Secondary Antibody Cross-Adsorption and Cross Reactivity
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.
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.
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.
- 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.
- 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.
For Research Use Only. Not for use in diagnostic procedures.