Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration

Membranes

Teresa Janas¹  Karolina Sapoń¹  Dominika Maziarz¹  Tadeusz Janas¹  AleksanderF. Sikorski² 

[1] Institute of Biology, University of Opole, Kominka 6, 45-032 Opole, Poland;Research and Development Center, Regional Specialist Hospital, 51-124 Wroclaw, Poland;

关键词: cholera toxin subunit B;    cholesterol;    exosomes;    gel chromatography;    GM1 ganglioside;    liposomes;   

DOI  :  10.3390/membranes10080172

来源: DOAJ

【 摘 要 】

We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 10⁸ isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 10⁸ to 6 × 10⁹ exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r² = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.

【 授权许可】

Unknown