Measurement of Human Interleukin 11

Frann Bennett1, JoAnn Gianotti1, Abbie Celniker1, Katherine J. Turner1, Steven C. Clark1

1 Genetics Institute, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.15
DOI:  10.1002/0471142735.im0615s18
Online Posting Date:  May, 2001
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Abstract

This unit describes an ELISA and a cell proliferation assay that can be used, respectively, to measure the protein level or biologic activity of human and murine interleukin 11 (IL‐11). The bioassay is based on the ability of IL‐11 to support growth of the B9‐11 cell line, a subline of B9 that has traditionally been used to measure levels of IL‐6. B9‐11 is substantially more responsive to IL‐11 than the T10 line used in older protocols. This new bioassay therefore provides improved sensitivity, with a detection limit of ˜20 pg/ml. An alternate procedure is provided that employs neutralizing antibodies in the cell proliferation bioassay to use to ensure that the activity of the desired molecule (IL‐11) is being measured in samples containing multiple cytokines. A describes maintenance of B9‐11 cells.

     
 
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Table of Contents

  • Basic Protocol 1: Enzyme‐Linked Immunosorbent Assay (ELISA) for Human IL‐11
  • Basic Protocol 2: Cell Proliferation Assay for IL‐11 Activity
  • Alternate Protocol 1: Detection of IL‐11 in Mixed Cytokine Samples
  • Support Protocol 1: Maintenance of B9‐11 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Enzyme‐Linked Immunosorbent Assay (ELISA) for Human IL‐11

  Materials
  • Coating antibody: 11h3/19.6.1 anti‐IL‐11 MAb (Genetics Institute; direct all requests to Biological Sample Request Coordinator)
  • recipeCoating buffer (see recipe)
  • recipeTHST (see recipe)
  • recipeTHSG (see recipe), 37°C
  • IL‐11 standard: dilute recipe1 µg/ml IL‐11 reference standard (see recipe) to 150 µg/ml in recipeTHST (store up to 1 month at −80°C)
  • 50% and 25% (v/v) pooled normal human serum/ recipeTHST (optional; use if testing human serum samples)
  • Samples to be assayed for IL‐11
  • Biotinylated llh3/15.6.13 anti‐IL‐11 MAb (Genetics Institute)
  • Avidin–alkaline phosphatase conjugate (Zymed)ELISA Amplification System (Life Technologies) containing: substrate for alkaline phosphatase: lyophilized NADPH amplifier for alkaline phosphatase: lyophilized alcohol dehydrogenase and diaphorase
  • Quench solution: 0.3 M H 2SO 4
  • EIA capture plate: 96‐well flat‐bottom microtiter plate (e.g., Costar)
  • 12‐channel pipettor
  • EIA plate washer (e.g., Dynatech)
  • Polypropylene micro test tubes racked in 8 × 12 format (e.g., Titertube from Bio‐Rad)
  • Acetate microtiter plate sealers (e.g., Costar)
  • EIA plate reader with 490‐nm filter (e.g., Vmax from Molecular Devices)
  • ELISA curve‐fitting software (e.g., SOFTmax from Molecular Devices)

Basic Protocol 2: Cell Proliferation Assay for IL‐11 Activity

  Materials
  • Complete RPMI‐5 medium ( appendix 2A)
  • recipe1 µg/ml human IL‐11 reference standard (see recipe)
  • Unknown sample(s) containing IL‐11 (but no other cytokines)
  • B9‐11 cell culture (see protocol 4)
  • Multichannel pipettor and tips
  • 96‐well round‐bottom tissue culture–treated microtiter plates with lids (e.g., Costar)
  • IEC HN‐SII centrifuge (or equivalent)
  • Additional reagents and equipment for determining number of viable cells by trypan blue exclusion ( appendix 3B), labeling cells and determining [3H]thymidine incorporation ( appendix 3D), and quantitation of interleukin activity (units 6.3 & 6.13)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Alternate Protocol 1: Detection of IL‐11 in Mixed Cytokine Samples

  Materials
  • B9‐11 cells, from Genetics Institute; direct all requests to Biological Sample Request Coordinator; cells available with prior permission of Dr. Bernard Klein, Institute for Molecular Genetics BP5051, 1919 Route de Mende, Montpellier, Cédex 1, France
  • Complete RPMI‐5 medium ( appendix 2A)
  • recipe1 µg/ml human IL‐11 (see recipe for IL‐11 reference standard)
  • 15‐ml conical centrifuge tube
  • 25‐cm2 tissue culture flask
  • IEC HN‐SII centrifuge (or equivalent)
  • Additional reagents and equipment for counting viable cells ( appendix 3B)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.
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Literature Cited

Literature Cited
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