This guide will help you choose the best secondary antibody for your application...

How to Choose the Best Secondary Antibody

Overview: Choosing the best secondary antibody and best secondary antibody conjugate is assay dependent. Much depends on the species that you are detecting along with the primary antibody that you have selected. This guide is designed to help you choose the best secondary antibody for your assay. 

What is a Secondary Antibody? Secondary Antibodies are antibodies that designed to detect other antibodies. Using a secondary antibody creates a signal enhancement for your assay which leads to greater sensitivity. Typically, an immunoglobulin (IgG/IgA/or IgM), called from one species is injected (immunized) into another animal species. The animal being immunized creates antibodies directed against the immunogen. For example, if Mouse IgG is injected into a Goat. The Goat has an immune response and makes anti-Mouse IgG’s. The secondary antibody is called Goat anti-Mouse IgG.  This secondary antibody can be used to detect Mouse host primary antibodies, like monoclonal antibodies. Commonly used secondary antibodies are Anti-Mouse, Anti-Rabbit, Anti-Rat, Anti-Hamster, and Anti-Guinea Pig.

7 Questions To Help You Choose The Best Secondary Antibody

1. What is the host species of the primary antibody?

You need a secondary antibody that reacts with antibodies from the host species of the primary antibody. For example: If you use a primary antibody raised in rabbits, you will need an anti-rabbit secondary antibody. If the primary antibody is raised in mice (most monoclonal primary antibodies), you will need an anti-mouse secondary antibody.

2. What host species do I need for the secondary antibody?

Secondary antibodies are made in many different species. Common hosts are Goats, Rabbits, Donkeys, and Chickens.  ***Choose a secondary host species that is different than primary antibody host. For example: If you use a Rabbit host primary antibody, you will want an anti-rabbit secondary that is not raised in rabbits. A good choice would be Goat/Donkey/or Chicken anti-Rabbit.  

3. What do I need to know about the isotype of the primary antibody?

The secondary antibody has to be directed against the class/isotype of the primary antibody. Primary antibodies that are polyclonal are generally raised in rabbits, goats, sheep or donkeys. They are generally an IgG isotype. The secondary antibody will typically be an anti-IgG H&L antibody. Check the certificate of analysis for the isotype.

Monoclonal primary antibodies are commonly raised in mice, occasionally in rabbits and rats. For example, if the primary monoclonal antibody is a mouse IgG1 subclass, you will need an anti-mouse IgG. You can use an antibody that recognizes all subclasses (H&L) or an IgG1 specific.

• Immunoglobulins: Classes, Subclasses, and Types:  
• Classes (aka isotypes): IgG (γ heavy chain), IgM (μ), IgA (α), IgE (ε), IgD (δ)
• Subclasses: IgG1 (γ1 heavy chains), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2)
• Types: κ light chain, λ light chain

4. Do I need a pre-absorbed secondary antibody?

Pre-absorbed antibodies are antibodies that have been processed to remove cross-reactivity to select animal species. It is recommended to use a secondary antibody that is pre-adsorbed to the species that you are detecting. For example, if you are studying human tissue samples, you want to choose a secondary antibody that won’t recognize human proteins. This could cause false positive results or high backgrounds.

Choose pre-absorbed antibodies when western blotting tissues and cells that also have high levels of immunoglobulins. Pre-adsorbed secondary antibodies are less likely to interact with endogenous immunoglobulins and which may reduce non-specific background.

If you are use two different secondary antibodies in the same assay (multiple labeling) you want to ensure that neither secondary antibody recognizes each other.

5. Should I use an affinity purified antibody or an IgG fraction?

The advantage of using affinity purified antibodies or IgG fractions will depend on the type of binding expected. Affinity purified antibodies give the lowest amount of non-specific binding whereas IgG fractions contain high affinity antibodies. In general, Ig Fractions can be used in turbidimetric assays. Most other immunoassays should use affinity purified antibodies. If you are uncertain choose a secondary antibody that has been affinity purified.  

6. Do I need a F(ab')2 fragment antibody?

F(ab')2 fragment antibodies are antibodies that have the Fc portion enzymatically removed. One advantage of F(ab)’2 fragments is that they eliminate non-specific binding between Fc portions of antibodies and Fc receptors on certain cells (i.e., macrophages, dendritic cells, neutrophils, NK cells and B cells). Another advantage is to penetrate tissues more efficiently due to their smaller size in IHC applications. Lastly, they do not interfere with anti-Fc mediated antibody detection. They are more costly so, you might want to choose a whole molecule antibody prior to choosing a F(ab)’2 fragment.

7. What conjugate do I need? Enzymatic or Fluorescent Detection?

The type of conjugation is also assay dependent. For enzymatic and biotin detection, e.g. in Western Blotting or ELISA, we suggest a secondary antibody conjugated to Horseradish Peroxidase, Alkaline Phosphatase, or Biotin.  Using a Biotin conjugated secondary antibody in conjunction with a labeled streptavidin (Streptavidin-Alkaline Phosphatase or Streptavidin-HRP) can produce a 4x signal amplification. If a laser light is involved, such as Flow Cytometry, ICC/IF or IHC, we suggest a fluorescently labeled secondary antibody (i.e., DyLight, FITC, TRITC, PE, etc.).

ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina