By Ryan Hamnett, PhD
Cross-adsorbed secondary antibodies reduce background in multiplexed experiments by eliminating cross-reactivity between secondary antibodies and off-target proteins. They are engineered for greater specificity by undergoing an additional purification step, leaving only highly specific antibodies remaining that are ideal for multiplexed experiments where cross-reactivity is an issue.
Cross-reactivity of secondary antibodies occurs in immunoassays when a secondary antibody binds to off-target proteins, primary antibodies, or endogenous antibodies within a sample (Figure 1).
Immunoglobulins from different species or different isotypes share conserved sequences and similar quaternary structure, meaning cross-reactivity against other antibodies is particularly common. These homologous epitopes can be found between antibodies from any species, but they are most frequently seen between closely related species such as mouse and rat.
Figure 1: Cross-reactivity in indirect IHC. The middle secondary antibody exhibits cross-reactivity, recognizing and binding to both the middle and right-hand primary antibodies. When visualizing this tissue, the orange fluorophore will therefore reflect the localization of both the middle and right-hand antigens.
Cross-adsorbed secondary antibodies exhibit reduced cross-reactivity because they have undergone a purification process that filters out antibodies that bind to off-target serum proteins and immunoglobulins.
For more information on secondary antibodies, see our page on How to Choose a Secondary Antibody.
Cross-adsorption is the process of making cross-adsorbed antibodies. Cross-adsorption removes antibodies that exhibit cross-reactivity against proteins from selected species (Figure 2).
Figure 2: Cross-adsorbed antibody production
Antibodies are passed through a column matrix containing immobilized proteins that are potentially cross reactive. These can include serum proteins from other species and off-target immunoglobin isotypes that share conserved sequences or structure with the target antibody. Non-specific antibodies will remain bound to the column, while highly specific cross-adsorbed secondary antibodies flow through to be collected.
Cross-adsorbed antibodies have several advantages over standard secondary antibodies, including:
The most common application of cross-adsorbed secondaries is in multiplexed immunoassays. For instance, consider an IHC experiment in which the primary antibodies are raised in rabbit and mouse. A goat-anti rabbit secondary antibody may recognize and bind to the mouse primary as well as the rabbit primary antibody. However, if the goat-anti rabbit secondary antibody has been cross-adsorbed against mouse antibodies, then it will show greater specificity for the rabbit primary and exhibit almost no cross-reactivity.
This same logic applies to potential off-target binding of endogenous immunoglobulins in the sample itself, which can be an issue with certain tissues such as lymphoid tissues.
Applications of cross-adsorbed secondary antibodies include:
Cross-adsorbed secondary antibodies are an excellent solution to non-specific binding and high background, particularly in multiplexed experiments, but they may be sub-optimal for some situations.
Because antibodies against more common epitopes are removed during the cross-adsorption process, cross-adsorbed antibodies have the potential for decreased sensitivity because fewer epitope targets will be recognized by the resultant pool.
Cross-adsorbed antibodies will also not solve all problems relating to high background or non-specific binding. Primary antibodies can still exhibit off-target binding, so even with cross-adsorption, a secondary antibody is only as good as its primary antibody partner. Likewise, equipment such as excitation and emission filters on a fluorescence microscope should be set to ensure optimal signal capture and exclusion of signals from other channels.
If cross-adsorbed antibodies are being used to target specific antibody isotypes, it is possible that some cross-reactivity will remain even when using cross-adsorbed antibodies. This is because different antibody isotypes, which are determined by their heavy chains, all have common light chains: either kappa or lambda.
As with any scientific reagent, choosing the secondary antibody that is best suited to the aims and setup of your experiment will give the best results. Below are some tips for choosing the best cross-adsorbed antibody:
| Cross-Adsorbed Antibody | Host | Label | Cross-Adsorbed Against |
|---|---|---|---|
| Anti-Human IgG H&L Antibody | Dk | FITC | Bo, Ch, Gt, Ms, Rb, Rt, Sh |
| Anti-Human IgG H&L Antibody | Gt | FITC | Bo, Ch, Gt, Ms, Rb, Rt, Sh |
| Anti-Human IgG H&L Antibody | Rb | FITC | Ms, Rt |
| Anti-Mouse IgG H&L Antibody | Dk | FITC | Bo, Ch, Gt, Hu, Rb, Rt, Sh |
| Anti-Mouse IgG H&L Antibody | Gt | FITC | Bo, Ch, Hs, Hu, Pi, Rb, Rt |
| Anti-Mouse IgG H&L Antibody | Rb | FITC | Hu, Rt |
| Anti-Rabbit IgG H&L Antibody | Gt | Cy3 | Bo, Ch, Hs, Hu, Ms, Pi, Rt |
| Anti-Guinea Pig IgG H&L Antibody | Gt | Cy3 | Bo, Ch, Hs, Hu, Ms, Rb, Rt, Sh |
| Anti-Rat IgG H&L Antibody | Gt | Cy3 | Bo, Ch, Hu, Ms, Rb, Sh |
| F(ab')2 Anti-Mouse IgG Fc Antibody | Gt | PE | Hu, Rt |
| F(ab')2 Anti-Mouse IgG F(ab')2 Antibody | Gt | PE | Hu, Rt |
| Anti-Human IgG H&L Antibody | Gt | Cy5 | Bo, Ch, Hs, Ms, Pi, Rb, Rt |
| Anti-Mouse IgG H&L Antibody | Gt | Cy5 | Bo, Ch, Hs, Hu, Pi, Rb, Rt |
| Anti-Chicken IgY H&L Antibody | Gt | Cy5.5 | Bo, Hs, Hu, Ms, Pi, Rb, Rt |
| Anti-Goat IgG H&L Antibody | Dk | Cy5.5 | Ch, Hu, Ms, Pi, Rb, Rt |
| Cross-Adsorbed Antibody | Host | Label | Cross-Adsorbed Against |
|---|---|---|---|
| Anti-Rabbit IgG H&L Antibody | Gt | HRP | Bo, Ch, Hs, Hu, Ms, Pi, Rt |
| Anti-Rabbit IgM Antibody | Gt | HRP | Hu, Ms, Rt |
| Anti-Human IgG H&L Antibody | Gt | HRP | Bo, Ch, Hs, Ms, Pi, Rb, Rt |
| F(ab')2 Anti-Human IgG H&L Antibody | Dk | HRP | Bo, Ch, Gt, Ms, Rb, Rt, Sh |
| F(ab')2 Anti-Human IgG Fc Antibody | Gt | HRP | Ms, Rt |
| F(ab')2 Anti-Human IgG F(ab')2 Antibody | Gt | HRP | Ms, Rt |
| Anti-Rat IgG H&L Antibody | Gt | AP | Bo, Ch, Hu, Ms, Rb, Sh |
| Anti-Mouse IgG H&L Antibody | Gt | AP | Bo, Ch, Hs, Hu, Pi, Rb, Rt |
| Cross-Adsorbed Antibody | Host | Label | Cross-Adsorbed Against |
|---|---|---|---|
| Anti-Chicken IgY H&L Antibody | Gt | Biotin | Bo, Hs, Hu, Ms, Pi, Rb, Rt |
| Anti-Guinea Pig IgG H&L Antibody | Gt | Biotin | Bo, Ch, Hs, Hu, Ms, Rb, Rt, Sh |
| Anti-Human IgG H&L Antibody | Gt | Biotin | Bo, Ch, Hs, Ms, Pi, Rb, Rt |
| Anti-Mouse IgG H&L Antibody | Gt | Biotin | Bo, Ch, Hs, Hu, Pi, Rb, Rt |
| Anti-Rat IgG H&L Antibody | Gt | Biotin | Bo, Ch, Hu, Ms, Rb, Sh |
