Secondary Antibody Cross-Adsorption and Cross Reactivity

• Why consider cross-adsorption when choosing a secondary antibody?
• What is cross-adsorption of secondary antibodies?
• How can cross-adsorption help my experiment?
• What should be considered before using a cross-adsorbed antibody?
• What are the limitations of cross-adsorption?

Are you performing these types of experiments?

• Multiple labeling experiments
• Working with multiple primary antibodies

Are you experiencing these types of issues?

• High background
• Non-specific binding
• Multiplexing cross-reactivity with other primary and secondary antibodies

If you answered yes to either of those questions, then you should consider your secondary antibody’s level of cross-adsorption. 

Let’s start by defining two important terms involved with secondary antibodies:

Cross-adsorption:  an optional purification process that filters out members that bind to off-target species of immunoglobulins (IgG). The process decreases species cross-reactivity and increases specificity.

Cross-reactivity:  when a secondary antibody binds to an unintended IgG (i.e., endogenous antibodies in the sample or one of the multiplexing primary antibodies used in a previous step), leading to high background and non-specific binding. 

While these two terms are related, they don’t mean the same thing. Cross-adsorption of a secondary antibody can prevent cross-reactivity in your experiments. So, if you are performing any of the above experiments or are experiencing any of those issues, a cross-adsorbed antibody might be for you.

Invitrogen secondary antibodies are available in highly cross-adsorbed and cross-adsorbed varieties. The difference between the two is the number of species that the antibody has been cross-adsorbed against. 

For example, consider the Invitrogen Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488. This goat anti-mouse IgG antibody has been cross-adsorbed against bovine IgG, goat IgG, rabbit IgG, rat IgG, and human IgG. Being cross-adsorbed against these species means that the antibody will not cross-react with those species—whether from a primary antibody or endogenous IgG. 

Therefore, if you are performing a multiplexing experiment with two different primary antibodies you could pick the cross-adsorbed secondaries that would give you the least chance for cross-reactivity.  For instance, if you have two primary antibodies—one is a mouse primary and the other is a human primary—the goat-anti-mouse highly cross-adsorbed secondary mentioned above won’t cross-react with your human primary antibody because the secondary has been cross-adsorbed against human IgG.

Advantages:

• Useful in multiplexing experiments where there is potential for cross-reactivity with other primary antibodies and secondary antibodies.
• Minimize non-specific binding and high background.
• Useful in IHC experiments with samples with endogenous immunoglobulins. A secondary antibody cross-adsorbed against the same species as the sample will reduce the chance of cross-reactivity.

Considerations:

• Potential decreased sensitivity in certain applications. When a secondary antibody is cross-adsorbed the polyclonal pool is filtered and unwanted immunoglobulins are removed. This leads to better specificity for the intended target, but less sensitivity because fewer targets are available.

Cross-adsorption does have its limits. Sometimes you will still have cross-reactivity issues even when using a cross-adsorbed antibody. Those limitations include:

• Primary antibody binding—if the primary antibody does not bind to your target of interest, no matter the amount of cross-adsorption, your secondary antibody will not provide accurate results.
• Equipment/filters—ensure that the equipment and filters you are using to visualize your experiment work with your antibodies.
• All immunoglobulins have the same light chain—no matter how many species a secondary antibody has been cross-adsorbed against all immunoglobulins have the same light chain that could still cause cross-reactivity issues.