The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse

Fred Finkelman1, Suzanne Morris1, Tatyana Orekhova2, David Sehy3

1 University of Cincinnati College of Medicine and Cincinnati Veterans Administration Medical Center, Cincinnati, Ohio, 2 University of Cincinnati College of Medicine, Cincinnati, Ohio, 3 BD Biosciences PharMingen, San Diego, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.28
DOI:  10.1002/0471142735.im0628s54
Online Posting Date:  August, 2003
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Because most cytokines are utilized, catabolized, or excreted shortly after they are produced, it has been difficult to directly measure in vivo cytokine production. Consequently, it has been necessary to infer in vivo cytokine secretion levels from the results of ex vivo assays of cytokine secretion, assays that measure tissue levels of cytokine mRNA, or assays that stain tissues for cytokine protein levels. Results of these assays provide important and useful information, but do not necessarily reflect in vivo cytokine secretion. To better determine in vivo cytokine production, the in vivo cytokine capture assay (IVCCA) was developed. IVCCA facilitates measurement of cytokines in serum by increasing their in vivo half‐lives. This increases the sensitivity of measurement of in vivo cytokine production 30‐ to 1,000‐fold. The first protocol described in this unit is for luminescence‐based ELISA, while the second is for an absorbance‐based method.

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Table of Contents

  • Basic Protocol 1: Measurement of Mouse In Vivo Cytokine Production by Luminescence
  • Alternate Protocol 1: Measurement of Mouse In Vivo Cytokine Production by Absorbance
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Measurement of Mouse In Vivo Cytokine Production by Luminescence

  • Biotin‐labeled anti‐cytokine capture antibodies (Table 6.28.2)
    Table 6.8.2   Materials   Antibody Combinations for Specific Cytokines using IVCCA   Antibody Combinations for Specific Cytokines using IVCCA

    Target cytokine Capture antibody c ELISA antibody
    IL‐2 Biotin‐JES6‐5H4 JES6‐1A12
    IL‐4 Biotin‐BVD4‐1D11 BVD6‐24G2.3
    IL‐6 Biotin‐MP5‐32C11 MP5‐20F3
    IL‐10 Biotin‐JES5‐16E3 JES5‐2A5
    IFN‐γ Biotin‐R4‐6A2 AN‐18
    TNF‐α Biotin‐TN3 G281‐2626

     aThe capture and ELISA antibodies are for the in vivo and in vitro portions of the protocol as outlined in Figure
     bAll antibodies are available from BD PharMingen.
     cInject only mAbs that lack preservatives, such as sodium azide (NaN 3), and that are low in endotoxin.
  • recipe1% autologous mouse serum (see recipe)
  • 2 µg/ml ELISA antibody (Table 6.28.2) in recipeTBS, pH 7.2 (see recipe)
  • recipeWashing buffer (see recipe)
  • recipeDilution buffer (see recipe)
  • recipeTBS, pH 7.2 (see recipe)
  • recipe100 ng/ml cytokine:anti‐cytokine mAb standards (see recipe)
  • 1 mg/ml streptavidin‐horseradish peroxidase (Pierce): aliquot and store up to 1 year at −20°C
  • recipeLuminescence substrate (see recipe)
  • 96‐well Costar high‐binding, flat‐bottomed, white, solid microtiter plates (Corning)
  • Standard 96‐well microplate (e.g., vinyl plates; Thermo Labsystems)
  • Luminescence microtiter‐plate reader (e.g., Fluoroskan Ascent FL, Labsystems)
  • Additional reagents and solutions for handling mice (unit 1.3), parenteral injections (unit 1.6), and blood collection and preparation of serum (unit 1.7)
NOTE: All antibodies, including biotinylated antibodies, are stored at −70°C and are stable under these conditions for at least one year.

Alternate Protocol 1: Measurement of Mouse In Vivo Cytokine Production by Absorbance

  • 1 mg/ml alkaline phosphatase‐streptavidin (Jackson ImmunoResearch)
  • recipeAbsorbance substrate (see recipe)
  • 96‐well Immulon II microtiter plate (Dynatech)
  • Absorbance‐based microtiter‐plate reader (e.g., Multiskan MS, Labsystems)
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Literature Cited

Literature Cited
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   Carter, L.L. and Swain, S.L. 1997. Single cell analyses of cytokine production. Curr. Opin. Immunol. 9:177‐182.
   Finkelman, F.D. and Morris, S.C. 1999. Development of an assay to measure in vivo cytokine production in the mouse. Int. Immunology. 11:1811‐181.
   Finkelman, F.D., Madden, K.B., Morris, S.C., Holmes, J.M., Boiani, N., Katona, I.M., and Maliszewski, C.R. 1993. Anti‐cytokine antibodies as carrier proteins: Prolongation of in vivo effects of exogenous cytokines by injection of cytokine—anti‐cytokine antibody complexes. J. Immunol. 151:1235‐124.
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