Abstract
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.
Highlights
Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders
We present an MS-based quantitative IGF1 assay that meets all of the above mentioned requirements through achieving these two goals: 1) To rigorously quantify IGF1 in human plasma samples at a rate of .1,000 samples/day, in order to factually benchmark time and economic considerations associated with translating such targeted mass spectrometric assays from research laboratories to clinical deployment, and 2) To accommodate IGF1 heterogeneity discovered from analysis of large populations in order to intelligently design IGF1 mass spectrometric assays that avoid error due to structural variants
An important feature of the quantitative IGF1 assay is the addition of the internal reference standard (IRS) at the beginning of the sample preparation
Summary
Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. 99% of the IGF1 is bound to IGFBPs (Insulin-like growth factor binding proteins), with 80% of circulating IGF1 in a ternary complex consisting of one molecule of IGF1, one molecule of IGFBP3, and one molecule of an acid labile subunit [4,8,9,10]. For over the past thirty years, IGF1 has been generally quantified using including radioimmunoassay (RIA), immuno-radiometric assay (IRMA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence [3,11,12]. These methods employ various sample preparation steps to disrupt and remove IGFBPs
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