Measurement of Antiviral Activity Induced by Interferons α, β, and γ

Stefanie N. Vogel1, Robert M. Friedman1, M. Michele Hogan2

1 Uniformed Services University of the Health Sciences, Bethesda, Maryland, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.9
DOI:  10.1002/0471142735.im0609s37
Online Posting Date:  May, 2001
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Abstract

The in this unit describes an assay for murine IFN‐induced antiviral activity and employs vesicular stomatitis virus (VSV) and IFN‐sensitive fibroblasts. describes the preparation of VSV cultures and the calculation of multiplicity of infection (MOI; i.e., concentration of viral particles required to infect cells). describes the antibody neutralization assay, which can be used to identify the bioactive species of IFN in a sample, or to test the potency of an antibody preparation against a particular species of IFN. 1 covers measurement of human IFN‐induced antiviral activity; while the steps are quite similar to , different viral and cell cultures are described.

     
 
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Table of Contents

  • Basic Protocol 1: Assay for Murine IFN–Induced Antiviral Activity
  • Support Protocol 1: Preparation of VSV Cultures
  • Support Protocol 2: Antibody Neutralization Assay
  • Alternate Protocol 1: Assay of Human IFN‐Induced Antiviral Activity
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Assay for Murine IFN–Induced Antiviral Activity

  Materials
  • recipeSupplemented Eagle minimum essential medium (EMEM)
  • recipeTrypsin solution
  • IFN‐sensitive L929 fibroblasts (see critical parameters)
  • Murine interferon reference standards IFN‐α, IFN‐β, IFN‐γ, and IFN‐αβ (available from Dr. C. Laughlin, National Institute of Allergy and Infectious Diseases, Bethesda, MD)
  • Vesicular stomatitis virus (VSV; Indiana strain; see caution, step )
  • recipeSupplemented Earles balanced salt solution (EBSS), 4°C
  • 5% (v/v) formalin
  • 0.05% (w/v) crystal violet in 20% ethanol
  • 100% methanol
  • 75‐cm2 straight neck tissue culture flasks (e.g., Falcon, Costar)
  • 8‐channel pipettor (fixed or adjustable to 50‐ and 100‐µl volumes; e.g., Costar octapette #4850, #4800)
  • 96‐well flat‐bottom microtiter plate (Falcon #3072 or Costar #3596)
  • 8‐well aspirator (Drummond; e.g., Thomas Scientific #7691‐R32)
  • Additional reagents and equipment for plaque assay (Vogel and Fertsch, ) and preparation of L929 fibroblasts (unit 6.10)

Support Protocol 1: Preparation of VSV Cultures

  Additional Materials
  • Vero cells (ATCC #CCL 81)
  • 150‐cm2 tissue culture flasks
  • Rocking platform

Support Protocol 2: Antibody Neutralization Assay

  Additional Materials
  • Encephalomyocarditis (EMC) virus (ATCC #VR 129B)
  • Human interferon reference standards (available from Dr. C. Laughlin, National Institute of Allergy and Infectious Diseases, Bethesda, MD)
  • Human diploid fibroblast (FS‐4; Havell and Vilcek, ), trisomy 21 human line (GM2504; Preble et al., ), or human lung carcinoma line (A549; ATCC #CCL 185)
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Figures

Videos

Literature Cited

Literature Cited
   Curry, R.C., Kiener, P.K., and Spitalny, G.L. 1987. A sensitive immunochemical assay for biologically active muIFN‐γ. J. Immunol. Methods 104:137‐142.
   Guyre, P.M., Morganelli, P.M., Fillian, F., Miller, R., and Bodwell, J.E. 1984. Virus‐free bioassay of human immune interferon by flow cytometric assay of Fc receptor modulation. Fed. Proc. 43:1830.
   Havell, E.A. and Spitalny, G.L. 1983. Production and characterization of anti‐murine interferon‐gamma sera. J. Interferon Res. 3:191‐198.
   Havell, E.A. and Vilcek, J. 1972. Production of high‐titered interferon in cultures of human diploid cells. Antimicrob. Agents Chemother. 2:476‐484.
   Preble, O.T., Black, R.J., Friedman, R.M., Klippel, J.H., and Vilcek, J. 1982. Systemic lupus erythematosis: Presence in human serum of an unusual acid‐labile leukocyte interferon. Science 216:429‐431.
   Rubenstein, S., Familletti, P.C., and Pestka, S. 1981. Convenient assay for interferons. J. Virol. 37:755‐758.
   Straub, S.X., Garry, F.R., and Magee, W.E. 1974. Interferon induction by poly(I):poly(C) enclosed in phospholipid particles. Infect. Immun. 10:783‐792.
   Vogel, S.N. and Fertsch, D. 1984. Endogenous interferon production by endotoxin‐responsive macrophages provides an autostimulatory differentiation signal. Infect. Immun. 45:417‐423.
   Vogel, S.N. and Fertsch, D. 1987. Macrophages from endotoxin‐hyporesponsive (Lpsd) C3H/ HeJ mice are permissive for vesicular stomatitis virus because of reduced levels of endogenous interferon: Possible mechanism for resistance to virus infection. J. Virol. 61:812‐818.
   Vogel, S.N., English, K.E., and O'Brien, A.D. 1982. Silica enhancement of murine endotoxin sensitivity. Infect. Immun. 38:681‐685.
   Vogel, S.N., Havell, E.A., and Spitalny, G.L. 1986. Monoclonal antibody–mediated inhibition of interferon‐γ–induced macrophage antiviral resistance and surface antigen expression. J. Immunol. 136:2917‐2923.
Key References
   Rubenstein et al., 1981. See above.
   Vogel et al., 1982, 1986. See above.
   Yeh, T.J., McBride, P.T., Overall Jr., J.C., and Green, J.A. 1982. Automated, quantitative cytopathic effect reduction assay for interferon. J. Clin. Microbiol. 16:413‐415.
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