Blocking Non-Specific Binding
Flow Cytometry Illustrated Assay
General Flow Cytometry Protocol
Isotype Controls – Uncovering Non-Specific Staining
Background: Successful detection of target molecules in immunoassays involves being able to distinguish specific antibody signal from background staining. Non-specific binding of antibodies to Fc receptors, endogenous enzymes, reactive epitopes, and other off-target effects contribute to background staining and skew experimental results. Thus, including negative controls are critical to identify the source of unwanted staining and to reduce the risk of false-positive results.
What is an isotype control? An isotype control is an antibody that maintains similar properties to the primary antibody but lacks specific target binding. Used in place of the primary antibody, this negative control helps determine the contribution of non-specific background to staining.
What should I consider when selecting and using an isotype control? Isotype controls should closely match the properties of the primary antibody and be used under identical experimental conditions to best determine the presence and extent of non-specific binding. Isotype controls should be from the same host species, class and subclass (isotype), and used at the same working concentration as the primary antibody. When using a labeled primary antibody, the isotype control should also have the same conjugate and ideally, with the equivalent label-to-antibody ratio.
How do I know if my primary antibody is specific after running an isotype control? During analysis, compare the signal intensity of the isotype control sample with the experimental sample, probed with the primary antibody (see example provided below). The isotype control should have negligible staining when background signal is not affected by Fc receptor binding, endogenous enzymes, and reactive epitopes. While this negative control exposes multiple factors that contribute to background staining, it does not provide confirmation of specific antibody-antigen binding.
What applications use an isotype control? Isotype controls are commonly used in flow cytometry experiments and immunohistochemistry (IHC). For these applications and other immunoassays including ELISAs, Western Blotting, and Immunocytochemistry (ICC), an isotype control antibody can also serve as a blocking or coating agent.
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Example Data Using an Isotype Control
Human testicular embryonic carcinoma cells were either stained using SSEA-4 Alexa Fluor® 647-conjugated mouse antibody [FAB1435R, orange] or the isotype control, mouse Alexa Fluor® 647-conjugated mouse IgG3 antibody [IC007R, blue]. The data suggest specific antibody staining is easily distinguished from background staining with the isotype control.