Secondary Antibody Selection Guide
antibodies-onlineSecondary antibodies bind to primary antibodies rather than directly to the target antigen. They are widely used for signal detection and amplification in methods including Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, ELISA and immunoprecipitation-based workflows.
Selecting the appropriate secondary antibody requires more than simply matching the species of the primary antibody. Important parameters include the primary antibody host species, immunoglobulin class or subclass, binding specificity, degree of cross-adsorption, antibody format and the detection label required for the application.
How to Select a Secondary Antibody
The following parameters should be considered when choosing a secondary antibody:
- Identify the host species of the primary antibody. A primary antibody raised in rabbit requires an anti-rabbit secondary antibody, while a mouse primary antibody requires an anti-mouse secondary antibody.
- Determine the immunoglobulin class and, where relevant, the subclass. For example, a mouse IgG1 monoclonal antibody can be detected with a general anti-mouse IgG secondary or with an IgG1 subclass-specific secondary antibody.
- Select the required antibody specificity. Depending on the experiment, this may be an IgG (H+L), heavy-chain-specific, Fc-specific, Fab-specific or subclass-specific secondary antibody.
- Consider cross-adsorption. Cross-adsorbed secondary antibodies can reduce unwanted binding to immunoglobulins from the sample species or to other primary antibodies used in multiplex experiments.
- Choose the appropriate antibody format. Whole IgG is suitable for many general detection applications, whereas F(ab')2 or Fab fragments can be advantageous in experiments involving Fc receptors, tissue penetration or specialized multiplex protocols.
- Select the conjugate according to the detection method. Common options include HRP, alkaline phosphatase, biotin and fluorescent dyes such as FITC, PE, Alexa Fluor and DyLight fluorophores.
- Confirm that the secondary antibody is validated or suitable for the intended application. Application requirements differ between Western blotting, ELISA, IHC, IF, flow cytometry and other techniques.
Important terminology: The target species or reactivity of a secondary antibody refers to the species in which the primary antibody was raised. The host species of the secondary antibody refers to the animal in which the secondary antibody itself was produced.
Antibody Class, Subclass and Binding Specificity
Immunoglobulin classes are defined by their heavy chains. In mammals, the major immunoglobulin classes are IgG, IgM, IgA, IgE and IgD. Their heavy chains are designated gamma, mu, alpha, epsilon and delta, respectively. Immunoglobulins additionally contain either kappa or lambda light chains.
The specificity of a secondary antibody determines which region of the primary antibody it recognizes. Selecting the appropriate specificity can reduce background and is particularly important in multiplex experiments.
- IgG (H+L): recognizes epitopes on both the heavy and light chains. These antibodies provide broad recognition and are commonly used for general immunodetection. Because light chains are shared between different immunoglobulin classes, H+L antibodies may also recognize other immunoglobulin classes from the same species.
- Heavy-chain-specific: recognizes the heavy chain of a defined immunoglobulin class, such as IgG, IgM or IgA. This is useful when discrimination between immunoglobulin classes is required.
- Fc-specific: recognizes epitopes within the Fc region of the primary antibody and avoids binding to Fab fragments.
- Fab-specific: recognizes the antigen-binding portion of the primary antibody and can be useful when Fc-specific interactions need to be avoided.
- Subclass-specific: distinguishes immunoglobulin subclasses such as mouse IgG1, IgG2a, IgG2b or IgG3. Subclass-specific secondary antibodies are particularly useful when several monoclonal primary antibodies from the same species are used in one experiment.
Cross-Adsorbed Secondary Antibodies
Immunoglobulins from different species share structurally related epitopes. As a consequence, a secondary antibody directed against immunoglobulins from one species may also recognize immunoglobulins from another species. This cross-reactivity can produce background staining or unintended detection of other antibodies present in an experiment.
Cross-adsorbed secondary antibodies are additionally purified against immunoglobulins or serum proteins from potentially cross-reactive species. This reduces unwanted species cross-reactivity and is especially useful for:
- multiplex immunofluorescence or immunohistochemistry,
- samples containing endogenous immunoglobulins,
- antibody-rich tissues or cell populations,
- experiments combining primary antibodies from several species, and
- indirect sandwich immunoassays in which several antibody species are present.
For tissue staining, the secondary antibody should ideally be cross-adsorbed against the species from which the tissue or cells originate. In multiplex experiments, each secondary antibody should additionally show minimal cross-reactivity with the species of the other primary antibodies.
However, extensive cross-adsorption against closely related species such as mouse and rat can reduce recognition of some epitopes. The required degree of cross-adsorption should therefore be matched to the experimental system.
Secondary Antibody Formats: Whole IgG, F(ab')2 and Fab
Secondary antibodies are available as whole immunoglobulins or as antibody fragments. The appropriate format depends on the sample and application.
| Format | Properties | Typical Considerations |
|---|---|---|
| Whole IgG | Divalent antibody containing Fab and Fc regions. | Suitable for most standard Western blot, ELISA, IF and IHC applications and generally provides strong signal. |
| F(ab')2 | Divalent fragment lacking the Fc region; generated by proteolytic cleavage below the hinge region. | Useful for cells or tissues containing Fc receptors and may improve accessibility in some samples because of its smaller size. |
| Fab | Monovalent antigen-binding fragment lacking the Fc region. | Useful for blocking strategies, reducing antibody-mediated crosslinking and specialized multiple-labeling protocols. |
Host Species of Secondary Antibodies
Mouse and rabbit are among the most frequently used host species for primary antibodies. Consequently, anti-mouse and anti-rabbit secondary antibodies are widely used. Secondary antibodies themselves can be raised in several host species, including goat, donkey, rabbit and other species.
The host species of the secondary antibody becomes particularly important in multiplex experiments. When possible, secondary antibodies directed against different primary antibody species can be selected from the same secondary host species. This minimizes the possibility that different secondary antibodies recognize one another.
If secondary antibodies from different host species must be combined, appropriate cross-adsorption against the other species used in the experiment should be considered.
Detection Labels and Conjugates
Secondary antibodies are available either unconjugated or conjugated to enzymes, fluorophores, biotin and other detection labels. The optimal conjugate depends on the experimental method, detection instrument, required sensitivity and whether single- or multiplex detection is performed.
- Horseradish peroxidase (HRP): widely used for chemiluminescent and colorimetric detection in Western blotting and ELISA. A large variety of substrates is available.
- Alkaline phosphatase (AP): provides an alternative enzymatic detection chemistry for colorimetric or chemiluminescent assays. The relative sensitivity of HRP and AP depends on the substrate, assay format and detection system rather than on the enzyme alone.
- Biotin: biotinylated secondary antibodies can be detected using avidin- or streptavidin-based systems and can provide additional signal amplification. Endogenous biotin should be considered when working with some tissues.
- Fluorophores: fluorescent conjugates enable direct optical detection and are particularly useful for immunofluorescence, flow cytometry and fluorescent Western blotting.
- Phycoerythrin (PE) and other fluorescent proteins: are frequently used in flow cytometry where high fluorescence intensity is advantageous.
For multiplex fluorescence experiments, fluorophores should be selected according to the excitation sources and detection channels of the instrument while minimizing spectral overlap between labels.
Learn more about protein labels and conjugates or compare commonly used fluorophores in our excitation and emission maxima guide.
Secondary Antibodies for Multiplex Experiments
Multiplex immunofluorescence and immunohistochemistry require additional attention to antibody specificity and cross-reactivity.
Where possible, primary antibodies from different host species should be selected. Each secondary antibody can then specifically recognize one primary antibody species. Secondary antibodies should be cross-adsorbed against the species of the other primary antibodies and, where relevant, against the species of the sample.
If several monoclonal primary antibodies from the same species are required, different immunoglobulin subclasses can sometimes be distinguished using subclass-specific secondary antibodies. Sequential staining strategies, Fab fragment blocking or directly conjugated primary antibodies provide additional options for same-species multiplex experiments.
Fluorescent labels should also be chosen with sufficient spectral separation to allow individual signals to be distinguished reliably.
Choosing Secondary Antibodies by Application
| Application | Common Detection Strategy | Important Selection Criteria |
|---|---|---|
| Western Blotting | HRP, AP or fluorescent conjugates | Match the primary antibody species and class. Fluorescent multiplex Western blots require spectrally distinct labels and appropriate cross-reactivity profiles. |
| ELISA | HRP, AP or biotin-based detection | Consider class specificity and possible interactions with other antibodies used in the assay. |
| Immunofluorescence (IF) | Fluorophore-conjugated secondary antibodies | Cross-adsorption against the sample species and other primary antibody species is especially important in multiplex experiments. |
| Immunohistochemistry (IHC) | Enzyme- or fluorophore-conjugated antibodies | Consider endogenous immunoglobulins, Fc receptors, sample species and multiplex compatibility. |
| Flow Cytometry | Fluorescent secondary antibodies | Choose fluorophores appropriate for the cytometer. Fc receptor blocking and/or F(ab')2 secondaries can be useful for Fc-receptor-rich cell populations. |
| Immunoprecipitation / IP-Western | Application-specific secondary detection | Conventional secondary antibodies may detect the heavy and light chains of the antibody used for immunoprecipitation, producing bands around the expected IgG chain molecular weights. |
Special Case: IP-Western Blotting
In IP-Western workflows, conventional secondary antibodies can recognize the denatured heavy and light chains of the immunoprecipitating antibody. These signals may interfere with detection of proteins migrating near approximately 50 kDa or 25 kDa.
Specialized secondary detection systems such as TrueBlot® are designed to reduce detection of denatured immunoprecipitating antibody.
Find the Right Secondary Antibody
The first selection criterion is usually the species in which the primary antibody was raised. Browse secondary antibodies by primary antibody host species below.
Secondary Antibodies by Primary Antibody Host Species
When working with hamster primary antibodies, determine whether the antibody originates from Armenian or Syrian hamster where possible, since secondary antibody recognition between hamster species may not be complete.
Secondary Antibodies by Conjugate
Secondary Antibodies by Application
Immunoglobulin Isoforms
Learn more about immunoglobulin classes and secondary antibodies designed to recognize specific immunoglobulin isotypes:
