TRIFic™ detection assay

Code Description Price Qty
EX101 TRIFic™ CD9 Exosome Assay, 96 wells £475.00
EX102 TRIFic™ CD63 Exosome Assay, 96 wells £475.00
EX103 TRIFic™ CD81 Exosome Assay, 96 wells £475.00
EX-P31 Wash Buffer (Concentrate 25x), 20 ml £45.00
TRIFic™ kit

Product description

The TRIFic™ exosome detection assay delivers clear and consistent data quickly by providing quantitative data from purified or unpurified samples, including direct measurement of exosomes in plasma in a convenient 96-well format. It is similar to an ELISA assay, but the target is detected directly with a Europium label without any enzymatic reaction. TRIFic™ exosome detection assays are being made available for widely used exosome markers, allowing for identification of CD9+/CD81+/CD63+ - EVs.

 

Assay principle

Exosomes typically have multiple copies of tetraspanin facing towards the attachment surface and additional marker molecules available for detection. Based on this property, 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 (see below). Subsequently, an identical monoclonal antibody (labeled with europium) is used for detection.

Exosomes provide an ideal structure to link CD9, CD63 or CD81 molecules and allow detection of the tetraspanin of interest. 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 tetraspanin protein even within unpurified complex biological samples, such as blood plasma and cerebrospinal fluid (CSF).

Protocol Overview

 

 

 

Applications

TRIFic™ assays are time-resolved immunofluorescence assays which utilize europium and have been developed to measure the abundance of CD9, CD63, or CD81 protein specifically associated with exosomes in biological fluids including urine, saliva, cell culture medium, cerebrospinal fluid (CSF), and blood plasma. The TRIFic™ detection assay is ideal for detection of specific markers of exosomes, for purified or unpurified samples.

Why Europium?

Europium is a fluorescent chelate which exhibits a large Stokes shift (— 290 nm) with no overlap between excitation and emission spectra and a very narrow (10 nm bandwidth) emission spectra at 615 nm. Additionally, their long fluorescence lifetimes (600-1000 ns is for Eu3+ compared with 5-100 ns for conventional fluorophores) allows use of microsecond time-resolved fluorescence (TRF) measurements, which reduce the observed background signals. The principle of the time-resolved fluorometric measurement is as follows: When a mixture of fluorescent compounds is excited with a short pulse of light, the excited molecules emit either short-or long-lived fluorescence. Although both types of fluorescence decay follow an exponential curve, short-lived fluorescence dissipates to zero in < 100 µs. If no measurements are taken during the first 100-200 is after excitation, all short-lived fluorescence background signals and scattered excitation radiation are completely eliminated, and the long-lived fluorescence signals can be measured with very high sensitivity. In practice, the only background signal observed when using europium chelate labels is that produced the nonspecific binding of the labelled reagents used. Abridged from Diamandis and Christopulous

 

 

TRIFic™ Assay Attributes

Simplicity

Sensitivity

Specificity

The assay is simple and reproducible

The assay uses europium labelling which generates time-resolved fluorescence (TRF) in which emission is delayed following excitation which eliminates signal from the excitation source. The Stokes shift is large and the efficiency of fluorescence is high. Together, these factors provide maximum sensitivity

By using the same antigen for both capture and detection, only antigens displayed in multiple copies from discrete structures are detected. This provides specificity for exosomes.

 

 

Product data

 

 

 

 

 

 

 

 
 

References

Agnieszka Kaminska, Mark Platt, Joanna Kasprzyk, Beata KuVnierz-Cabala, Agnieszka Gala-Budzinska, Olga Wofnicka, Benedykt R. Jany, Franciszek Krok, Wojciech Piekoszewski, Marek Kufniewski, et al. (2016). Urinary Extracellular Vesicles: Potential Biomarkers of Renal Function in Diabetic Patients. Journal of Diabetes Research Volume 2016, Article ID 5741518.