| Isotype Controls: Isotype controls are a type of negative control designed to measure the level of non-specific background signal caused by primary antibodies, based upon the tissue type of the sample. Non-specific binding of primary antibodies are typically caused by the following reasons: Non-specific Fc receptor binding to cells Non-specific antibody interactions with cellular proteins or fluorochromes Non-specific binding of the antibody or fluorochromes to cellular components Cell autofluorescence Usually, the background signal is the result of immunoglobulins binding non-specifically to Fc receptors present on the cell surface. For example, antibodies raised in mice, particularly those of the IgG2a isotype, will bind strongly to some human leukocytes regardless of the test antibody specificity. In this case you would need a mouse IgG2a isotype control for use with human cells or tissues. Isotype controls are most commonly used in flow cytometry experiments, but may also be used in immunohistochemistry. Selecting an Isotype Control: Typically, an isotype control is matched to the host species and isotype of your specific primary antibody, the heavy chain (IgA, IgG, IgD, IgE, or IgM), subclass and light chain (kappa, lambda) class of the primary antibody, the same fluorochrome (PE, APC, etc.), and have the same F:P ratio. F:P is a measurement of how many fluorescent molecules are present on each antibody. When using directly labeled primary antibodies, it is also necessary to make sure that the isotype control is conjugated to the same fluorochrome or label as the test antibody. It is not sufficient to use one with a spectrally similar fluorochrome. Non-specific Binding: To avoid non-specific binding, you also need to block Fc receptors on cell types, such as spleen cells, with FcR blocking reagents. Isotype controls should be used at the same concentration/dilution as the primary antibody. Isotype controls are mainly used to determine if the staining is real. Apart from isotype controls, unstained cells should always be included in the experimental set-up to monitor autofluorescence. Applications for Isotype Controls: Although isotype controls are mainly used in flow cytometry, they can be used as standard blocking agents and protein coating agents for other applications including immunofluorescence, immunocytochemistry, western blotting, and ELISA. Fusion Partners: Spleen cells from BALB/c mice were fused with cells from the X63/Ag-8653 mouse myeloma cell line. Applications: Flow Cytometry: Neat Optimal working dilutions to be determined by researcher. Flow Cytometry: Use 10ul of the suggested working dilution to label 10e6 cells or 100ul whole blood. |
