Standardisation of a procedure for quantifying surface antigens by indirect immunofluorescence.

Quantitative indirect immunofluorescence (QIIF) methods used to measure absolute numbers of surface-expressed antigens have produced conflicting results [Marchant, A., Duchow, J., Delville, J., Goldman, M., 1992. Lipopolysaccharide induces up-regulation of CD14 molecule on monocytes in human whole blood. 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. Quantitation of surface CD14 on human monocytes and neutrophils. Journal of Leukocyte Biology 61, 721-728.]. The aim of this study was to standardise a flow cytometric method using the quantitative indirect immunofluorescence kit (QIFIkit, Dako, Denmark) for quantifying surface-expressed bovine classical major histocompatibility complex (MHC) class I molecules. The importance of accurately titrating antibodies in this procedure and using live cell gates is already accepted. However, little work has been carried out in optimising cell washes to remove excess antibody, or to study the influence of cell numbers used in the assay. In addition, information on the binding properties of each antibody is required in order to make accurate measurements. This study demonstrates that a number of critical parameters must be established prior to using this method for accurate numerical assessment of cell surface-expressed molecules.