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Kidney Week

Abstract: SA-PO919

Development and Validation of an Enzyme-Linked Immunosorbent Assay (ELISA)-Based Method for Quantitative Evaluation of Urinary Extracellular Vesicles as Biomarkers for CKD

Session Information

Category: Pathology and Lab Medicine

  • 1800 Pathology and Lab Medicine

Authors

  • Nishimura, Tatsuya, Department of Pediatrics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  • Hirakawa, Yosuke, Division of Nephrology and Endocrinology, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  • Takizawa, Keiichi, Department of Pediatrics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  • Kajiho, Yuko, Department of Pediatrics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  • Kanda, Shoichiro, Department of Pediatrics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  • Harita, Yutaka, Department of Pediatrics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Background

Urinary extracellular vesicles (uEVs) are emerging as promising biomarkers for various diseases. Our previous work highlighted the potential of MUC1 expression levels on uEVs as an indicator for chronic kidney disease (CKD) in children (iScience 2022). We have developed an ELISA-based method for the quantitative assessment of uEVs (STAR Protocols, 2023; ASN 2023). Despite the existence of several techniques for uEVs purification on ELISA plates, their optimization remains incomplete. Furthermore, storage and processing of urine samples suitable for uEVs-ELISA have not been established.

Methods

We generated monoclonal antibodies against MUC1 and evaluated their performance across two uEVs purification methods (Tim4 and CD9 antibodies) on ELISA plates. Selecting the optimal antibody and plate combination, we analyzed urine samples from 160 adult patients (83 males) with varying kidney functions. The median age was 50 years, and the median estimated glomerular filtration rate (eGFR), calculated using serum creatinine, was 57.6 ml/min/1.73m^2.

Results

Among the developed monoclonal antibodies, the most sensitive one, in conjunction with both Tim4 and CD9 antibodies for uEVs purification on ELISA plates, effectively captured variations in MUC1 levels in uEVs. The CD9 plate method more accurately mirrored clinical outcomes. The area under the curve (AUC) values from receiver operating characteristic (ROC) curve analysis for predicting the presence of eGFR <60 or <45, standardized with urine creatinine value, were 0.912 and 0.928, respectively. This predictive accuracy was consistent across gender-specific analyses. This method has been found to provide stable results when storing urine at 4°C for up to a week and over a long period when stored at -80°C.

Conclusion

Given the stability of urine for uEVs-ELISA analysis, this system offers a non-invasive approach, demonstrating significant potential as a clinical biomarker for adult CKD patients as well as children.