title: Development of a rapid streptavidin capture-based assay for the tyrosine phosphorylated CSF-1R in peripheral blood mononuclear cells.
abstract: A novel assay was developed to measure ratio of p-FMS (phospho FMS) to FMS using the Meso Scale Discovery(®) (MSD) technology and compared to the routinely used, IP-Western based approach. The existing IP-Western assay used lysed PBMCs (Peripheral Blood Mononuclear Cells) that were immunoprecipitated (IP) overnight, and assayed qualitatively by Western analysis. This procedure takes three days for completion. The novel IP-MSD method described in this paper employed immunoprecipitation of the samples for one hour, followed by assessment of the samples by a ruthenium labeled secondary antibody on a 96-well Streptavidin-coated MSD plate. This IP-MSD method was semi-quantitative, could be run in less than a day, required one-eighth the volume of sample, and compared well to the IP-Western method. In order to measure p-FMS/FMS, samples from healthy volunteers (HV) were first stimulated with CSF-1(Macrophage colony-stimulating factor) to initiate the changes in the phosphotyrosyl signaling complexes in FMS. The objective of the present work was to develop a high throughput assay that measured p-FMS/FMS semi-quantitatively, with minimal sample requirement, and most importantly compared well to the current IP-Western assay.
authors: 1. Janssen Pharmaceutical Companies of Johnson & Johnson 1400 McKean Rd, Spring House, PA 19477, USA.
authors: Shalini
authors: S
authors: Chaturvedi
authors: Elayne
authors: E
authors: Dell
authors: Derick
authors: D
authors: Siegel
authors: Gregory
authors: G
authors: Brittingham
authors: Shobha
authors: S
authors: Seetharam
mesh_terms: D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D010766:Phosphorylation; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000097:blood*; D019809:Streptavidin / Q000737:chemistry*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D019809:Streptavidin; D016186:Receptor, Macrophage Colony-Stimulating Factor
keywords: CSF-1; Colony stimulating factor - 1 receptor; IP-MSD assay