PromarkerD 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. A 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. Processing 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. An 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.

中文翻译：

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用于糖尿病肾病临床预测的稳健多重免疫亲和质谱分析 (PromarkerD)

PromarkerD 是一种用于预测糖尿病肾病 (DKD) 的新型蛋白质组学衍生血液测试。该测试基于一种算法，该算法将三种血浆蛋白生物标志物（CD5L、APOA4 和 IBP3）的测量与三个临床变量（年龄、HDL-胆固醇和 eGFR）相结合。该测定的初始形式使用血浆样品的免疫耗竭，然后是靶向质谱（MRM-LCMS）。本研究的目的是将现有的测定转化为与更高通量和稳健的临床应用兼容的免疫亲和方法。以 96 孔格式开发了一种新优化的基于免疫亲和性的测定，其中 MRM 测量使用低流量 LCMS 方法进行。评估了测定的稳定性、重现性和精密度。在一个 100 人的队列中进行了免疫亲和方法和原始免疫耗竭方法之间的直接比较。随后，在两个独立实验室中对优化的免疫亲和方法进行了实验室间研究。通过切换到免疫亲和珠捕获方法，与更快、更强大的微流 LCMS 系统相结合，大大简化了血浆样品的处理。处理时间从 7 天减少到 2 天，色谱从 90 分钟减少到 8 分钟。温度和时间差异处理的生物标志物稳定性通过了验收标准。所有生物标志物的日内/日间测试重现性和精度均在 11% CV 以内。新免疫亲和方法的 PromarkerD 测试结果证明与原始免疫耗竭方法具有极好的相关性 (R = 0.96)。免疫亲和测定成功转移到第二个实验室 (R = 0.98)，证明了该方法的稳健性和方法转移的简易性。开发并优化了免疫亲和捕获靶向质谱分析。它显示出与从原始免疫耗竭方法获得的结果具有统计学可比性的结果，并且在部署到独立实验室时也能够提供可比的结果。采用研究级分析并优化到临床级工作流程，可以深入了解具有免疫亲和质谱基础的多重生物标志物测量的未来。在目前的格式中，PromarkerD 免疫亲和测定有可能对糖尿病肾病的预测产生重大影响，从而使患者受益。