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Avoiding false discovery in biomarker research.

Pranali Patel1, Uros Kuzmanov2,3, Seema Mital4,5,6

  • 1Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.

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Summary

Commercial ELISA kits for SIRPA may yield false positives in pediatric cardiac injury biomarker studies. Robust protein verification is crucial to prevent inaccurate biomarker discovery.

Keywords:
Biomarker discoveryELISA assayMass spectrometrySIRPAValidation

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Area of Science:

  • Biomarker Discovery
  • Cardiovascular Research
  • Pediatric Surgery

Background:

  • Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker on cardiomyocytes derived from human embryonic stem cells.
  • Investigating SIRPA as a potential circulating biomarker for cardiac injury in pediatric patients undergoing open heart surgery.

Purpose of the Study:

  • To evaluate the utility of circulating SIRPA levels as a biomarker for cardiac injury in pediatric patients.
  • To assess the reliability of commercially available SIRPA ELISA kits for this purpose.

Main Methods:

  • Serum samples from pediatric patients (n=48) undergoing cardiac surgery and controls (n=6) undergoing non-cardiac surgery were analyzed for SIRPA levels using ELISA kits from two manufacturers.
  • Protein identity was verified using Western blot and Mass Spectrometry (MS) on ELISA kit calibrators.
  • Recombinant human SIRPA (rhSIRPA) was also tested on the ELISA kits.

Main Results:

  • Post-operative SIRPA concentrations were significantly higher in pediatric cardiac surgery patients compared to controls, as detected by both ELISA kits.
  • Western blot analysis of ELISA kit calibrators failed to identify SIRPA.
  • Mass Spectrometry analysis of ELISA kit calibrators detected albumin and inflammatory markers, but not SIRPA.

Conclusions:

  • Commercially available ELISA kits for SIRPA provide false-positive results.
  • Rigorous protein characterization is essential to validate biomarker findings and avoid erroneous conclusions when using commercial immunoassays.