Cross-adsorbed secondary antibodies are polyclonal antibodies that are manufactured with an additional purification step to filter out members that bind to off-target species of immunoglobulin (IgG). The process decreases species cross-reactivity and increases specificity. Depending on the experimental need there are cross-adsorbed and highly cross-adsorbed secondary antibody varieties. 

Read the following information to learn more about the definition of cross-adsorption, what the advantages of cross-adsorption are, and what limitations are involved when considering a cross-adsorbed antibody to improve your experimental results.



Why consider cross-adsorption when choosing a secondary antibody?

Are you performing these types of experiments?

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. 


What is cross-adsorption of secondary antibodies?

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. 


How can cross-adsorption help your experiment?

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.

Immunofluorescent analysis of glial fibrillary acidic protein (GFAP) in E18 Sparague Dawley primary cortical neuronal cells containing astrocytes. The cells were fixed with 4% formaldehyde for 15 min., permeabilized with 0.25% Triton X-100 in PBS for 10 min, and blocked with 3% BSA in PBS for 30 min at RT. Cells were stained with a GFAP mouse monoclonal antibody (Cat. No. MA5-12023) at a dilution of 1:200 in 3% BSA in PBS for 1 hr at RT, and then incubated with Invitrogen Alexa Fluor Plus 488 goat anti-mouse IgG secondary antibody (Cat. No. A32723) at a dilution of 1:1000 for 1 hr. at RT. Nuclei were stained with Hoechst 33342 (Cat. No. H3570). The image contains overlay of GFAP (green) and nuclei (blue). Images were taken on a Zeiss LSM 710 confocal microscope at 40X magnification.


What should be considered before using a cross-adsorbed antibody?

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.

What are the limitations of cross-adsorption?

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.