Measurement of Mouse and Human Interleukin 10

Tim Mosmann1

1 University of Alberta, Edmonton, Alberta
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.14
DOI:  10.1002/0471142735.im0614s18
Online Posting Date:  May, 2001
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Interleukin 10 (IL‐10) is a multifunctional cytokine produced by T cells and other cell types. It regulates activities of a variety of the cells of the immune system and may play a crucial role in cross‐regulation of different types of T cells. Two methods of quantitating IL‐10 are provided in this unit. The first is an enzyme‐linked immunosorbent assay (ELISA) using a pair of monoclonal antibodies specific for mouse IL‐10. This method has the advantages of high specificity and insensitivity to other cytokines present in test samples. The second method is a bioassay which has the advantages that both human and mouse IL‐10 are detected and that functional protein is measured. Both methods have similar sensitivity.

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Table of Contents

  • Basic Protocol 1: Mouse IL‐10‐Specific ELISA
  • Alternate Protocol 1: Cytokine‐Synthesis Inhibition Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Mouse IL‐10‐Specific ELISA

  • Coating antibody: purified anti‐IL‐10 MAb (JES5‐2A5 rat IgG1 anti‐mouse IL‐10; Pharmingen)
  • IL‐10 standard (Pharmingen)
  • Complete RPMI‐10 ( appendix 2A)
  • Blocking solution: 10% FCS in PBS ( appendix 2A), filter‐sterilized and stored at 4°C
  • Wash buffer: 0.05% Tween 20 in PBS, stored at room temperature 1 to 2 weeks
  • Biotinylated second antibody (SXC1 anti‐mouse IL‐10; Pharmingen)
  • recipeSecond antibody buffer
  • Horseradish peroxidase (HRPO)‐conjugated streptavidin (Jackson Immunoresearch #016‐030‐084)
  • recipeSubstrate solution
  • Stop solution: 0.2 M citric acid, stored at room temperature
  • 12‐channel pipettor
  • 96‐well round‐bottom polyvinyl flexible microtiter plates (Dynatech #001‐010‐2401 or Falcon #3911)
  • 96‐well polystyrene microtiter plates
  • ELISA plate washer (e.g., Elcawash from Elcatech)Multiwell scanning spectrophotometer (preferably dual wavelength; e.g., VMAX, Molecular Devices)
  • Curve‐fitting software (e.g., Softmax from Molecular Devices or DeltaSoft from Biometallics; see appendix 55)

Alternate Protocol 1: Cytokine‐Synthesis Inhibition Assay

  Additional Materials
  • 40 ng/ml IL‐10 standard (Pharmingen; diluted just before use)
  • Resting antigen‐ or allo‐specific mouse helper T (T H1) cells (e.g., LB2‐1, ATCC #CRL 8629, patent #4798789; or see unit 3.13 for production of T H1 cell clones)
  • Mouse spleen cells syngeneic with T H1 cell line (e.g., C57BL/6 for LB2‐1; see also unit 3.1) and Table 97.80.4711
  • Antigen: chicken red blood cells (RBC; Colorado Serum), washed in RPMI‐10 immediately before use (for LB2‐1 cells)
  • Anti‐mouse IL‐4 MAb (Pharmingen; or 11B11, ATCC #HB188; or Table 97.80.4711)
  • Anti‐TGF‐β antibody (R & D Systems)
  • Interleukin 2 (see Table 97.80.4711 for suppliers)
  • Curve‐fitting software (e.g., Assay Zap from Biosoft; see appendix 55)
  • Humidified 5% CO 2, 37°C incubator
  • Additional reagents and equipment for washing, irradiating, and counting mouse spleen cells (units 3.1 & 3.12 and 3.NaN, respectively) and for quantitating IFN‐γ (unit 6.8)
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Literature Cited

Literature Cited
   Fiorentino, D.F., Bond, M.W., and Mosmann, T.R. 1989. Two types of mouse T helper cell IV: TH2 clones secrete a factor that inhibits cytokine production by TH1 clones. J. Exp. Med. 170:2081‐2095.
   Fiorentino, D.F., Zlotnik, A., Vieira, P., Mosmann, T.R., Howard, M., Moore, K.W., and O'Garra, A. 1991. IL‐10 acts on the antigen‐presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444‐3451.
   Go, N.F., Castle, B.E., Barrett, R., Vieira, P., Kastelein, R., Dang, W., Mosmann, T.R., Moore, K.W., and Howard, M. 1990. Interleukin 10 (IL‐10) a novel B cell stimulatory factor: Unresponsiveness of “XID” B cells. J. Exp. Med. 172:1625‐1631.
   Hsu, D‐H., de Waal Malefyt, R., Fiorentino, D.F., Dang, M‐N., Vieira, P., De Vries, J., Spits, H., Mosmann, T.R., and Moore, K.W. 1990. Expression of interleukin‐10 activity by Epstein‐Barr virus protein BCRFI. Science 250:830‐832.
   MacNeil, I., Suda, T., Moore, K.W., Mosmann, T.R., and Zlotnik, A. 1990. Interleukin 10: A novel cytokine growth cofactor for mature and immature T cells. J. Immunol. 145:4167‐4173.
   Moore, K.W., Vieira, P., Fiorentino, D.F., Trounstine, M.L., Khan, T.A., and Mosmann, T.R. 1990. Homology of cytokine synthesis inhibitory factor (IL‐10) to the Epstein‐Barr virus gene BCRFI. Science 248:1230‐1234.
   Mosmann, T.R. and Coffman, R.L. 1989. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv. Immunol. 46:111‐147.
   Mosmann, T.R., Schumacher, J.H., Fiorentino, D.F., Leverah, J., Moore, K.W., and Bond, M.W. 1990. Isolation of MAbs specific for IL‐4, IL‐5, and IL‐6, and a new TH2‐specific cytokine, Cytokine Synthesis Inhibitory Factor (CSIF, IL‐10), using a solid phase radioimmunoadsorbent assay. J. Immunol. 145:2938‐2945.
   Thompson‐Snipes, L., Dhar, V., Bond, M.W., Mosmann, T.R., Moore, K.W., and Rennick, D. 1991. Interleukin 10: A novel stimulatory factor for mast cells and their progenitors. J. Exp. Med. 173:507‐510.
   Vieira, P., de Waal‐Malefyt, R., Dang, M.‐N., Johnson, K.E., Kastelein, R., Fiorentino, D.F., de Vries, J.E., Roncarolo, M.‐G., Mosmann, T.R., and Moore, K.W. 1991. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein‐Barr virus open reading frame BCRF1. Proc. Natl. Acad. Sci. U.S.A. 88:1172‐1176.
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