This was based on the assumption that an appropriate isotype control would display the same degree of nonspecific binding as the specific antibody of interest. In the past decades, isotype controls have been widely used to control for nonspecific antibody binding. To further investigate this, we included both serum and purified IgG as blocking reagents in our analyses. Nonetheless, it has been stated that the concentration of Ig in serum is too low for sufficient blocking, and therefore it was recommend to use purified IgG 8. Serum is another regularly used blocking reagent. They clearly found human Ig superior to goat Ig, the latter showing virtually no effect 7. Sedlmayer and colleagues have compared goat and human immunoglobulins (Ig), regarding the ability to block nonspecific binding of mouse isotype controls to activated human monocytes. However, in another study, this blocking reagent did not completely block nonspecific binding of antibodies to mouse monocytes 6. In this study, the authors used commercially available rat-anti-mouse CD16/32 antibody for Fc-blocking. One or more of these receptors are expressed by all leukocytes except for T cells, with highest expression on monocytes and macrophages 4, 5.Īs nicely demonstrated by Kuonen and colleagues, failing to block nonspecific binding of antibodies to mouse CD11b + myeloid cells resulted in erroneous results when performing immunophenotyping by flow cytometry 2.
Nonspecific binding of antibodies to leukocytes is mainly due to binding to Fc receptors (FcRs, CD16, CD32, CD64) 1- 3. Besides coming from electronic noise of the instrument, background fluorescence primarily originates from autofluorescence, spectral overlap in multicolour assays, and nonspecific antibody binding to the cells of interest 1. A clear separation of populations (a high resolution index or staining index) may be obtained by proper titration of antibodies and by minimizing background fluorescence. In flow cytometry, the ability to differentiate between “positive” and “negative” events is of major importance. © 2016 International Society for Advancement of Cytometry Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Previously, isotype controls have been widely used in flow cytometry assays.
Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages.
A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results.
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