Pu•MA System ELISA: Mouse Tumor Necrosis Factor-α

Introduction

Mouse TNF-α (Tumor Necrosis Factor-Alpha), also termed Cachectin, Cytotoxic Factor (CF), CTX, Hemorrhagic Factor, Macrophage-Derived Cytotoxic Factor, and Macrophage Cytotoxic Factor (MCF), is a potent multifunctional cytokine that exerts regulatory, inflammatory and cytotoxic effects on a wide range of normal lymphoid and non-lymphoid cells and tumor cells. TNF-α is produced by a wide variety of immune and epithelial cell types. TNF-α regulates lymphoid tissue development through control of apoptosis. It also promotes inflammatory responses by inducing the activation of vascular endothelial cells and macrophages. TNF-α is a key cytokine in the development of several inflammatory disorders, illuminating it as an essential molecule investigated in a plethora of small animal studies.

Assay Overview

The workflow for the PuMA System cytokine immunoassay is shown in Figure 1. All assay reagents are prepared in advance and then loaded into PuMA System flowchips using single or 8-channel pipettes. The wells are conveniently located on standard 384 multiwell plate spacings. The flowchips are now ready to be loaded into the PuMA System for “hands-off” processing. The ELISA steps are automatically performed by the system using pre-loaded protocols. Once the assay is complete, absorbance results are read on your Plate Reader.

Pu•MA System Complete Workflow: 2-3 hours “hands-off”

Figure 1. Schematic Pu•MA System assay workflow.

The adaption of existing ELISA kits and/or antibody pairs is straightforward with the PuMA System. Assay buffers optimized for the PuMA System flowchips are provided for sample and antibody dilutions. Blocking solutions and wash buffers are also provided. The TNF-α assays are adapted from BioLegend ELISA Max Kits (p/n 430901). The reagents required for the assay are shown in the table.

Assay Procedure

Assay reagents were prepared according to the dilutions shown in the Table using PuMA Assay Buffer (PAB). TNF-α standards were reconstituted according to instruction provided by BioLegend. A 1:2 serial dilution series of Mouse TNF-α Standard was prepared starting at 100 pg/well using PAB. 20 μl of each reagent was added to the appropriate wells (see Fig 3) except for the Stop Solution where 10 μl was dispensed. Four replicates were run per concentration. The flowchips were loaded into a PuMA System and processed using the TNF-α Assay Protocol. Plates were read within 5 min of completion on an absorbance plate reader at 450nm (Tecan Spectrafluor Plus).

Name Reagent Source
Capture Ab Mouse TNF-α Capture Antibody (1:100) BL
Block Pu•MA Blocking Buffer PFI
Sample Mouse TNF-α Standard BL
1°Ab Mouse TNF-α Primary Antibody (1:100) BL
2°Ab Avidin-HRP (1:5k) BL
Wash Pu•MA Wash Buffer PFI
Substrate FAST Substrate PFI
Stop Pu•MA Stop Solution PFI

Pu•MA System Mouse TNF-α Standard Curve

Figure 2. Response of Cytokine Standards for ELISA run on Pu•MA System. ELISA antibody pairs and standards were obtained from BioLegend. Absorbance was read at 450 nm. OD values shown are background subtracted.

How It Works

The Pu•MA Flowchip and System uses established antibody pairs to perform an automated ELISA. All assay reagents are loaded into reservoirs and then moved one at a time through the “Assay Channel” by the Pu•MA System. Preloaded protocols execute all fluid transfer and incubation steps The system incorporates patented valve-less fluidic switching (VLFS) to precisely control fluid movement in a flowchip. Use of microfluidics reduces
both incubation times and reagent volumes.

Reagent Loading Setup

  • Low sample/reagent usage: < 20 μl/well)
  • Enhanced kinetics: Faster Assay Results
  • Effi cient fl uid removal: Less Wash Required

Figure 3. Pu•MA Flowchip reagent loading setup.

Microfluidic Assay Workflow

PuMA System

Conroy

  • Compact benchtop system
  • Easy top-loading of flowchips
  • Precision pneumatic control

Software

PuMa Software

  • Touchscreen-driven interface
  • Preloaded assay protocols
  • Simple Select and Run operation

Reagents & Flowchips

Reagents and Flowchips

  • Validated immunoassays
  • Open platform for existing Ab pairs
  • Works with standard pipettors & tips