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Secondary Antibody Selection Guide

In selecting a secondary antibody, a number of factors should be considered -

Host of the primary antibody

The secondary antibody should be directed to the host species of the primary antibody. For example, if your primary antibody is raised in mouse, you will need an anti-mouse secondary antibody.

Isotype / class / subclass of the primary antibody

The secondary antibody should also be directed against the isotype of the primary antibody.

Monoclonal primary antibodies are commonly raised in mouse, rat, hamster, and rabbit. For example, if the isotype of your primary monoclonal antibody is a mouse IgM, you will need a secondary antibody that is specific to mouse IgM (anti-mouse IgM).

Polyclonal primary antibodies are typically raised in rabbit, goat, sheep, and donkey and are generally IgG isotypes. For example, if the isotype of your primary polyclonal antibody is a rabbit IgG, you will need a secondary antibody that is specific to rabbit IgG (anti-rabbit IgG). An anti-rabbit IgG(H+L) which reacts with the heavy and light chains of IgG as well as the light chains of other immunoglobulins is typically used.

Summary of immunoglobulin classes and subclasses -
Immunoglobulin Classes - IgG, IgM, IgA, IgD, IgE
Mouse Immunoglobulin Subclasses - IgG1, IgG2a/IgG2c, IgG2b, IgG3
Human Immunoglobulin Subclasses - IgG1, IgG2, IgG3, IgG4, IgA1, IgA2
Rat Immunoglobulin Subclasses - IgG1, IgG2a, IgG2b, IgG2c
Hamster Immunoglobulin Subclasses - IgG1, IgG2, IgG3
Light Chains - Kappa, Lambda
Heavy Chains - IgG (gamma/γ), IgM (mu/μ), IgA (alpha/α), IgD (delta/δ), IgE (epsilon/ε)

Species Cross-Adsorptions / Pre-Adsorptions

Cross-adsorption, or pre-adsorption, of a secondary antibody indicates that antibody has undergone a step during the manufacturing process to remove reactivity to a particular species, a non-target Ig, or non-target Ig region. This increases specificity and reduces non-specific binding due to potential cross-reactivities. For example, if your samples are human tissues, select a secondary antibody that has been cross-adsorbed against human Ig or serum proteins (SP). This will help ensure that your secondary antibody exhibits minimal cross-reactivity to the human tissue.

Whole Molecule vs Fragmented Antibodies

Secondary antibodies are available in whole molecule and fragmented formats. Fragmented secondary antibodies (F(ab’)2 and Fab fragments) lack the Fc portion and are often preferred for use with samples in which Fc receptors may be present as they can reduce non-specific binding/background. Additionally, their small size allows for better penetration of tissues.

Conjugates

The choice of conjugate is dependent on the application and the technology utilized for detection (colorimetric, chemiluminescent, or fluorescent). For western blotting and ELISA applications, enzyme-conjugated secondary antibodies such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are typically utilized and both can be detected using colorimetric and chemiluminescent platforms. For flow cytometry or immunofluorescence applications, a secondary antibody conjugated to a fluorochrome is required (e.g., Alexa Fluor®, FITC, TRITC, APC, PE).
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