anti human msln  (R&D Systems)

Journal: eBioMedicine

Article Title: Tumour extracellular vesicle surface Protein-mRNA integration assay for early detection of epithelial ovarian cancer

doi: 10.1016/j.ebiom.2025.105884

Figure Lengend Snippet: Schematic illustration of the Epithelial Ovarian Cancer (EOC) Extracellular Vesicle (EV) Surface Protein-mRNA Integration (SPRI) Assay for early detection of EOC . (a) The EOC EV Surface Protein-mRNA Integration (SPRI) Assay was carried out via a two-step workflow––Step 1: Click chemistry-mediated enrichment of EOC EVs by EV Click Beads, and Step 2: Quantification of enriched EOC EVs by detecting the two reference mRNAs through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Three trans-cyclooctene (TCO)-grafted EOC-specific antibodies, i.e., TCO-anti-folate receptor alpha (FRα), TCO-anti-mesothelin (MSLN), and TCO-anti-trophoblast cell-surface antigen 2 (TROP2) were prepared to enable click chemistry-mediated enrichment of EOC EV subpopulations. Each EOC EV subpopulation was then subjected to RT-qPCR quantification of the two reference mRNAs, i.e., MPP1 and ACTB . The signals from MPP1 and ACTB reflect the amount of EOC EVs. (b) Clinical study design flowchart. First, plasma samples from eligible participants were collected from a training cohort comprising 39 patients with EOC, 4 with borderline ovarian tumour, 30 with benign ovarian masses (BOM), and 45 healthy donors (HD). All samples were subjected to EOC EV SPRI Assay to quantify the six subpopulations of EOC EVs (i.e., FRα + EOC EVs- MPP1 , MSLN + EOC EVs- MPP1 , TROP2 + EOC EVs- MPP1 , FRα + EOC EVs- ACTB , MSLN + EOC EVs- ACTB , and TROP2 + EOC EVs- ACTB ). Subsequently, the EOC EV SPRI Score was generated by logistic regression analysis and cross-validated by leave-one-out cross validation. Finally, the diagnostic performance of EOC EV SPRI Score was validated in an independent validation cohort comprising 42 EOC, 31 BOM, and 45 HD.

Article Snippet: Following blocking with 2% donkey serum (Jackson ImmunoResearch Cat# 017-000-001) for 30 min, cells were incubated overnight at 4 °C with the following primary antibodies: anti-human FRα (R & D Systems Cat# MAB5646, RRID: AB_2278620 ), anti-human MSLN (R & D Systems Cat# MAB32652, RRID: AB_2147798 ), or anti-human TROP2 (R & D Systems Cat# AF650, RRID: AB_2205667 ).

Techniques: Biomarker Discovery, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Clinical Proteomics, Generated, Diagnostic Assay

Journal: eBioMedicine

Article Title: Tumour extracellular vesicle surface Protein-mRNA integration assay for early detection of epithelial ovarian cancer

doi: 10.1016/j.ebiom.2025.105884

Figure Lengend Snippet: Validation of the three EOC EV surface protein markers using EOC cell lines and EOC tissue microarray (TMA) . (a) Representative IF micrographs illustrating expression of FRα, MSLN, and TROP2 in OVCAR3 and OVCAR8 EOC cell lines. Blue: DAPI; red: Cy5; green: FITC. Scale bar, 10 μm. (b) Representative haematoxylin and eosin (H&E) and immunohistochemistry (IHC) images displaying expression of the three EOC EV surface protein markers on EOC TMA slides. Scale bar, 200 μm. (c) Heatmap summarising IHC staining intensity and positive percentages for each marker across EOC TMA. Pie charts depicting the percentage of (d) all-stage EOC samples and ( e ) earlier-stage EOC samples (stage I-II) categorised by IHC staining intensity of strong (3+), moderate (2+), weak (1+), and negative (0) for the three EOC EV surface protein. Bar charts summarising the percentage of IHC positive staining for each surface protein marker and their combinations in (d) all-stage EOC samples and ( e ) earlier-stage EOC samples.

Article Snippet: Following blocking with 2% donkey serum (Jackson ImmunoResearch Cat# 017-000-001) for 30 min, cells were incubated overnight at 4 °C with the following primary antibodies: anti-human FRα (R & D Systems Cat# MAB5646, RRID: AB_2278620 ), anti-human MSLN (R & D Systems Cat# MAB32652, RRID: AB_2147798 ), or anti-human TROP2 (R & D Systems Cat# AF650, RRID: AB_2205667 ).

Techniques: Biomarker Discovery, Microarray, Expressing, Immunohistochemistry, Marker, Staining

Journal: eBioMedicine

Article Title: Tumour extracellular vesicle surface Protein-mRNA integration assay for early detection of epithelial ovarian cancer

doi: 10.1016/j.ebiom.2025.105884

Figure Lengend Snippet: Characterisation of EOC cell line-derived EVs and linearity study of EOC EV SPRI Assay using synthetic plasma samples . (a) Representative transmission electron microscopy (TEM) images of click chemistry mediated immobilisation of TCO-anti-FRα, TCO-anti-MSLN, and TCO-anti-TROP2 labelled OVCAR3 EVs, visualised by immunogold staining with anti-CD63-grafted gold nanoparticles (gold arrows). Scale bar, 100 nm. (b) Linearity study showing correlation between EV concentration and reference mRNA (i.e., MPP1 and ACTB ) expression in OVCAR8 and OVCAR3 EVs. Two replicates were examined for each group. Data are presented as mean ± SD. R 2 was calculated using simple linear regression. (c) Workflow for linearity study of the EOC EV SPRI Assay using synthetic plasma samples spiked with OVCAR3 EVs. The EVs were enriched using EV Click Beads in conjunction with each of the three TCO-grafted antibodies, i.e., TCO-anti-FRα, TCO-anti-MSLN, or TCO-anti-TROP2. RT-qPCR detection of the ACTB mRNA served as a surrogate for enriched OVCAR3 EV concentrations (d–i) Dynamic linearity ranges of ACTB signal for the EOC EV subpopulations enriched by TCO-grafted EOC EV-specific antibodies. Two replicates were examined for each group. Data are presented as mean ± SD. R 2 was calculated using simple linear regression.

Article Snippet: Following blocking with 2% donkey serum (Jackson ImmunoResearch Cat# 017-000-001) for 30 min, cells were incubated overnight at 4 °C with the following primary antibodies: anti-human FRα (R & D Systems Cat# MAB5646, RRID: AB_2278620 ), anti-human MSLN (R & D Systems Cat# MAB32652, RRID: AB_2147798 ), or anti-human TROP2 (R & D Systems Cat# AF650, RRID: AB_2205667 ).

Techniques: Derivative Assay, Clinical Proteomics, Transmission Assay, Electron Microscopy, Staining, Concentration Assay, Expressing, Quantitative RT-PCR

Journal: eBioMedicine

Article Title: Tumour extracellular vesicle surface Protein-mRNA integration assay for early detection of epithelial ovarian cancer

doi: 10.1016/j.ebiom.2025.105884

Figure Lengend Snippet: EOC EV SPRI Score for detecting EOC in the training cohort . (a) Workflow for the EOC EV SPRI Assay applied to plasma samples from the training cohort, which included 39 patients with EOC, 4 with borderline ovarian tumours, 30 with benign ovarian masses (BOM), and 45 healthy donors (HD). (b) Heatmaps summarising six subpopulations of EOC EVs (i.e., FRα + EOC EVs- MPP1 , MSLN + EOC EVs- MPP1 , TROP2 + EOC EVs- MPP1 , FRα + EOC EVs- ACTB , MSLN + EOC EVs- ACTB , and TROP2 + EOC EVs- ACTB ) in the training cohort. (c – h) Significantly higher signals in patients with EOC compared to the combined BOM and HD control group in the training cohort were observed in the subpopulations of FRα + EOC EVs- MPP1 , MSLN + EOC EVs- MPP1 , TROP2 + EOC EVs- MPP1 , FRα + EOC EVs- ACTB , MSLN + EOC EVs- ACTB , and TROP2 + EOC EVs- ACTB . Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 (unpaired Student's t-tests). (i) The EOC EV SPRI Score was generated using a stepwise logistic regression. Significantly higher EOC EV SPRI Scores were observed in patients with EOC compared to the combined BOM and HD controls in the training cohort, with an optimal cutoff of −0.61. Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 (unpaired Student's t-test). (j) Receiver operating characteristic (ROC) curve illustrating the diagnostic performance of EOC EV SPRI Score in distinguishing EOC from the combined BOM and HD control group in the training cohort. (k) ROC curve showing the performance of EOC EV SPRI Score after leave-one-out cross-validation (LOOCV).

Article Snippet: Following blocking with 2% donkey serum (Jackson ImmunoResearch Cat# 017-000-001) for 30 min, cells were incubated overnight at 4 °C with the following primary antibodies: anti-human FRα (R & D Systems Cat# MAB5646, RRID: AB_2278620 ), anti-human MSLN (R & D Systems Cat# MAB32652, RRID: AB_2147798 ), or anti-human TROP2 (R & D Systems Cat# AF650, RRID: AB_2205667 ).

Techniques: Clinical Proteomics, Control, Generated, Diagnostic Assay, Biomarker Discovery

Journal: eBioMedicine

Article Title: Tumour extracellular vesicle surface Protein-mRNA integration assay for early detection of epithelial ovarian cancer

doi: 10.1016/j.ebiom.2025.105884

Figure Lengend Snippet: EOC EV SPRI Score for distinguishing EOC from BOM and HD controls in the validation cohort . (a) Heatmaps summarising MPP1 mRNA signals across FRα + EOC EVs, MSLN + EOC EVs, and TROP2 + EOC EVs. (b–d) Significantly higher MPP1 mRNA signals in the subpopulations of EOC EVs were observed in patients with EOC compared to the combined BOM and HD control group. The signal is represented as 40—Ct value. Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 (unpaired Student's t-tests). (e) Boxplot illustrating EOC EV SPRI Scores in EOC versus BOM and HD controls at a cutoff of −0.61. Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 (unpaired Student's t-test). (f) ROC curve showing the diagnostic performance of the EOC EV SPRI Score in distinguishing EOC from BOM and HD.

Article Snippet: Following blocking with 2% donkey serum (Jackson ImmunoResearch Cat# 017-000-001) for 30 min, cells were incubated overnight at 4 °C with the following primary antibodies: anti-human FRα (R & D Systems Cat# MAB5646, RRID: AB_2278620 ), anti-human MSLN (R & D Systems Cat# MAB32652, RRID: AB_2147798 ), or anti-human TROP2 (R & D Systems Cat# AF650, RRID: AB_2205667 ).

Techniques: Biomarker Discovery, Control, Diagnostic Assay