A sensitive solid-phase assay for identification of class A macrophage scavenger receptor ligands using cell lysate.
In order for macrophages to perform their numerous homeostatic, immunological and tissue remodeling functions they are required to express a broad repertoire of cell-surface receptors. These receptors are particularly important for their host-defense functions in the recognition of foreign pathogens. Delineation of the particular functions of specific receptors requires the identification of ligands recognized by the receptor. We have developed a sensitive, high throughput, solid-phase assay for the detection of ligands for the class A macrophage scavenger receptor (SR-A). Post-nuclear cell lysate from murine bone marrow-derived macrophages is used as a source of receptor and specific ligand binding to SR-A is detected with a monoclonal antibody for SR-A. This assay has been used effectively to identify protein ligands for SR-A on the surface of the bacterium Neisseria meningitidis (Peiser, L. et al. [Peiser, L., Makepeace, K., Pluddemann, A., Savino, S., Wright, J.C., Pizza, M., Rappuoli, R., Moxon, E.R., Gordon, S., 2006. Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay. Infect. Immun. 74, 5191-5199]). In this paper we describe the method in detail and define the specific variables governing the assay.
Plüddemann
Annette
A
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
Neyen
Claudine
C
Gordon
Siamon
S
Peiser
Leanne
L
D000818:Animals; D000911:Antibodies, Monoclonal; D001412:Bacillus subtilis / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002458:Cell Fractionation; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009980:Influenza A virus / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009345:Neisseria meningitidis / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D051117:Scavenger Receptors, Class A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D008024:Ligands; D051117:Scavenger Receptors, Class A