Monoclonal antibodies to equine CD14

Abstract

CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 ( rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine CD14. One mAb, clone 105, was used for further characterization of CD14 + cells in peripheral blood mononuclear cells (PBMC). Phenotyping indicated that the majority of the CD14 + PBMC were non-B/non-T-cells. Magnetic cell sorting enriched CD14 + cells to >95% as detected by flow cytometry. Differential cell counts on Wright's-stained cytospin smears of CD14 + cell fractions demonstrated that 49–73% of them were monocytes. The discrepancy between CD14 + cells detected by flow cytometric analysis and monocytes based on morphologic criteria suggests that some of the equine CD14 + PBMC are lymphoid cells. The mAbs to equine CD14 provide new tools for cellular analysis and CD14 + cell isolation in horses.