| Autor: |
Visnovitz T; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Osteikoetxea X; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Sódar BW; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Mihály J; Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary., Lőrincz P; Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary., Vukman KV; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Tóth EÁ; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Koncz A; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary., Székács I; Nanobiosensorics Laboratory MTA-EK-MFA, Budapest, Hungary., Horváth R; Nanobiosensorics Laboratory MTA-EK-MFA, Budapest, Hungary., Varga Z; Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary., Buzás EI; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.; MTA-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary. |
| Abstrakt: |
The field of extracellular vesicles (EVs) is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of EV samples have not been completely solved. Currently, EV samples are standardised on the basis of their protein content sometimes combined with determination of the particle number. However, even this combined approach may result in inaccuracy and overestimation of the EV concentration. Lipid bilayers are indispensable components of EVs. Therefore, a lipid-based quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the EV field. In this study, we set the goal to improve the previously reported sulfo-phospho-vanillin (SPV) lipid assay. We introduced an aqueous phase liposome standard (DOPC) to replace the purified lipid standards in organic solvents (used commonly in previous studies). Furthermore, we optimised the concentration of the vanillin reagent in the assay. We found that elimination of organic solvents from the reaction mixture could abolish the background colour that interfered with the assay. Comparison of the optimised assay with a commercial lipid kit (based on the original SPV lipid assay) showed an increase of sensitivity by approximately one order of magnitude. Thus, here we report a quick, reliable and sensitive test that may fill an existing gap in EV standardisation. When using the optimised lipid assay reported here, EV lipid measurements can be more reliable than protein-based measurements. Furthermore, this novel assay is almost as sensitive and as easy as measuring proteins with a simple BCA test. |
