Measurement of Mouse and Human Interferon γ

Robert D. Schreiber1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.8
DOI:  10.1002/0471142735.im0608s37
Online Posting Date:  May, 2001
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Abstract

This unit describes two protocols that can be used to quantitate interferon γ (IFN‐γ), an important modulatory lymphokine that regulates natural, cell‐mediated, and humoral immunity by eliciting a number of biological responses in many different cell types. Depending upon the specificity of the reagents used, the assays will detect either human or murine IFN‐γ. The is an enzyme‐linked immunosorbent assay (ELISA) based on IFN‐γ's unique antigenic structure. The is a functional activity assay based on IFN‐γ's ability to induce major histocompatibility (MHC) class II antigens (also termed Ia antigens) on responsive cells. The advantage of the ELISA protocol is its specificity, while the advantage of the MHC protocol is its sensitivity.

     
 
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Table of Contents

  • Basic Protocol 1: IFN‐γ‐Specific ELISA
  • Alternate Protocol 1: MHC Class II Antigen Induction Assay
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: IFN‐γ‐Specific ELISA

  Materials
  • recipePurified IFN‐γ‐specific MAb (Genzyme #1598‐00, anti‐human IFN‐γ; #1222‐00, anti‐murine IFN‐γ)
  • recipeCarbonate coating buffer
  • recipeIFN‐γ standard (Genzyme #HG‐IFN, human IFN‐γ; #MG‐IFN, murine IFN‐γ)
  • recipeSample buffer
  • recipeELISA wash buffer (dilute 10× stock to 1× just before use)
  • Goat or rabbit polyclonal anti‐IFN‐γ‐serum (Genzyme)
  • Peroxidase‐labeled anti‐goat or anti‐rabbit immunoglobulin (IgG; U.S. Biochemical #1144H, rabbit anti‐goat IgG; #1251H, goat anti‐rabbit IgG)
  • recipeSubstrate buffer
  • recipePeroxidase substrate solution
  • Multichannel pipettor
  • 96‐well flat‐bottom Immulon 2 ELISA plates with adhesive covers (Dynatech Laboratories #011‐010‐3450)
  • 96‐well V‐bottom microtiter plates
  • Multiwell scanning spectrophotometer with 414‐nm filter

Alternate Protocol 1: MHC Class II Antigen Induction Assay

  Additional Materials
  • Complete medium: RPMI‐10 containing 7.5% Na 2CO 3 for COLO‐205 cells or DMEM‐10 for WEHI‐3 cells ( appendix 2A)
  • Human COLO‐205 cells or murine WEHI‐3 cells (ATCC #CC l222 and #TIB 68, respectively)
  • recipeWash buffer with and without 1% (w/v) normal mouse serum, heat‐inactivated (56°C, 30 min)
  • Biotinylated MAb specific for either MHC class II antigen (HLA‐DR) or murine MHC class II antigen (I‐Ad)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • Streptavidin–fluorescein isothiocyanate (FITC)
  • 12‐well tissue culture dishes (25‐mm diameter), sterile
  • 12 × 75–mm polypropylene tubes
  • Tabletop centrifuge
  • Additional reagents and equipment for flow cytometry (units 5.1 5.3)
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Figures

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Literature Cited

Literature Cited
   Buchmeier, N.A. and Schreiber, R.D. 1985. Requirement of endogenous interferon‐γ production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. U.S.A. 82:7404‐7408
   King, D.P. and Jones, P.P. 1983. Induction of Ia and H‐2 antigens on a macrophage cell line by murine interferon. J. Immunol. 131:315‐318
   Le, J. and Vilcek, J. 1984. Lymphokine‐mediated activation of human monocytes: neutralization by monoclonal antibody to interferon‐γ. Cell. Immunol. 85:278‐283
  Linscott's Directory of Immunological and Biological Reagents. 1990‐1991. Sixth edition. Mill Valley, Calif.
   Pfizenmaier, K., Bartsch, H., Scheurich, P., Seliger, B., Ucer, U., Vehmeyer, K., and Nagel, G.A. 1985. Differential γ‐interferon response of human colon carcinoma cells: Inhibition of proliferation and modulation of immunogenicity as independent effects of γ‐interferon on tumor cell growth. Cancer Res. 45:3503‐3509
   Schreiber, R.D., Hicks, L.J., Celada, A., Buchmeier, N.A., and Gray, P.W. 1985. Monoclonal antibodies to murine gamma interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol. 134:1609‐1618
Key References
   Dijkmans, R. and Billiau, A. 1988. Interferon γ: A master key in the immune system. Curr. Opinion Immunol. 1:269‐274.
   Schreiber, R.D. and Celada, A. 1985. Molecular characterization of interferon γ as a macrophage activating factor. Lymphokines 11:87‐118.
   Trinchieri, G. and Perussia, B. 1985. Immune interferon: A pleiotropic lymphokine with multiple effects. Immunol. Today 6:131‐136.
   Vilcek, J., Gray, P.W., Rinderknecht, E., and Sevastopoulos, C.G. 1985. Interferon γ: A lymphokine for all seasons. Lymphokines 11:1‐32.
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