Measurement of Human and Mouse Interleukin 18

Tomohiro Yoshimoto1, Hiroko Tsutsui1, Haruki Okamura1, Kenji Nakanishi1

1 Hyogo College of Medicine, Hyogo, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.26
DOI:  10.1002/0471142735.im0626s44
Online Posting Date:  November, 2001
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IL‐18, originally designated as interferon‐γ (IFN‐γ)‐inducing factor (IGIF), is a pleiotropic cytokine secreted by activated macrophages and Kupffer cells. The major activity associated with this cytokine is induction of IFN‐γ production from T cells, B cells, and NK cells, especially in collaboration with IL‐12. IL‐18 is synthesized without a signal peptide and must be enzymatically cleaved to become active. Therefore, it is important to determine whether the produced IL‐18 is an active or precursor form. This unit describes functional assays for measurement of bioactive human and mouse IL‐18 and ELISAs for measurement of murine and human IL‐18 proteins. The functional assays are based on the induction of IFN‐γ production by IL‐18. The ELISA measures the concentration of human or mouse IL‐18. Using a combination of monoclonal antibodies against human or mouse IL‐18, the proform and/or mature form of IL‐18 can be detected by ELISA.

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Table of Contents

  • Basic Protocol 1: IFN‐γ Induction Assay to Quantitate Mouse and Human IL‐18
  • Basic Protocol 2: ELISA for Human and Mouse IL‐18
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: IFN‐γ Induction Assay to Quantitate Mouse and Human IL‐18

  • Mice (BALB/c or C57BL/6 strain)
  • Anti‐asialo GM1 antibody (Wako)
  • Mouse IL‐12 (e.g., R & D Systems)
  • Mouse IL‐18 reference standard (commercially available from MBL International)
  • recipeComplete RPMI‐10 medium (see recipe)
  • Unknown sample for assay of IL‐18
  • Mouse or human IFN‐γ ELISA kits (e.g., R & D Systems)
  • 17 × 100‐mm polystyrene tubes
  • 96‐well flat‐bottom microtiter plates with lids (e.g. Costar)
  • 24‐well plates (e.g., Falcon)
  • Multichannel and repeating pipettors
  • Additional reagents and supplies for intravenous injection of mice (unit 1.6) and preparing mouse splenic T cells (unit 3.2)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.

Basic Protocol 2: ELISA for Human and Mouse IL‐18

  • recipeELISA coating antibody (see recipe) appropriate for human or mouse IL‐18
  • Coating buffer: PBS ( appendix 2A), pH 7.4
  • Washing buffer: Dulbecco's phosphate‐buffered serum (D‐PBS; e.g., Life Technologies) containing 0.01% (v/v) Tween 20; store up to 1 week at room temperature
  • Blocking solution: Dulbecco's phosphate‐buffered serum (D‐PBS; e.g., Life Technologies) containing 1% (w/v) bovine serum albumin (BSA); store up to 4 weeks at 4°C
  • IL‐18 reference standard (commercially available from MBL International)
  • Unknown sample containing IL‐18
  • recipeAssay buffer (see recipe)
  • recipeConjugation buffer (see recipe)
  • Peroxidase conjugated rat anti‐human IL‐18 monoclonal antibody (159‐12B) or anti‐mouse IL‐18 monoclonal antibody (93‐10C): available from MBL International
  • Chromogenic substrate for detecting peroxidase: e.g., tetramethylbenzidine (TMB)/H 2O 2 (Becton Dickinson), prepared according to manufacturer's instructions
  • Stop solution appropriate for substrate employed (e.g., 2 N H 2SO 4)
  • Multichannel and repeating pipettors
  • 96‐well EIA plates with plate sealers (e.g., EIA/RIA plate from Costar)
  • ELISA plate reader (e.g., Benchmark, Bio‐Rad) with appropriate filter for peroxidase substrate employed
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Literature Cited

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Key Reference
   Okamura, H. et al. 1995. See above.
  An original paper of IL‐18.
   Nakanishi, K. et al., 2001b. See above.
  Excellent reviews of all facts of IL‐18 biology, biochemistry, and molecular biology.
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