TRIFic™ detection assay
The TRIFic™ exosome assay is similar to an ELISA assay, however, there are some significant differences. Unlike an ELISA assay, there is no enzymatic reaction. Rather, the target is detected directly with a Europium label. TRIFic™ exosome assays quickly provide quantitative data from purified or unpurified samples, including direct measurement of exosomes in plasma in a convenient 96-well format. TRIFic™ exosome assays are being made available for widely used markers of exosomes; the tetraspanin proteins CD9, CD63 and CD81. TRIFic™ exosome assays deliver clear, consistent data.
In the TRIFic™ exosome assay, the same antibody is used for binding of target to the assay plate and for detection. The assay consists of a monoclonal antibody (labeled with biotin) bound to a streptavidin coated plate which captures protein present on the surface of exosomes (Figure 1). Subsequently, an identical monoclonal antibody (labeled with Europium) is used for detection. Because the capture and detection antibody are identical, they require two linked copies of the same epitope for a signal to be detected. Exosomes provide an ideal structure to link CD9 molecules and allow detection of CD9 in this assay. Exosomes typically have multiple copies of CD9 facing towards the attachment surface and additional CD9 molecules available for detection. Any non specific binding of capture and detection antibodies is unlikely to generate a signal. Using a Europium fluorophore provides high levels of sensitivity for the assay, which is able to detect small changes in the abundance of the target CD9 protein even within unpurified complex biological samples, such as blood plasma and cerebrospinal fluid.
Schematic for TRIFic™ exosome assay.
- Biotinylated antibody is bound to streptavidin coated assay plates.
- Biological samples are added. Exosomes and any free antigen are captured by the antibody.
- Europium-labeled antibody is added and binds specifically to exosome antigen. The epitopes of bound monomers are already occupied and not detected. Samples are read on a time-resolved fluorescence plate reader.
- Simplicity of the assay results in a high degree of reproducibility.
- Europium is used for detection, providing a high degree of sensitivity.
- Only antigens displayed in multiple copies on a support within the sample (as with tetraspanins on exosomes) are detectable with the assay, resulting in increased specificity.