Measurement of Human and Mouse Interleukin‐12

Maurice K. Gately1, Richard Chizzonite1, David H. Presky1

1 Hoffmann‐La Roche Inc., Nutley, New Jersey
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.16
DOI:  10.1002/0471142735.im0616s15
Online Posting Date:  May, 2001
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Abstract

This unit describes functional assays for measurement of bioactive IL‐12 and ELISAs for measurement of IL‐12 protein. The functional assays are based on the ability of IL‐12 to stimulate proliferation of PHA‐activated T lymphoblasts (“PHA blasts”). The ELISAs are technically simpler to perform than the functional assays, but cannot distinguish bioactive from inactive cytokine.

     
 
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Table of Contents

  • Basic Protocol 1: Antibody‐Capture Bioassay for IL‐12 Activity
  • Alternate Protocol 1: Lymphoblast Proliferation Assay for IL‐12 Activity
  • Support Protocol 1: Preparation of PHA‐Activated Human Lymphoblasts
  • Basic Protocol 2: ELISA for Human and Mouse IL‐12 p75 Heterodimer
  • Alternate Protocol 2: ELISA for Human and Mouse IL‐12 p40 Subunit
  • Support Protocol 2: Conjugation of Antibody with Horseradish Peroxidase
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Antibody‐Capture Bioassay for IL‐12 Activity

  Materials
  • Coating antibody: rat monoclonal anti–human IL‐12 antibody 2‐4A1 or rat monoclonal anti–mouse IL‐12 antibody 5C3 (both available from R. Chizzonite, Hoffmann‐La Roche; see appendix 55)
  • Coating buffer: use recipe in unit 7.12 but adjust to pH 9.5
  • Blocking solution: 1% (w/v) BSA in Dulbeccos phosphate‐buffered saline with Ca+2 and Mg+2 (D‐PBS; Life Technologies)
  • recipeIL‐12 reference standard (see recipe)
  • recipeSupplemented medium (see recipe)
  • Unknown sample containing IL‐12
  • 2 × 105 cell/ml PHA‐activated human lymphoblast suspension (see protocol 3)
  • Multichannel and repeating pipettors
  • 96‐well EIA plates with lids (e.g., Nunc Immuno‐plate MaxiSorp)
  • 17 × 100–mm polystyrene tubes
  • Additional reagents and equipment for labeling cells and determining [3H]thymidine incorporation ( appendix 3D) and for quantitation of interleukin activity (units 6.3 & 6.13)

Alternate Protocol 1: Lymphoblast Proliferation Assay for IL‐12 Activity

  • 4 × 105 cell/ml PHA‐activated human lymphoblast suspension (see protocol 3)
  • 96‐well flat‐bottom microtiter plates with lids (e.g., Costar)

Support Protocol 1: Preparation of PHA‐Activated Human Lymphoblasts

  Materials
  • recipeSupplemented medium (see recipe)
  • 10 mg/ml phytohemagglutinin‐P (PHA‐P; Difco Laboratories; reconstitute lyophilized material according to manufacturer's instructions)
  • Human recombinant IL‐2 (Table 97.80.4711)
  • 75‐cm2 tissue culture flasks
  • 15‐ml conical centrifuge tube
  • Additional reagents and equipment for collecting human peripheral blood cells ( appendix 3F), isolating human PBMC (unit 7.1), and counting viable cells ( appendix 3B)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Basic Protocol 2: ELISA for Human and Mouse IL‐12 p75 Heterodimer

  Materials
  • recipeELISA coating antibody (see recipe) appropriate for human or mouse IL‐12 heterodimer
  • Coating buffer: use recipe in unit 7.12 but adjust to pH 9.5
  • recipeWash buffer (see recipe)
  • recipeBlocking solution (see recipe)
  • recipeIL‐12 reference standard (see recipe)
  • Unknown sample containing IL‐12
  • recipeAssay buffer (see recipe)
  • Rat monoclonal anti–human IL‐12 p40 subunit antibody 4D6 (available from R. Chizzonite, Hoffmann‐La Roche) or rat monoclonal anti–mouse Il‐12 p40 subunit antibody 5C3 (available from D. Presky, Hoffman‐La Roche), conjugated with HRPO (see protocol 6)
  • Chromogenic substrate for detecting HRPO (e.g., K‐Blue Substrate, ELISA Technologies), prepared according to manufacturer's instructions
  • Stop solution appropriate for substrate employed (e.g., 1 M H 3PO 4 for K‐Blue Substrate)
  • Multichannel and repeating pipettors
  • 96‐well EIA plates with plate sealers (e.g., Immulon II, Dynatech)
  • ELISA plate reader (e.g., Vmax, Molecular Dynamics) with appropriate filter for HRPO substrate employed

Alternate Protocol 2: ELISA for Human and Mouse IL‐12 p40 Subunit

  Materials
  • Antibody to be conjugated (0.5 to 4 mg/ml stock solution)
  • 50 mM NaHCO 3 (pH 8.0; adjust with 10 N NaOH), 4°C
  • Horseradish peroxidase (HRPO; purity grade 1, Boehringer‐Mannheim)
  • 0.1 M sodium periodate (107 mg NaIO 4 dissolved in 5 ml distilled H 2O)
  • 0.5 M ethylene glycol (0.15 ml ethylene glycol dissolved in 5 ml of distilled H 2O)
  • 5 mM sodium acetate (pH 4.5), 4°C
  • 0.4 M NaHCO 3, pH 10.5 (adjust with 10 N NaOH)
  • 0.1 M sodium borohydride (37 mg NaBH 4 dissolved in 10 ml distilled H 2O)
  • 0.154 M sodium chloride
  • recipeStock buffer (see recipe)
  • Dialysis membranes (15,000 MWCO, 16‐mm diameter and 50,000 MWCO, 22‐mm diameter)
  • Additional reagents and equipment for dialysis ( appendix 3H)
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Trinchieri, G. 1994. See above.
  An excellent review of all facets of IL‐ 12 biology, biochemistry, and molecular biology.
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