A secondary antibody is used to detect an unconjugated primary antibody that has bound to a target antigen. Secondary antibodies conjugated to enzymes and labels are key components of detection systems – selection of an optimum secondary antibody can improve staining and reduce false positive or negative staining.
AbD Serotec’s range of secondary reagents has been carefully selected to provide optimum quality and flexibility.
Secondary antibodies are available in many formats and are useful in a wide range of applications, including flow
cytometry (FITC & RPE), immunocytochemistry (HRP & alkaline phosphatase) and Western blotting (HRP & Biotin).
There are a number of reasons why you may need to use a secondary antibody, including:
If there is no conjugated primary antibody available.
Your primary antibody isn’t conjugated to the required fluorochrome/enzyme.
You need to increase the sensitivity of your staining procedure.
Our Recommended Secondary Antibodies
For most secondaries, we offer a choice of monoclonal or polyclonal antibodies suitable to meet these needs, including whole IgG molecules, or F(ab’)2 fragments.
All secondary reagents in our “STAR” range are affinity-purified, and have been carefully optimized for specificity and titer. Several of our anti-mouse, anti-rat, and anti-hamster IgG reagents have also been carefully adsorbed against rat and mouse immunoglobulins to avoid cross-species reactivity.
Secondary antibodies for use with Human antibodies.
Secondary antibodies for use with Bovine antibodies.
Secondary antibodies for use with Canine antibodies.
Secondary antibodies for use with Porcine antibodies.
Secondary antibodies for use with Caprine antibodies.
Secondary antibodies for use with Equine antibodies.
Secondary antibodies for use with Cavia antibodies.
Secondary antibodies for use with Chicken antibodies.
Secondary antibodies for use with Feline antibodies.
Secondary antibodies for use with Rabbit antibodies.
Secondary antibodies for use with Mouse antibodies.
Secondary antibodies for use with Rat antibodies.
AbD Serotec manufacturers and distributes high quality immunological reagents with almost thirty years of experience, and a portfolio of fifteen thousand primary antibodies (monoclonal and polyclonal antibodies) and related products.
View our range of primary antibodies.
Things To Consider When Selecting The Secondary Antibody
Choosing the correct secondary antibody is critical for obtaining precise results. It is important that the following is taken into account:
The Host That the Primary Antibody Was Developed In
For immunohistochemistry, the primary antibody should be raised in a species as phylogenetically different as possible to the species of your sample, to avoid cross-reactivity of the secondary antibody.
Your Application Will Affect Your Label
I.e. for ELISA and immunoblotting, enzyme-linked secondaries are the most popular choice, whereas for flow cytometry you need to choose a secondary with a fluorochrome attached. For cell or tissue staining, either choose HRP, alkaline phosphatase, or a fluorescent-linked secondary antibody.
Class/Subclass of Antibody
This is mainly important for monoclonal antibodies, where the secondary antibody should match the class/subclass of the primary antibody. Since polyclonal antibodies (e.g. goat, rabbit, sheep, donkey) are usually IgG class immunoglobulins, anti-IgG secondary antibodies may be used.
Is An Adsorbed Secondary Antibody Available?
This will reduce non-specific background staining, which can be very important when working with mouse and rat tissues. Beware when working with closely homologous species.
Is a F(ab’)2 Fragment Necessary?
When working with some immune tissues or cells that contain a lot of Fc receptors, it helps to choose a F(ab’)2 fragment to eliminate non-specific binding. Alternatively, block Fc receptors via an absorption step, using purified IgG from the host species of your secondary antibody.