Validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of endostatin levels in mice as a biomarker of developing glomerulonephritis

PLoS One. 2019 Aug 12;14(8):e0220935. doi: 10.1371/journal.pone.0220935. eCollection 2019.


Endostatin, the C-terminal fragment of type XVIII collagen, was shown to be one of the most potent endothelial cell-specific inhibitors of angiogenesis. As altered circulating endostatin concentration is associated with impaired kidney function, new tools for measuring endostatin in rodents may be helpful to further investigate and understand its role within kidney disease progression. A novel and commercially available ELISA for the quantification of mouse and rat endostatin was developed and validated according to international quality guidelines including the parameters specificity, robustness, accuracy, dilution linearity, precision, limit of detection (LOD) and lower limit of quantification (LLOQ). Endostatin and blood urea nitrogen (BUN) concentration were measured in mice with a glomerulonephritis phenotype. The validation revealed that within the range of 0.5-32 nmol/L the immunoassay is robust and highly specific for the measurement of rodent endostatin with high sensitivity (LOD 0.24 nmol/L, LLOQ 0.5 nmol/L) and good reproducibility (intra- and inter-assay CV <10%). Also accuracy and dilution linearity were within the range of acceptance. BCL2 transgenic and ETV6/RUNX1;BCL2 double transgenic mice develop a glomerulonephritis phenotype over time, which was displayed by staining of kidney sections. Even before full manifestation of disease serum endostatin concentration rises significantly, whereas BUN levels just slightly increase. This newly developed and commercially available ELISA provides a reliable and accurate tool for the quantification of mouse and rat endostatin and may give new perspectives in the investigation of the role of endostatin as an important and early biomarker for reduced kidney function. Measurement of endostatin concentration is recommended to be used as a superior biomarker for chronic kidney disease compared to BUN.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Biomarkers / blood
  • Endostatins / blood*
  • Enzyme-Linked Immunosorbent Assay
  • Glomerulonephritis / blood*
  • Glomerulonephritis / genetics
  • Mice
  • Mice, Transgenic


  • Biomarkers
  • Endostatins

Grants and funding

This work was funded from the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement number 305436 (STELLAR, partly financing the position of JW. Financial support for this project was also provided by the St. Anna Kinderkrebsforschung, Children’s Cancer Research Institute, Vienna, Austria (, financing the position of EB and PA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Antibody Lab GmbH provided support in the form of salaries for authors (JW, EG, AB) and research material, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.