Validation of monoclonal anti-PKC isozyme antibodies for flow cytometry analyses in human T cell subsets and expression in cord blood T cells

Abstract

T cells from neonates (cord blood) with a tendency to develop allergic diseases express low PKCζ levels. More extensive investigations into PKC isozyme levels in T cell subsets and changes during neonatal T cell maturation are hampered by limitations of Western blot analyses. We have undertaken to validating the specificity of commercially available antibodies marketed for flow cytometry to measure PKCα, βI, βII, δ, ε, η, θ, ζ, ι/λ and μ. Western blot analyses of human peripheral blood mononuclear cell (PBMC) lysates demonstrated that some antibodies were unsuitable for flow cytometry assays. A panel of antibodies with the desirable specificity and reliability in the flow cytometry assay were identified using both PBMC and whole blood assays. The results showed that all PKC isozymes were expressed in CD4⁺ and CD8⁺ T cells, monocytes and neutrophils. Murine lymphocytes showed similar patterns of expression. A major finding was that 35.2% and 38.5% of cord blood samples have low PKCζ (≤the 5th percentile of adult levels) in the CD4⁺ and CD8⁺ subsets, respectively, consistent with the incidence of allergy development in the population. Furthermore, these low PKCζ levels 'normalised' within 24 h after initiation of maturation of these cells in culture, providing a 'window of opportunity' for altering PKCζ levels.