Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™
Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein. The soluble form of PD-L1 (sPD-L1) and PD-L1 derived from small extracellular vesicles (sEVPD-L1) are promising cancer biomarkers. While sEVPD-L1 in particular may contribute to immune evasion and is associated with a poor prognosis,...
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MDPI AG
2025-07-01
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| Series: | Current Issues in Molecular Biology |
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| author | Kyo Okita Hasumi Arita Keita Sudo Teruki Yoshimura Etsuro Ito |
| author_facet | Kyo Okita Hasumi Arita Keita Sudo Teruki Yoshimura Etsuro Ito |
| author_sort | Kyo Okita |
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| description | Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein. The soluble form of PD-L1 (sPD-L1) and PD-L1 derived from small extracellular vesicles (sEVPD-L1) are promising cancer biomarkers. While sEVPD-L1 in particular may contribute to immune evasion and is associated with a poor prognosis, it exists only in trace amounts, making it difficult to detect using conventional enzyme-linked immunosorbent assay (ELISA) methods. Therefore, we developed an ultrasensitive detection method, TN-cyclon™. The TN-cyclon™ method combines sandwich ELISA with enzyme cycling amplification. We applied TN-cyclon™ to measure recombinant PD-L1 protein and sEVPD-L1 in serum samples from cancer patients and healthy donors. Recombinant PD-L1 protein was measured with an ultrasensitive detection limit of 0.172 pg/mL. In clinical specimens, sEVPD-L1 levels were significantly higher in lung cancer patients than in healthy donors, whereas sPD-L1 levels measured with a conventional ELISA did not differ significantly between groups. Our results demonstrated that the TN-cyclon™ method exhibits a 20-fold increase in sensitivity compared to a conventional ELISA. Although this is a pilot study, our new assay enables the detection of very low concentrations of sEVPD-L1 in serum that can be used to evaluate the predictive and prognostic performance of sEVPD-L1 in lung cancer patients in future studies. |
| format | Article |
| id | doaj-art-87d94d090dfe40c0bdf01143a9b5a692 |
| institution | Kabale University |
| issn | 1467-3037 1467-3045 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | MDPI AG |
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| series | Current Issues in Molecular Biology |
| spelling | doaj-art-87d94d090dfe40c0bdf01143a9b5a6922025-08-20T03:58:31ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452025-07-0147756410.3390/cimb47070564Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™Kyo Okita0Hasumi Arita1Keita Sudo2Teruki Yoshimura3Etsuro Ito4Department of Biology, TWIns, Waseda University, Shinjuku, Tokyo 162-0056, JapanDepartment of Biology, TWIns, Waseda University, Shinjuku, Tokyo 162-0056, JapanDepartment of Biology, TWIns, Waseda University, Shinjuku, Tokyo 162-0056, JapanR&D Department, BioPhenoMA Inc., Waseda University Entrepreneurship Center, Shinjuku, Tokyo 169-0051, JapanDepartment of Biology, TWIns, Waseda University, Shinjuku, Tokyo 162-0056, JapanProgrammed death-ligand 1 (PD-L1) is an immune checkpoint protein. The soluble form of PD-L1 (sPD-L1) and PD-L1 derived from small extracellular vesicles (sEVPD-L1) are promising cancer biomarkers. While sEVPD-L1 in particular may contribute to immune evasion and is associated with a poor prognosis, it exists only in trace amounts, making it difficult to detect using conventional enzyme-linked immunosorbent assay (ELISA) methods. Therefore, we developed an ultrasensitive detection method, TN-cyclon™. The TN-cyclon™ method combines sandwich ELISA with enzyme cycling amplification. We applied TN-cyclon™ to measure recombinant PD-L1 protein and sEVPD-L1 in serum samples from cancer patients and healthy donors. Recombinant PD-L1 protein was measured with an ultrasensitive detection limit of 0.172 pg/mL. In clinical specimens, sEVPD-L1 levels were significantly higher in lung cancer patients than in healthy donors, whereas sPD-L1 levels measured with a conventional ELISA did not differ significantly between groups. Our results demonstrated that the TN-cyclon™ method exhibits a 20-fold increase in sensitivity compared to a conventional ELISA. Although this is a pilot study, our new assay enables the detection of very low concentrations of sEVPD-L1 in serum that can be used to evaluate the predictive and prognostic performance of sEVPD-L1 in lung cancer patients in future studies.https://www.mdpi.com/1467-3045/47/7/564immune checkpointlung cancerPD-L1small extracellular vesicleultrasensitive ELISA |
| spellingShingle | Kyo Okita Hasumi Arita Keita Sudo Teruki Yoshimura Etsuro Ito Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ Current Issues in Molecular Biology immune checkpoint lung cancer PD-L1 small extracellular vesicle ultrasensitive ELISA |
| title | Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ |
| title_full | Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ |
| title_fullStr | Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ |
| title_full_unstemmed | Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ |
| title_short | Establishment of an Assay with Ultrahigh Sensitivity for Detecting sEV-Derived PD-L1 as a Serum Biomarker for Lung Cancer—A Pilot Study Using TN-cyclon™ |
| title_sort | establishment of an assay with ultrahigh sensitivity for detecting sev derived pd l1 as a serum biomarker for lung cancer a pilot study using tn cyclon™ |
| topic | immune checkpoint lung cancer PD-L1 small extracellular vesicle ultrasensitive ELISA |
| url | https://www.mdpi.com/1467-3045/47/7/564 |
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