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Mouse Interferon-Gamma

Introduction

Mouse IFN-γ (Interferon-gamma), known as type II or immune interferon, plays an essential role in modulating the immune response. IFN-γ acts primarily in immune regulation and anti-microbial/anti-viral host defense. IFN-γ is involved in a variety of roles from upregulating antigen presenting molecules to functioning as an anti-inflammatory mediator through promoting regulatory T cell development. IFN-γ is activated when the IFN-γ receptor I and II bind, thus activating the JAK-STAT pathway. It is produced by a variety of immune cells such as T cells and Natural Killer cells. IFN-γ activity is also associated with pregnancy, obesity, allergies, autoimmune diseases, as well as cancer. IFN-γ is particularly significant in tumor autoimmunity and immunoevasion.

Assay Overview

The workflow for the Pu•MA System cytokine immunoassay is shown in the figure below. All assay reagents are prepared in advance and then loaded into Pu•MA 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 Pu•MA 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.

  • Low-volume assays (10-20 μl)
  • Simple “hands-off” operation
  • Open platform – no expensive plates

Assay Procedure

The adaption of existing ELISA kits and/or antibody pairs is straightforward with the Pu•MA System. Assay buffers optimized for the Pu•MA System flowchips are provided for sample and antibody dilutions. Blocking solutions and wash buffers are also provided. The IFN-γ assays are adapted from BioLegend ELISA Max Kits (p/n 430801).

Assay reagents were prepared according to the dilutions shown in the Table using Pu•MA Assay Buffer (PAB). IFN-γ standards were reconstituted according to instruction provided by BioLegend. A 1:2 serial dilution series of Mouse IFN-γ 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 Pu•MA System and processed using the IFN-γ Assay Protocol. Plates were read within 5 min of completion on an absorbance plate reader at 450nm (Tecan Spectrafluor Plus).

Pu•MA System Mouse IFN-γ Standard Curve

Pu•MA System ELISA

PuMA System ELISA

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.

Name Reagent Source
Capture Ab Mouse IFN-γ Capture
Antibody (1:50)
BL
Block Pu•MA Blocking Buffer PFI
Sample Mouse IFN-γ Standard BL
1°Ab Mouse IFN-γ Primary
Antibody (1:50)
BL
2°Ab Avidin-HRP (1:5K) BL
Wash Pu•MA Wash Buffer PFI
Substrate FAST Substrate PFI
Stop Pu•MA Stop Solution PFI

Microfluidic Assay Workflow

Schematic of PuMA system ELISA

PuMA System

Pu•MA System

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

PuMA System Software

Software

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

Reagents and Flowchips

Reagents & Flowchips

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