Generation of Human Neutrophil Gelatinase-Associated Lipocalin Monoclonal Antibodies for Use in ARCHITECT® Assay
| Autor: | Joan D. Tyner, David J. Hawksworth, Bailin Tu, Bryan C. Tieman, A. Scott Muerhoff, Susan E. Brophy, Robert Ziemann |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
medicine.drug_class
Immunology Antibody Affinity Enzyme-Linked Immunosorbent Assay Lipocalin Monoclonal antibody law.invention Antibodies Monoclonal Murine-Derived Epitopes Mice Immune system Lipocalin-2 law Proto-Oncogene Proteins medicine Animals Humans Immunology and Allergy Gelatinase Hybridomas biology medicine.diagnostic_test Surface Plasmon Resonance Molecular biology Lipocalins Kinetics Titer Biochemistry Immunoassay biology.protein Recombinant DNA Antibody Epitope Mapping Acute-Phase Proteins Protein Binding |
| Zdroj: | Hybridoma. 31:436-442 |
| ISSN: | 1557-8348 1554-0014 |
| DOI: | 10.1089/hyb.2012.0055 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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