: Robert
authors.initials: R
authors.lastname: Ziemann
mesh_terms: D000209:Acute-Phase Proteins / Q000276:immunology*; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007700:Kinetics; D000071068:Lipocalin-2; D054834:Lipocalins / Q000276:immunology*; D051379:Mice; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000276:immunology*; D020349:Surface Plasmon Resonance
chemical_list: D000209:Acute-Phase Proteins; D058846:Antibodies, Monoclonal, Murine-Derived; D000939:Epitopes; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins