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\begin{document}

\title{Tetraspanin immunoassay for the detection of extracellular vesicles and
renal cell carcinoma}



\author[1]{Misba Khan}%
\author[1]{Md. Khirul Islam}%
\author[1]{Mafiur Rahman}%
\author[2]{Bert Dhondt}%
\author[3]{Ileana  Quintero}%
\author[3]{Maija  Puhka}%
\author[1]{Panu M.  Jaakkola}%
\author[1]{Urpo Lamminmäki}%
\author[1]{Janne Leivo}%
\affil[1]{University of Turku}%
\affil[2]{Affiliation not available}%
\affil[3]{University of Helsinki}%


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  \date{May 23, 2024}


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\begin{abstract}
Half of patients with renal cell carcinoma (RCC) will develop
metastases. The disease is likely to be curable at early stages but
incurable when metastatic. New and non-invasive biomarkers are needed
for the diagnosis of RCC. Extracellular vesicles (EVs) are considered
promising new biomarker targets for the diagnosis of various diseases.
Our study aimed to develop an EV-based assay for the detection of RCC
using a highly sensitive nanoparticle-aided time-resolved fluorescence
immunoassay (NP-TRFIA). To confirm that the tetraspanins were located on
EVs, we used size exclusion chromatography to separate EV- and PE
(protein-enriched)-fractions from RCC4 and 786-O RCC cancer cell lines
and HEK293. EV- and PE-fractions were quantified using NP-TRFIA assays
established for tetraspanins CD9, CD63, CD81, and CD151. Tetraspanin
biomarkers were further measured from RCC cell culture medium as well as
serum samples of RCC (n=14), benign (n=17), and healthy (n=9)
individuals. Among the tetraspanins, CD63 showed 3-5-fold higher
expression on EVs derived from RCC4 and 786-O cell lines compared to
those from the HEK293. A sandwich CD63-CD63 assay demonstrated
significant discrimination of RCC patients from benign (p=0.0003), and
healthy (p= 0.005) individuals, respectively. Similarly, the CD81-CD81
assay also enabled significant separation of RCC patients compared to
benign (p=0.014), and healthy (p= 0.003) controls, respectively. This
result suggests that RCC cell lines and serum of RCC patients show
higher amounts of CD63- and CD81-enriched EVs compared to controls.
Detection of these tetraspanin-enriched EVs using our NP-TRFIA approach
may play a vital role in the detection of RCC.%
\end{abstract}%



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\verb`20112023_Khan_Manuscript.docx`  available at \url{https://authorea.com/users/702611/articles/688521-tetraspanin-immunoassay-for-the-detection-of-extracellular-vesicles-and-renal-cell-carcinoma}

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