Measurement of Interleukin‐13

Angus Lauder1, Andrew N.J. McKenzie1

1 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.18
DOI:  10.1002/0471142735.im0618s46
Online Posting Date:  February, 2002
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Abstract

This unit describes two protocols that can be used to quantitate interleukin‐13 (IL‐13). The enzyme‐linked immunosorbent assay (ELISA) has the advantage of being highly specific for human IL‐13 and does not recognize other cytokines present in the sample. A bioassay is also presented based on the ability of IL‐13 to stimulate the growth of the B9 plasmacytoma cell line. The bioassay method can be used to detect both mouse and human IL‐13. B9 cells are dependent on IL‐6 for growth and will also respond to IL‐4. Thus, although B9 cells can readily be used to quantify IL‐13 in the absence of other cytokines, neutralizing antibodies must be incorporated into the bioassay when other cytokines are present in the sample.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Human and Mouse IL‐13 Using ELISA
  • Alternate Protocol 1: Detection of Mouse or Human IL‐13 by B9/Hybridoma Bioassay
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Detection of Human and Mouse IL‐13 Using ELISA

  Materials
  • Coating antibody: purified anti‐human IL‐13 MAb (JES10‐35G12 rat IgG 2a; available from ATCC with permission from DNAX) or anti‐human monoclonal antibody clone MAB213 (R & D Systems). For murine IL‐13 ELISA, use anti‐mouse monoclonal antibody clone MAB413 (R & D Systems).
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 ng/ml IL‐13 standard (PeproTech)
  • Complete RPMI‐10 medium ( appendix 2A)
  • Test samples
  • Blocking solution: 20% (v/v) FBS (heat inactivated 1 hr at 56°C) in PBS (filter sterilize and store at 4°C)
  • Wash buffer: 0.05% (v/v) Tween 20 in PBS (store ≤2 weeks at room temperature)
  • Biotin‐conjugated second antibody (JES10‐2E10 anti‐human IL‐13; from ATCC with permission from DNAX; unit 5.3) or biotinylated anti‐human polyclonal antibody clone BAF213 (R & D Systems). For murine IL‐13 ELISA, use biotinylated anti‐mouse polyclonal antibody clone BAF413 (R & D Systems).
  • Second antibody dilution buffer: 1% BSA (w/v)/0.05% Tween 20 (v/v) in PBS
  • Horseradish peroxidase (HRPO)–conjugated streptavidin (Jackson Immunoresearch)
  • recipeSubstrate solution (see recipe)
  • 96‐well round‐bottom polyvinyl microtiter plates (Dynatech)
  • Microtiter plate washer (e.g., Elcawash from Elcatech) or plastic squirt bottle
  • Microtiter plate reader (Dynatech)

Alternate Protocol 1: Detection of Mouse or Human IL‐13 by B9/Hybridoma Bioassay

  • B9 cells in log‐phase growth (unit 6.6)
  • 96‐well, flat‐bottom polystyrene tissue culture plate, sterile
  • 15‐ml screw‐cap centrifuge tube, sterile
  • V‐bottom reservoir trays, sterile
  • Additional reagents and equipment for B9 cell maintenance (unit 6.6), cell counting ( appendix 3A), quantitation of DNA synthesis by MTT colorimetric assay (unit 6.3), and labeling cells for [3 H]thymidine uptake ( appendix 3D)
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Literature Cited

Literature Cited
   Brown, K.D., Zurawski, S.M., Mosmann, T.R., and Zurawski, G. 1989. A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocytes and fibroblast‐derived inflammatory agents, growth factors, and indicators of various activation processes. J. Immunol. 142:679‐687.
   de Waal Malefyt, R., Figdor, C., Huijbens, R., Mohan‐Peterson, S., Bennett, B., Culpepper, J., Dang, W., Zurawski, G., and de Vries, J.E. 1993. Effects of IL‐13 on phenotype, cytokine production, and cytotoxic function of human monocytes. J. Immunol. 151:6370‐6381.
   Doherty, T.M., Kastelein, R., Menon, S., Andrade, S., and Coffman, R.L. 1993. Modulation of murine macrophage function by interleukin‐13. J. Immunol. 151:7151‐7160.
   Emson, C.L., Bell, S.E., Jones, A., Wisden, W., and McKenzie, A.N.J. 1998. Interleukin (IL)‐4‐independent induction of immunoglobulin (Ig)E, and perturbation of T cell development in transgenic mice expressing IL‐13. J. Exp. Med. 188:399‐404.
   Grunig, G., Warnock, M., Wakil, A.E., Venkayya, R., Brombacher, F., Rennick, D.M., Sheppard, D., Mohrs, M., Donaldson, D.D., Locksley, R.M., and Corry, D.B. 1998. Requirement for IL‐13 independently of IL‐4 in experimental asthma. Science 282:226‐2263.
   Kapp, U., Yeh, W.C., Patterson, B., Elia, A.J., Kagi, D., Ho, A., Hessel, A., Tipsword, M., Williams, A., Mirtsos, C., Itie, A., Moyle, M., and Mak, T.K. 1999. Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed‐Sternberg cells. J. Exp. Med. 189:1939‐1945.
   Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y.F., Miyazono, K., Urabe, A., and Takaku, F. 1989. Establishment and characterization of a unique human cell line that proliferates dependently on GM‐CSF, IL‐3, or erythropoietin. J. Cell. Physiol. 140:323‐334.
   Lai, Y.H. and Mosmann, T.R. 1999. Mouse IL‐13 enhances antibody production in vivo and acts directly on B cells in vitro to increase survival and hence antibody production. J. Immunol. 162:78‐87.
   McKenzie, A.N.J., Culpepper, J.A., de Waal Malefyt, R., Briere, F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B.G., Menon, S., de Vries, J.E., Banchereau, J., and Zurawski, G. 1993. Interleukin‐13, a novel T cell‐derived cytokine that regulates human monocyte and B cell function. Proc. Natl. Acad. Sci. U.S.A. 90:3735‐3739.
   McKenzie, G.J., Fallon, P.G., Emson, C.L., Grencis, R.K., and McKenzie, A.N.J. 1999. Simultaneous disruption of interleukin (IL)‐4 and IL‐13 defines individual roles in T helper cell type 2‐mediated responses. J. Exp. Med. 189:1565‐1572.
   Minty, A., Chalon, P., Derocq, J.‐M., Dumont, X., Guillemot, J.‐C., Kaghad, K., Labit, C., Leplatois, P., Liauzun, P., Miloux, B., Minty, C., Casellas, P., Loison, G., Lupker, J., Shire, D., Ferrara, P., and Caput, D. 1993. Interleukin‐13: A novel human lymphokine regulating inflammatory and immune responses. Nature 362:248‐250.
   Montaner, L.J., Doyle, A.G., Collin, M., Herbein, G., Illei, P., James, W., Minty, A., Caput, D., Ferrar, P., and Gordon, S. 1993. Interleukin‐13 inhibits human immunodeficiency virus type 1 production in primary blood‐derived human macrophages in vitro. J. Exp. Med. 178:743‐747.
   Punnonen, J., Aversa, G., Cocks, B.G., McKenzie, A.N.J., Menon, S., Zurawski, G., de Waal Malefyt, R., and de Vries, J.E. 1993. Interleukin‐13 induces interleukin‐4‐independent IgG4 and IgE synthesis and CD23 expression by B cells. Proc. Natl. Acad. Sci. U.S.A. 90:3730‐3735.
   Skinnider, B.F., Elia, E.J., Gascoyne, R.D., Trumper, L.H., Von Bonin, F., Kapp, U., Patterson, B., Snow, B.E., and Mak, T.K. 2001. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed‐Sternberg cells of Hodgkin lymphoma. Blood 97:250‐255.
   Tomkinson, A., Duez, C., Cieslewicz, G., Pratt, J.C., Joetham, A., Shanafelt, M.C., Gundel, R., and Gelfand, E.W. 2001. A murine IL‐4 receptor antagonist that inhibits IL‐4‐ and IL‐13‐induced responses prevents antigen‐induced airway eosinophilia and airway hyperresponsiveness. J. Immunol. 166:5792‐5800.
   Wills‐Karp, M., Luyimbazi, J., Xu, X., Schofield, B., Neben, T.Y., Karp, C.L., and Donaldson, D.D. 1998. Interleukin 13: Central mediator of allergic asthma. Science 282:225‐2261.
   Zurawski, S.M., Vega, Jr. F., Huyghe, B., and Zurawski, G. 1993. Receptors for interleukin‐13 and interleukin‐4 are complex and share a novel component that functions in signal transduction. EMBO J. 12:3899‐3905.
Key Reference
   McKenzie et al., A.N.J. 1993. See above.
  Describes cloning and initial characterization of human IL‐13.
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