| Autor: |
Oran PE; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Trenchevska O; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Nedelkov D; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Borges CR; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Schaab MR; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Rehder DS; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Jarvis JW; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Sherma ND; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Shen L; Center for Innovations in Medicine, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America., Krastins B; Thermo Fisher Scientific, The Biomarkers Research Initiatives in Mass Spectrometry Center, Cambridge, Massachusetts, United States of America., Schwenke DC; Phoenix VA Health Care System, Phoenix, Arizona, United States of America; College of Nursing & Health Innovation, Arizona State University, Phoenix, Arizona, United States of America., Reaven PD; Phoenix VA Health Care System, Phoenix, Arizona, United States of America., Nelson RW; Molecular Biomarkers Laboratory, Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America. |
| Abstrakt: |
Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications. |
