This protocol automates chemiluminescent protein detection and quantitation from mouse tissue extracts or plasma samples using electroluminescence (ECL) technology on the Meso Scale Discovery (MSD) Sector S600 device accessible on the Transcriptic platform. This device expands our biomarker quantitation assay capabilities by adding ECL alongside more traditional ELISA immunoassays.
The MSD platform utilizes electrochemiluminescence (ECL) technology for robust, multiplexed protein analysis of a large selection of clinically relevant biomarkers / targets spanning sample types (cell supernatant, serum, plasma, whole blood, tissues) and species (mouse, human, etc.), requiring as little as 10 ul of sample per well (384 well). ECL technology enables biomarker profiling with high sensitivity, low background, a broad dynamic detection range up to 6 log-fold, and assay flexibility and customization. The Sector S600 accommodates 96 and 384 well plates as well as the ability to multiplex up to 10 analytes in one well for 96 format (or up to 4 analytes in one well for 384 format) to drive high throughput biomarker discovery. Commercial kits are available as validated pre-coated V-PLEX panels or custom coat your own assays using MSD approved antibodies (U-PLEX) or your own (U-PLEX Development Packs). A full range of kits can be found at https://www.mesoscale.com/en/products_and_services/assay_kits. Transcriptic has currently validated the protocol with mouse liver tissue extracts or plasma to automate high-throughput multiplexed immunoassays on the Transcriptic Robotic Cloud Lab as described below. Biomarker quantitation for other input types or analytes can be onboarded on request by contacting [email protected].
50 mg biopsies of whole mouse livers homogenized and extracted on the KingFisher Flex Purification System (Thermo) were quantitated using the Pierce BCA Protein Assay Kit (Thermo) and profiled for cytokine protein levels using the simple 3-step mouse V-PLEX Proinflammatory Panel 1 Kit (MSD). 25ul samples and kit standards were analyzed in duplicate using a 96-format 10-plex assay plate across the following 10 cytokines important for inflammation and immune function: IFN-α, IL-1α, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL- 12p70, and TNF-α. Plates pre-coated with 10 capture antibodies per well (10-spot MULTI-SPOT) were loaded with 50 ul sample per well (25 ul sample diluted 1:2 using Diluent 41), sealed to prevent evaporation and incubated at ambient with shaking (750 rpm) for 2 hours. Next, excess sample was washed away using 150 ul Wash Buffer per well 3x followed by the addition of 25 ul detection antibody per well. After another 2 hour shaking incubation at ambient, the plate was washed, exposed to 150 ul per well of 2X Read Buffer T, and ready for ECL detection on the Sector S600. This protocol captures the entire immunoassay workflow from sample prep to multiplexed biomarker / cytokine quantitation and profiling on the Transcriptic platform.
To generate liver tissue extracts, 50 mg biopsies of whole mouse livers were homogenized and collected for protein analysis on the KingFisher Flex Purification System (Thermo). Experimental samples and kit standards were assessed in duplicate for 10 inflammatory cytokines using the mouse V-PLEX Proinflammatory Panel 1 Kit (MSD).
Figure 1. Multiplexed (10-plex) cytokine concentration quantitated across a broad dynamic range using ECL technology on the Sector S600 (MSD) device with murine V-PLEX Pro-Inflammatory Panel 1 Kit (MSD). Samples are measured in duplicate.