Methods of selecting isotype controls in flow experiments

Antibodies sometimes bind certain intracellular components due to non-specific protein-protein interactions, bind lipids due to hydrophobic interactions, and may also bind some cell surface antibody receptors. For example, antibodies of the IgG subtype bind cell surface Fc receptors such as CD16, CD32, and CD64. Ideally, non-specific binding of all types can be blocked by proteins (BSA, milk, or animal serum.) Binding of Fc receptors, on the other hand, can be blocked by pre-incubation with immunoglobulin-containing serum. So an antibody's non-specific interactions and binding to the Fc receptor can be compared to an antibody of the same subtype of an unrelated antigen, i.e. an isotype control. So how do you choose an isotype control when doing a flow-through experiment?

Generally, the antibody is selected to be of exactly the same species origin, the same isotype and subchain, and the same fluorescent labeling as the components of the primary antibody. For example, if the source of anti-human CD56 antibody with FITC label is Mouse IgG1,κ chain, then its isotype control should be FITC labeled Mouse IgG1,κ chain.

If the antibody is in combination form, such as purified primary antibody + fluorescently labeled secondary antibody, then the isotype control of the primary antibody should be selected. For example, the purified antibody of CD86 is composed of Mouse IgG1,κ chain, and its isotype control is purified Mouse IgG1,κ chain, if the secondary antibody used in the experiment is PE-labeled anti-mouse IgG1. Then the staining method is as follows ------- Sample Tube: Purified CD86 + PE-labeled anti-mouse IgG1 (secondary antibody) + sample, Iso-control Tube: Purified Sample tube: purified CD86 + PE-labeled anti-mouse IgG1 (secondary antibody) + sample, Isotype control tube: purified Mouse IgG1 (κ chain) + PE-labeled anti-mouse IgG1 (secondary antibody) + sample.

If no exact matching isotype control is found, the order of preference should be subchain different - isotype different, while maintaining the same source, the same fluorescent labeling, and the same immunoglobulin class. For example, the source of a certain antibody is Mouse IgG2а,κ chain. If an identical isotype control cannot be found, then the same fluorescently labeled Mouse IgG2а is preferred; if the appropriate fluorescein-labeled Mouse IgG2а cannot be found either, then the same fluorescently labeled Mouse IgG2b is preferred; and if Mouse IgG2b is still not found, then the same fluorescently labeled Mouse IgG2 as isotype control.

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