Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

PromarkerD as an immunoaffinity mass spectrometry assay for diabetic kidney disease (#441)

Scott Bringans 1 , Kirsten Peters 1 , Tammy Casey 1 , Richard Lipscombe 1 , Sarah Thomas 1 , Stephen R Pennington 2 , Orla Coleman 2 , Holger A Ebhardt 2
  1. Proteomics International, Broadway, Nedlands, WA, Australia
  2. Atturos, Dublin, Ireland

The PromarkerD assay for diabetic kidney disease was originally developed as a multi-protein targeted mass spectrometry assay directly from depleted (top 14 proteins) digested human plasma. This assay has been adapted to an ELISA format and more recently an immunoaffinity mass spectrometry assay. The immunoaffinity method utilises bead-based antibody binding for the specific PromarkerD protein biomarkers in single multiplex capture step. The captured protein biomarkers are reduced, alkylated and digested in situ on the beads with injection onto a microflow LCMS system for targeted mass spectrometry. The results obtained with the immunoaffinity method applied to a 100-person cohort were compared to the original direct plasma digestion method with correlation between the two methods confirmed by Bland and Altman plot analysis. To test the robustness of the process between laboratories the assay was also performed in laboratories in Australia and Ireland using the same samples. The advantages of the immunoaffinity technology developed for this assay are three-fold. Firstly, increased throughput of analysis with a 96 well based robotic handling capable system which also minimises human intervention and handling. Secondly, samples that are injected onto the LCMS system are in a much cleaner form than a crude plasma digest which enhances sensitivity and reduces machine down time due to less frequent source cleaning and minimal LC blockages (in micro flow format). Thirdly, the immunoaffinity method has been designed to be available as a simple technology transfer process to partner laboratories that have LCMS capability. These advantages may make such an immunoaffinity-MS technology approach a superior choice for multi-protein biomarker diagnostic assays.