Development of a Rapid Streptavidin Capture-Based Assay for the Tyrosine Phosphorylated CSF-1R in Peripheral Blood Mononuclear Cells

Shalini Chaturvedi^1✉, Elayne Dell¹, Derick Siegel¹, Gregory Brittingham², Shobha Seetharam¹

1. Janssen Pharmaceutical Companies of Johnson & Johnson 1400 McKean Rd, Spring House, PA 19477, USA
2. Department of Biochemistry and Molecular Biology, Gettysburg College, 300 North Washington Street, Gettysburg, PA 17325, USA

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) License. See http://ivyspring.com/terms for full terms and conditions.

Citation:
Chaturvedi S, Dell E, Siegel D, Brittingham G, Seetharam S. Development of a Rapid Streptavidin Capture-Based Assay for the Tyrosine Phosphorylated CSF-1R in Peripheral Blood Mononuclear Cells. Int J Biol Sci 2013; 9(10):1099-1107. doi:10.7150/ijbs.7268. Available from https://www.ijbs.com/v09p1099.htm

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.

Keywords: Colony stimulating factor - 1 receptor, IP-MSD assay, CSF-1