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

  • 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
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Key Reference
   McKenzie et al., A.N.J. 1993. See above.
  Describes cloning and initial characterization of human IL‐13.
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