Standardisation of a procedure for quantifying surface antigens by indirect immunofluorescence

Quantitative indirect immunofluorescence (QIIF) methods used to measure abs olute numbers of surface-expressed antigens have produced conflicting resul ts [Marchant, A., Duchow, J., Delville, J., Goldman, M., 1992. Lipopolysacc haride induces up-regulation of CD14 molecule on monocytes in human whole b lood. European Journal of Immunology 22, 1663-1665; Antal-Szalmas, P., van Strijp, J.A.G., Weersink, A.J.L., Verhoef, J., van Kessel, K.P.M., 1997. Qu antitation of surface CD14 on human monocytes and neutrophils. Journal of L eukocyte Biology 61, 721-728.]. The aim of this study was to standardise a flow cytometric method using the quantitative indirect immunofluorescence k it (QIFIkit, Dako, Denmark) for quantifying surface-expressed bovine classi cal major histocompatibility complex (MHC) class I molecules. The importanc e of accurately titrating antibodies in this procedure and using live cell gates is already accepted. However, little work has been carried out in opt imising cell washes to remove excess antibody, or to study the influence of cell numbers used in the assay. In addition, information on the binding pr operties of each antibody is required in order to make accurate measurement s. This study demonstrates that a number of critical parameters must be est ablished prior to using this method for accurate numerical assessment of ce ll surface-expressed molecules. (C) 1999 Elsevier Science B.V. All rights r eserved.