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BMC Biochemistry

, 17:17

Chemical biology

Abstract

BackgroundHuman tyrosine-protein phosphatase non-receptor type substrate 1α SIRPA is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery.

ResultsPaired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery controls were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein rhSIRPA was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry MS. Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected.

ConclusionsWe conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.

KeywordsELISA assay Validation Biomarker discovery SIRPA Mass spectrometry Electronic supplementary materialThe online version of this article doi:10.1186-s12858-016-0073-x contains supplementary material, which is available to authorized users.

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Autor: Pranali Patel - Uros Kuzmanov - Seema Mital

Fuente: https://link.springer.com/







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