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Abstract: FR-PO166

Performance of Soluble Klotho Assays in Clinical Samples of Kidney Disease

Session Information

Category: Bone and Mineral Metabolism

  • 402 Bone and Mineral Metabolism: Clinical

Authors

  • Neyra, Javier A., University of Kentucky Medical Center, Lexington, Kentucky, United States
  • Moe, Orson W., University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Pastor, Johanne Virginia, University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Gianella, Fabiola, University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Sarnak, Mark J., Tufts Medical Center, Boston, Massachusetts, United States
  • Ix, Joachim H., UCSD, San Diego, California, United States
  • Drew, David A., Tufts Medical Center, Boston, Massachusetts, United States
Background

There is considerable interest in investigating soluble Klotho as a biomarker in patients with different types and severity of kidney diseases. Unfortunately, there remains uncertainty regarding the best method to measure soluble Klotho in human serum samples.

Methods

Using human serum samples obtained from several clinical cohorts with a wide range of kidney function, we measured soluble Klotho using a commercial enzyme linked immunosorbent assay (ELISA - IBL America) as well as with an immunoprecipitation-immunoblot (IP-IB) assay utilizing a synthetic antibody with high affinity and specificity for Klotho. Recovery of spiking with known amounts of exogenous Klotho was tested. A subset of samples was analyzed with and without the addition of a protease inhibitor cocktail at time of collection or after first freeze/thaw cycle.

Results

The IP-IB assay was superior to the ELISA at recovery of exogenous Klotho (81-115% vs. 60-81%) across the spectrum of kidney function. The IP-IB and ELISA assay were modestly correlated (R = 0.28, p = 0.01). Klotho concentrations by IP-IB were highly correlated with eGFR (R=0.80, p<0.001) in comparison to the commercial ELISA, which exhibited minimal correlation with eGFR (R=0.18, p=0.12) [Figure 1]. Use of a protease inhibitor cocktail neither improved nor impaired performance of the IP-IB assay; however, a subsequent freeze-thaw cycle resulted in a significant reduction in Klotho recovery and dissipated the correlation between Klotho levels and eGFR. With the ELISA, use of a protease inhibitor cocktail resulted in an increase of intra-subject variability.

Conclusion

The IP-IB assay is preferable to the commercial ELISA to measure soluble Klotho concentrations in never-thawed serum samples with varying severity of kidney disease.

Funding

  • NIDDK Support