ELISA

This protocol automates colorimetric protein detection from mouse tissue extracts or biological fluids using enzyme-linked immunosorbent assay (ELISA) technology to drive biomarker profiling and quantitation on the Transcriptic platform.

OVERVIEW

Enzyme-linked immunosorbent assay (ELISA) is a plate-based ligand-binding assay routinely used for protein detection and quantitation in drug discovery workflows. Capture antibodies immobilized to 96 well plates bind specific analytes (including cytokines, chemokines, proteases, growth factors, and more) within sample input types ranging from biological fluids (plasma, serum) to processed mouse tissue biopsies. The binding of streptavidin-HRP to biotin-labeled detection antibodies produces a colorimetric signal detectable by a microplate spectrophotometer. Commercial ELISA kits supplied by R&D Systems enable sensitive, reproducible biomarker detection, and are available as ready-to-use Quantikine Colorimetric Sandwich ELISA kits or custom coat your own DuoSet ELISA Development Systems. A full range of kits can be found at https://www.rndsystems.com/products/elisas. Transcriptic has currently validated this protocol with mouse liver tissue extracts and plasma using DuoSet Development kits on 15+ analytes to automate high-throughput ELISAs on the Transcriptic Robotic Cloud Lab as described below. Biomarker quantitation for other input types, analytes or kits can be onboarded on request by contacting [email protected]. For multiplex biomarker profiling needs, please refer to our article on our MSD capabilities.

METHODOLOGY

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 R&D Systems’ DuoSet ELISA Development Systems. 96-well flat-bottomed plates were patterned and incubated for 16h at ambient with selected capture antibodies. Kit provided standards and samples were transferred to coated ELISA plates and assayed in triplicate. Following washes, incubations, application of detection antibodies and colorimetric detection reagents, the optical density of each well at 450 nm was detected in a microplate reader (Tecan M200). Plates were also read at optical density 540 nm to correct for background. This protocol captures the entire ELISA workflow from sample prep to colorimetric protein detection and quantitation on the Transcriptic platform.

RESULTS

To generate liver tissue extracts, 50 mg biopsies of whole mouse livers were homogenized and collected for protein analysis. Experimental samples were tested in triplicate alongside standards in a 96-well plate format ELISA patterned with a selected capture antibody (Mouse IL-17F is demonstrated below in Figure 1) using R&D Systems DuoSet ELISA Development Systems.

Figure 1. Representative kit standards for R&D Systems DuoSet ELISA Development Systems were processed in triplicate on the Transcriptic platform in 96-well microplates patterned with a capture antibody targeting mouse IL-17F. The optical densities of each well at 450 nm and 540 nm were measured in a microplate reader. Data shown was analyzed using Four Parameter Logistic (4PL) Regression and plotted after subtracting out the background reading at 540 nm.

Currently Transcriptic offers the following validated R&D Systems DuoSet ELISA Development Systems for use on our platform. Biomarker quantitation for other input types, analytes or kits can be onboarded on request by contacting [email protected].

Mouse:

  • IL-4
  • TNFa
  • IFNg
  • IL-2
  • IL-6
  • IL-10
  • IL-12
  • IL-17a
  • IL-1b
  • IL-5
  • IL-17f
  • IL-22
  • CCL20
  • TIMP-1
  • Osteopontin