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Development of ELLA Approach to Measure Kidney Injury Molecule‐1
Author(s) -
Krandel Jessica,
Scheetz Marc H.,
Gilchrist Annette
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02466
Subject(s) - nephrotoxicity , polyclonal antibodies , medicine , urinary system , chemistry , kidney , acute kidney injury , monoclonal antibody , biomarker , biotinylation , antibody , pharmacology , immunology , biochemistry
Antibiotics with high nephrotoxic potential, such as polymyxins, are being used with increased frequency because of antibiotic resistance and few novel antibiotic agents. Dose escalation of polymyxins is limited by the kidney injury. The objective of this study was to develop a novel ELISA‐based assay using the ELLA instrument in an effort to increase sensitivity and decrease the amount of time required to detect sensitive biomarkers such as Kidney Injury Molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein expressed on the proximal convoluted tubule, shed into the urine and blood, and can be detected by ELISA as soon as 6 hours after cell injury, while remaining undetectable in persons with healthy kidneys. The FDA has qualified KIM‐1 for pre‐clinical and clinical study of drug‐induced nephrotoxicity. The ELLA platform has been reported to be more sensitive than MAGPIX for several biomarker assays such as IL‐1beta, PD‐L1, IL‐4, and TNF‐alpha. Improved sensitivity for KIM‐1 is needed for a blood assay as concentrations are considerably lower than the urinary assay counterpart. The ELLA platform is also reported to have better reproducibility given the reduction in manual steps. Each cartridge is manually loaded and then run hands‐off for approximately 1.5 hours. Using the ELLA open cartridge system we examined several commercially‐available monoclonal and polyclonal antibodies to human KIM‐1. Capture antibodies were digoxigenin‐labeled and detection antibodies were biotinylated. A standard curve was created by spiking diluent with known concentrations of rhKIM‐1. Two diluents were tested – SD13 (Biotechne) and PBS. Curves for rhKIM‐1 were run ranging from a minimum of 0.49 pg/mL to a maximum of 10000 pg/mL. Quality control urine and plasma samples of known concentrations were included with runs of unknown urine samples from patients treated with vancomycin. Results were analyzed using Graphpad Prism. We determined the optimum concentration for the detection and capture antibodies, and identified in which position monoclonal and polyclonal antibodies performed better, providing the most sensitive and accurate assay. Currently measurement of KIM‐1 is done using an ELISA or MAGPIX approach. Thus, we compared the ELLA assay to a MAGPIX assay and found that the ELLA is quicker and provides a lower LLOQ. Further development of the ELLA approach may provide a test that can be rapidly employed in a hospital setting for blood or urine to examine KIM‐1 levels and identify patients at risk for acute kidney injury. Support or Funding Information Funding: Kenneth A. Suarez Fellowship from Midwestern University, Biotechne, Intramural grant from Midwestern University (Chicago College of Pharmacy Pilot Grant, awarded to Dr. Gilchrist)