Applications of Secondary Antibodies - Flow Cytometry
Flow cytometry is a powerful technique for measuring and analyzing the physical characteristics of single particles in solution as they travel past a beam of light. Properties such as relative size and fluorescence can be measured, and fluorescently tagged antibodies enable cells to be interrogated for multiple proteins and molecular dynamics. Isotype controls are used for experiment validation and analysis of results.
Indirect Flow Cytometry
Flow cytometry can be performed directly, using conjugated primary antibodies, or indirectly, using a conjugated secondary antibody to bind an unconjugated primary. Indirect flow cytometry allows the choice of a wide range of probe molecules, enabling the user to match the desired probe with any primary antibody. Secondary antibody conjugates can improve a flow cytometry experiment by preserving the active site of the primary antibody, and by signal amplification (see Figure 1 B).
Figure 1. Direct (A) and indirect (B) flow cytometry.
Secondary Antibody Format for Flow Cytometry
The format of secondary antibody can impact the success of an experiment. In addition to whole molecule IgG, Jackson ImmunoResearch offers fragments of secondary antibodies.
F(ab’)2 Fragments
F(ab’)2 fragments are generated by proteolysis of the whole IgG to yield a divalent fragment containing two Fab arms and no Fc domain. When used to stain tissue or cells, the F(ab’)2 secondary antibodies can help to avoid background caused by off-target binding. The absence of an Fc region prevents F(ab’)2 antibodies from being captured by Fc receptors expressed on cell surfaces. Please note that if a primary antibody is trapped by an Fc receptor, the F(ab’)2 secondary antibody will detect the off-target binding, so blocking is critical.
FabuLight™ secondary antibodies are created by papain digestion of IgG, followed by removal of Fc fragments. These monovalent Fab fragments are specific for the Fc region of primary antibodies, so they don’t interact with the primary’s antigen-binding region. Conjugated FabuLights are convenient for labeling primary antibodies prior to incubation with an experimental sample, saving incubation and wash steps. Like F(ab’)2 fragments, these Fab fragments can minimize background staining due to Fc receptor binding.
The choice of fluorescent dye conjugate depends on a number of experimental variables.
Instrument capabilities. Consider excitation capabilities (excitation wavelengths or laser colors), and which filters are available.
Experimental sample. It may be necessary to consider autofluorescence or the expression of recombinant fluorescently tagged proteins, which may preclude using fluorophores with spectral overlap.
Sensitivity required. Several fluorophores may have similar excitation and emission spectra, but differences in inherent brightness can result in one fluorophore showing a larger population shift. For example, Alexa Fluor® 488 is brighter than FITC.
Degree of color separation required. For multiple labeling, the dye panel choices will be constrained by the equipment available. To achieve good color separation it is important to look at the emission overlap of the fluorophores in the dye panel. Panel developing tools are available online which can help build dye panels specific to any instrument.
Flow cytometry with JIR Secondary Antibodies
Fluorescent dyes from UV to far red can be used for flow cytometry, depending on instrument capabilities. Jackson ImmunoResearch offers a range of fluorescent dye conjugates spanning the spectrum, making it possible to design a flow cytometry dye panel that accommodates instrument capabilities and recombinant proteins incorporated in the experiment. These secondary antibody conjugates can be found listed in the tables of Whole IgG and F(ab’)2 Fragments.
Figure 2. Comparison of direct and indirect flow cytometry methods. Human peripheral blood gated lymphocytes after ammonium chloride lysis of erythrocytes were analyzed for CD3 expression using direct or indirect flow cytometry. Comparison of mean fluorescence showed that the indirect method produced a brighter signal (22,973) compared to the direct method (8,985). (Experiment performed on BD FACSCelesta).
Biotin-SP Conjugates for Flow Cytometry
Jackson ImmunoResearch offers Biotin-SP-conjugated secondary antibodies in both whole IgG and F(ab’)2 format. Biotin-SP conjugates require the use of fluorescently labeled streptavidin for visualization.
Controls are essential to validate an experiment and interpret results. ChromPure™ proteins are purified from the serum of non-immunized animals and are appropriate experimental controls. An isotype control is a negative control which estimates the non-specific binding of an antibody. Isotype controls are antibodies which match the host species and class of antibodies used in the experiment but are not directed against the antigen of interest. An isotype control should be conjugated with the same reporter molecule as the specific antibody.
Isotype Controls for Flow Cytometry
An isotype control for direct immunofluorescence will be conjugated to the same fluorophore as the primary antibody. For polyclonal primary antibodies (e.g. rabbit or goat primaries), conjugated ChromPure purified proteins are good experimental controls. Conjugated ChromPure proteins can also be used as controls for monoclonal primaries, though it may be preferable to use a control that is the same subclass as the monoclonal.
Indirect immunofluorescence may require isotype controls for both the primary and secondary antibodies
Unconjugated ChromPure purified proteins can be used as experimental controls for unlabeled polyclonal primary antibodies. Some users choose a control that is the same subclass as their monoclonal primary antibody, but the mixed subclass ChromPure proteins may also be acceptable controls.
ChromPure purified proteins conjugated with the same reporter molecule as the labeled secondary antibody are good isotype controls. The isotype control should have the same format (whole molecule or antibody fragment) as the secondary antibody.
Suggested Dilution Ranges for Flow Cytometry Applications
The dilutions suggested in the following table are presented as ranges because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The optimal working dilution should be determined empirically for each application.
Product
Dilution
Unconjugated whole IgG and F(ab’)2 secondary antibodies
10 - 20 µg / ml
Unconjugated Fab secondary antibodies
20 - 40 µg / ml
All Alexa Fluor®, DyLight, and Cy3 Conjugates [IgG, F(ab’)2, Fab]
1:100 - 1:800
Brilliant Violet™ Conjugates [IgG]
1:50 - 1:200
AMCA, Cy2, FITC, TRITC, RRX, and Texas Red Conjugates [IgG, F(ab’)2, Fab]
1:50 - 1:200
Cy5 Conjugates [IgG]
1:100 - 1:400
Phycoerythrin and Allophycocyanin Conjugates [IgG, F(ab’)2, Streptavidin]
1:50 - 1:200
PerCP Conjugates [IgG, F(ab’2, Streptavidin]
1:25 - 1:100
Biotin-SP Conjugates [IgG, F(ab’)2, Fab]
1:200 - 1:1,000
All Alexa Fluor®-, DyLight 405-, and Cy3-Conjugated Streptavidin
