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Direct vs indirect antibody labeling for flow cytometry

What is meant by indirect detection?

When secondary antibodies are used as the detection reagent in an assay, the detection process is referred to as being indirect. This is because two different antibody reagents are required for visualization of the target of interest – an antigen-specific primary antibody, and an anti-species secondary antibody. The secondary antibody is typically conjugated to a detection moiety such as an enzyme or a fluorescent dye.

Indirect detection vs direct antibody labeling for flow cytometry

Comparison of indirect and direct cellular staining. An antibody against CD45RC was purchased in unconjugated form from a commercial supplier, and was conjugated using a Lightning-Link® R-PE antibody labeling kit according to the kit protocol. Cells were stained indirectly, using an R-PE-labeled secondary antibody (bottom left), and in parallel were stained directly with the labeled CD45RC antibody (bottom right). In the schematic images, CD45RC is illustrated in red, the anti-CD45RC antibody is shown in blue, and the R-PE-labeled secondary antibody is depicted in grey.

Why might indirect detection produce misleading assay results?

Although the flow cytometry data above shows similar levels of staining for both the indirect and direct methods, in many cases labeled secondary antibodies can be a source of unwanted background staining. This can occur if the secondary antibodies cross-react with endogenous immunoglobulins on the cells or with immunoglobulins in the antibody diluent, and can also result from cross-species reactivity with other labeled secondary antibodies during a multiplexing experiment.

How can the use of secondary antibodies be eliminated?

By using a directly-labeled primary antibody, secondary antibodies can be avoided altogether, however in many cases the directly conjugated primary antibody reagents required are unavailable commercially. Furthermore, methods of antibody labeling can be technically challenging and require specialist knowledge of chemical modification techniques. To overcome these issues and allow researchers to quickly and easily perform direct antibody labeling in-house, Expedeon offers Lightning-Link®.

Lightning-Link® for direct antibody labeling

Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups. The product range includes kits for labeling antibodies with almost 40 different fluorophores, covering the spectrum from UV to far infra-red, and is ideally suited for applications such as flow cytometry which rely on an immunofluorescent readout.

To produce the conjugate, the antibody is simply pipetted into a vial of lyophilized Lightning-Link® mixture containing the label of interest, incubated, and is then ready for use.

The benefits of Lightning-Link® include:

✓ Quick and easy to use

✓ Requires only 30 seconds hands-on time

✓ No separation steps involved so 100% of the antibody or protein is recovered

✓ Possibility to label from as little as 10ug to a gram or more of antibody

✓ Highly reliable technology – cited in well over 300 references to date, approximately one third of which detail the use of Lightning-Link for flow cytometry

To find out more about Lightning-Link®, why not watch our video?

Lightning-Link® for multi-color flow cytometry

To save on both time and precious sample material, many flow cytometry experiments are designed to detect multiple readouts at once. Although it is possible to use secondary antibodies to achieve this, by their very nature these reagents impose a limitation, since primary antibodies from the same host species cannot be detected simultaneously. Direct antibody labeling provides a simple solution to this problem; provided the panel of fluorophores is carefully selected to avoid spectral overlap, it is possible to produce an extremely sophisticated multiplexing experiment.

The power of Lightning-Link® for multi-color flow cytometry is clearly demonstrated in a publication by Robinson et al., who used eight different conjugates to undertake detailed analysis of oligodendroglial cell populations in murine brains.Why not visit our multi-color flow cytometry page to find out more?

The flow cytometry experiments which were performed by Robinson et al incorporated several Lightning-Link® tandem dyes. The inclusion of tandem dyes in a flow cytometry experiment is an effortless way of increasing the quantity of readouts, and is especially beneficial if the number of excitation lasers is a limiting factor.

Lightning-Link® tandem dye labeling kits
PerCP/Cy5.5
PE/Texas Red®
PE/Atto594
PE/Cy5
PE/Cy5.5
PE/Cy7
APC/Cy5.5
APC/Cy7

To aid you in the selection of suitable fluorophores for your assay, we have compiled the maximum absorption and emission wavelengths, Stokes shift and extinction co-efficient data for all our Lightning-Link® fluorescent labels in our downloadable fluorescence table.

For more information on Lightning-Link®, or any of our other products, please contact us.