Multiplexed immunodetection has traditionally involved using fluorophore-labeled secondary antibodies to visualize primary antibody binding to target biomolecules. But as multiplexed panels have grown larger, secondary antibody cross-reactivities have made panel design increasingly difficult. In this article, we explain how labeled primary antibodies overcome the problem of secondary antibody cross-reactivity and highlight some additional advantages of using them for scientific research.
Secondary antibody cross-reactivity refers to the non-specific binding of labeled secondary antibody reagents to biomolecules that are not antibodies of the target species or isotype. These include other primary and secondary antibodies being used in the same experiment, whereby off-target binding can easily be misinterpreted as a positive result.
Directly labeled primary antibodies overcome the problem of secondary antibody cross-reactivity by removing the need for secondary reagents. Provided primary antibodies are chosen that have been proven to have high specificity and selectivity for their target antigens, off-target binding can be avoided for greater confidence in immunoassay data.
Immunostaining techniques such as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry often employ indirect detection methods. First, target-specific primary antibodies are used to recognize and bind the antigens of interest, then any unbound antibodies are washed away. Next, the bound primary antibodies are visualized using species-specific secondary antibodies labeled with detection moieties such as enzymes or fluorescent dyes.
Using labeled primary antibodies for direct detection streamlines immunostaining workflows by reducing the number of protocol steps and decreasing hands-on time. This provides faster time to results while also improving experimental reproducibility by removing one of the variables—the secondary antibody—from the process.
Designing a multiplexed immunostaining panel involves painstaking antibody selection. Not only must antibody cross-reactivity be prevented, but fluorophores must be carefully selected to ensure they are compatible with one another and with the platform that will be used to measure them.
Where secondary antibodies are used for detection, primary antibodies must be raised in different host species or be of different isotypes for them to be combined in the same experiment. This places significant constraints on panel size since only a finite number of host species and isotypes exist.
In contrast, directly labeled primary antibodies allow multiple antibodies from the same host species to be used together. As well as increasing the number of readouts that can be generated from precious sample material, this approach provides a more like-for-like comparison of target expression by enabling different biomolecules to be evaluated at the same time and under identical immunoassay conditions.
When selecting fluorophore-conjugated antibodies for research, it is recommended that brighter fluorophores be paired with less abundant targets (and vice versa), and that dyes with distinct emission profiles be chosen to avoid spectral overlap. Spectral analyzers and panel selection tools are widely available to streamline panel design.
Antibody selection for IHC presents unique challenges since tissue samples often contain endogenous immunoglobulins. These can give rise to false positive results when they are recognized by secondary antibody reagents, a problem that becomes especially apparent when using primary antibodies raised in the same host species as the sample material.
One way of tackling this issue is to include an additional blocking step in immunostaining protocols, such as using a mouse-on-mouse blocking reagent that enables anti-mouse secondaries to be used when studying mouse tissue. However, this adds an extra step into already lengthy workflows and introduces another potential source of experimental variability.
Directly labeled primary antibodies streamline species-on-species detection by removing the need for either a secondary antibody incubation step or additional blocking. Moreover, multiple primary antibodies from a shared host species can be used simultaneously for deeper insights into target expression.
In recent years, directly labeled primary antibodies have become more widely available, providing researchers with increased flexibility when multiplexing target detection. But because it would be impossible for antibody manufacturers to provide off-the-shelf versions of every antibody conjugated to every possible fluorophore, some companies offer a bespoke conjugation service.
A main advantage of customized conjugation is that it can be tailored to a project’s exacting needs rather than providing a ‘best-fit’ solution. It can also be more cost-effective where directly labeled primary antibodies are required at scale.
Biorbyt offers a broad portfolio of directly labeled primary antibodies, including antibodies conjugated to FITC and CF® dyes. These are complemented by an antibody conjugation service for even greater experimental flexibility. For more information visit biorbyt.com