Abstract
BackgroundPromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application.MethodsA newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories.ResultsProcessing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer.ConclusionsAn immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients.
Highlights
PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD)
A hybrid test that harnesses the selectivity of antibody capture and the multiplexing capability of multiple reaction monitoring (MRM) targeted mass spectrometry offers the potential to convert what may only be suitable as a ‘research’ test into an assay more suited to high throughput clinical applications
The conversion of the PromarkerD assay to an immunoaffinity based mass spectrometry test necessitated specific antibodies for each of the three biomarker proteins (CD5L, Apolipoprotein A4 (APOA4), and Insulinlike growth factor-binding protein 3 (IBP3))
Summary
PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The advent of increasingly sensitive mass spectrometers and the abundance of techniques for quantifying large proteome datasets has had the effect of producing panels of biomarkers that can be combined in a model to determine a disease state or progression Examples of such tests are the FDA-cleared OVA1 diagnostic test that combines levels of 5 protein biomarkers to determine a score that assists a clinician in the assessment of the patient’s risk of ovarian cancer [7, 8] and the Vectra DA test that utilises 15 protein biomarkers and clinical variables to measure inflammation caused by rheumatoid arthritis [9]. A hybrid test that harnesses the selectivity of antibody capture and the multiplexing capability of multiple reaction monitoring (MRM) targeted mass spectrometry offers the potential to convert what may only be suitable as a ‘research’ test into an assay more suited to high throughput clinical applications
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