Immunoenzymetric Assay of Mouse and Human Cytokines Using NIP‐Labeled Anti‐Cytokine Antibodies

John S. Abrams1

1 DNAX Research Institute, Palo Alto, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.20
DOI:  10.1002/0471142735.im0620s13
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a general method for immunoenzymetric assay of mouse and human cytokines. The technique is based on use of two anti‐cytokine monoclonal antibodies (MAbs), each specific for a spatially distinct determinant on the cytokine. One of these is a coating antibody, and the other is a derivatized detecting antibody. A support protocol describes chemical labeling of anti‐cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. Another support protocol describes a method for producing horseradish peroxidase (HRPO)‐conjugated J4, a rat IgG1 anti‐NIP monoclonal antibody that confers the anti‐NIP specificity to the HRPO detection system used in the immunoenzymetric assay. The method described in this unit has allowed successful measurement of different cytokines in a wide variety of clinical samples including conditioned medium, serum and plasma, ascites, amniotic fluid, and bronchoalveolar lavage fluid.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Immunoenzymetric Assay for Cytokines Using NIP‐ and HRPO‐Labeled Antibodies
  • Support Protocol 1: Preparation of NIP‐Labeled Anti‐Cytokine Antibodies
  • Support Protocol 2: Preparation of HRPO‐Conjugated Anti‐NIP Monoclonal Antibody
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Immunoenzymetric Assay for Cytokines Using NIP‐ and HRPO‐Labeled Antibodies

  Materials
  • Coating antibody specific to cytokine of interest at appropriate concentration (Table 6.20.1) in recipe1× PBS (see recipe)
    Table 6.0.1   MaterialsMaterials and Conditions for Cytokine Immunoenzymetric Assay

    Cytokine a High stand. conc. b (ng/ml) Plate type c Coating Ab Detecting Ab
    Designation Conc. (µg/ml) Source Designation Source
    Human
    IL‐2 5 2 MQ1‐17H12 5 A1 Polyclonal anti‐IL‐2 IgG GN, PE f
    IL‐3 5 1 BVD8‐3G11 1 A3 BVD3‐1F9 A3
    IL‐4 5 1 8D4.8 2 PG MP4‐25D2 A2, EN, PG
    IL‐5 5 1 JES1‐39D10 2 A1, PG, PM JES1‐5A10 A1, PG, PM
    IL‐6 5 2 MQ2‐39C3 5 A1 MQ2‐13A5 A1
    IL‐7 10 2 BVD10‐40F6 2 A1, PG, PM BVD10‐11C10 A1, PG, PM
    IL‐10 10 2 JES3‐9D7 5 A1, EN, PG, PM JES3‐12G8 A1, EN, PG, PM
    IL‐13 50 2 JES10‐35G12 5 A1 JES10‐2E10 A1
    G‐CSF 50 2 BVD13‐3A5 5 A3 BVD11‐37G10 A3
    GM‐CSF 5 1 BVD2‐23B6 5 A2, PG, PM BVD2‐21C11 A2, PG, PM
    IFN‐γ 25 2 A35 5 A3 B27 A3
    TNF 25 2 MP9‐20A4 5 A3 GMO1‐1782 A3
    Mouse
    IL‐2 5 2 JES6‐1A12 2 A1, EN, PG, GN, BI JES6‐5H4 A1, EN, PG, BI
    IL‐3 5 2 MP2‐8F8 5 A1, EN, PG, GN, OS, UBI MP2‐43D11 A1, EN, PG
    IL‐4 5 2 BVD4‐1D11 1 A1, EN, PG, BI BVD6‐24G2 A1, EN, PG, BI
    IL‐5 10 2 TRFK5 5 A1, EN, PG, GN, UBI TRFK4 A1, EN, PG, GN
    IL‐6 5 2 MP5‐20F3 5 A1, EN, PG, GN, BI, OS, UBI MP5‐32C11 A1, EN, PG, BI
    IL‐9 5 2 D9302C12.10 5  — g PLH1‐19E2 A3
    IL‐10 5 2 JES5‐2A5 5 A1, EN, PG, GN, BI, UBI SXC1 A1, EN, PG, BI
    GM‐CSF 5 2 MP1‐22E9 5 A1, EN, PG, GN, OS, UBI MP1‐31G6 A1, EN, PG
    IFN‐γ 5 2 R46A2 5  — h AN18  — j
    LIF 10 2 MP8‐10C8 5 A3 MP8‐15F2 A3
    M‐CSF 10 2 5A1 5  — k D24  — k
    TNF 10 2 MP6‐XT3 5 A1, EN, PG, BI MP6‐XT22 A1, EN, PG, BI, UBI

     aCytokines are available from various suppliers—e.g., Amgen Biologicals, Biosource International, and Genzyme. See appendix 55 for addresses and phone numbers of suppliers.
     bI.e., highest concentration in standard curve; 2‐fold serial dilutions are then performed. See Critical Parameters for details on obtaining and storing cytokine standards.
     c(1) Immulon 1 microtiter plates, (2) Immulon 2 microtiter plates (both from Dynatech). Used only in robotic assays where rigid plates are required; otherwise use 96‐well U‐bottom PVC plates (Dynatech) for all cytokines.
     dAbbreviations: A1, ATCC Patent Deposit (Budapest Treaty; permission from DNAX Research Institute required to acquire material); A2, ATCC General Deposit (no permission required); A3, future ATCC Patent Deposit; BI, Biosource International; EN, Endogen; GN, Genzyme; OS, Oncogene Science; PE, Peprotech; PG, Pharmingen; PM, Promega; UBI, Upstate Biotechnology. See appendix 55 for addresses and phone numbers of suppliers.
     eThe ATCC (A1, A2, and A3) provides monoclonal cell lines rather than purified Abs.
     fThe author uses an in‐house polyclonal anti‐IL‐2 IgG as the detecting Ab for human IL‐2. As the commercial Abs have not been used by the author, they should be tested empirically.
     gFor information, contact J. Van Snick, Ludwig Institute for Cancer Research, Catholic University of Louvain, Brussels, Belgium.
     hSpitalny and Havel, for information contact G.L. Spitalny at Targetech, Inc., Meriden, Connecticut.
     JSlade and Langhorne, ; for information contact J. Langhorne at Max Planck Institute for Immunology, Fribourg, Germany.
     kLockeshwar and Lin, ; for information contact B.L. Lockeshwar at University of Miami, Florida.
  • Sample to be assayed for cytokine
  • recipeImmunoassay diluent (see recipe)
  • Cytokine standard (see Critical Parameters and Table 6.20.1) stored in 500‐µl aliquots at −80°C
  • Cytokine calibrator (e.g., conditioned medium from stimulated T cell clone containing known amount of cytokine; optional; store in aliquots at −80°C)
  • recipe1× wash buffer (see recipe)
  • Detecting antibody specific to cytokine of interest (Table 6.20.1) labeled with NIP (see protocol 2)
  • recipeDetecting buffer (see recipe)
  • HRPO‐conjugated anti‐NIP monoclonal antibody (see protocol 3)
  • recipeABTS substrate working solution (see recipe)
  • Multichannel pipettor and tips
  • Assay plates (96‐well U‐bottom microtiter plates; see Table 6.20.1)
  • Preparation plates (any 96‐well microtiter plates)
  • Spin‐X microfiltration device (0.45 µm nylon filter; Costar)
  • Microtiter plate sealers (e.g., Dynatech)
  • Microtiter plate reader with computer interface (e.g., Vmax or Emax from Molecular Devices)
  • Curve‐fitting software (e.g., Softmax from Molecular Devices)

Support Protocol 1: Preparation of NIP‐Labeled Anti‐Cytokine Antibodies

  Materials
  • Detecting antibody appropriate to cytokine of interest (Table 6.20.1)
  • recipe1× PBS (see recipe)
  • recipeNIP‐O‐Su solution (see recipe)
  • Dialysis tubing (e.g., Spectra/Por, MWCO 12,000 to 14,000; Spectrum)
  • 0.22‐µm filters
  • Additional reagents and equipment for dialysis ( appendix 3H)

Support Protocol 2: Preparation of HRPO‐Conjugated Anti‐NIP Monoclonal Antibody

  • 10 mg/ml horseradish peroxidase (HRPO; Type VI; Sigma) in recipesodium acetate solution (see recipe)
  • 75 mM sodium metaperiodate (prepare fresh)
  • 1.6 M ethylene glycol
  • recipeBorate‐buffered saline (see recipe)
  • 10 mg/ml J4 (rat IgG1 MAb anti‐NIP; ATCC Budapest Treaty Patent Deposit, or Promega) in recipeborate‐buffered saline (see recipe)
  • recipeSodium cyanoborohydride solution (see recipe)
  • 1 µg/ml NIP‐labeled MAb in recipe1× PBS
  • 13 × 100–mm borosilicate glass tubes
  • Dialysis tubing (e.g., Spectra/Por, MWCO 12,000 to 14,000; Spectrum)
  • Additional reagents and equipment for dialysis ( appendix 3H)
NOTE: Contact DNAX Research Institute for permission to acquire items from ATCC Budapest Treaty Patent Deposit.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abrams, J.S., Roncarolo, M.‐G., Yssel, H., Andersson, U., Gleich, G.J., and Silver, J. 1992. Strategies of anti‐cytokine monoclonal antibody development: Immunoassay of IL‐10 and IL‐5 in clinical samples. Immunol. Rev. 127:5‐24.
   Butterfield, J.H., Leifeman, K.M., Abrams, J.S., Silver, J.E., Bower, J., Gonchorff, N., and Gleich, G.J. 1992. Elevated serum levels of interleukin‐5 in patients with the syndrome of episodic angioedema and eosinophilia. Blood 79:688‐692.
   Chan, D.W. 1987. General principle of immunoassay. In Immunoassay: A Practical Guide (D.W. Chan and M.T. Perlstein, eds.) pp. 4‐5. Academic Press, Orlando, Fla.
   Gotlieb, W.H., Abrams, J.S., Watson, J.M., Velu, T., Berek, J.S., and Martinez‐Masa, O. 1992. Presence of IL‐10 in the ascites of patients with ovarian and other intra‐abdominal cancers. Cytokine 4:385‐390.
   Heyborne, K.D., McGregor, J.A., Henry, G., Witkin, S.S., and Abrams, J.S. 1994. Interleukin‐10 in amniotic fluid at midtrimester: Immune activation and suppression in relaton to fetal growth. Am. J. Obstet. Gynecol. 171:55‐59.
   Hirayama, K., Abrams, J.S., Quinn, J.J., and Harn, D.A. 1994. Heterogeneity of antigen‐specific CD4+ T cell clones from a patient with Schistosomiasis mansoni. Parasite Immunol. 16:561‐569.
   Johnson, G.F. 1987. Data reduction techniques for immunoassay. In Immunoassay: A Practical Guide (D.W. Chan and M.T. Perlstein, eds.) pp. 129‐147. Academic Press, Orlando, Fla.
   Limaye, A.P., Abrams, J.S., Awadzi, K., Francis, H.F., Silver, J.E., Ottesen, E.A., and Nutman, T.B. 1991. Interleukin‐5 and the post‐treatment eosinophilia in patients with onchocerciasis. J. Clin. Invest. 88:1418‐1421.
   Lokeshwar, B.L. and Lin, H. 1988. Development and characterization of monoclonal antibodies to murine macrophage colony‐stimulating factor. J. Immunol. 141:483‐488.
   Sander, B., Hoiden, I., Andersson, U., Moller, E., and Abrams, J.S. 1993. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen: Cytokine detection by immunoassay and intracellular immunostaining. J. Immunol. Methods 166:201‐214.
   Sedgwick, J.B., Calhoun, W.J., Gleich, G.J., Kita, H., Abrams, J.S., Schwartz, L.B., Volovitz, B., Ben Yaakov, M., and Busse, W.W. 1991. Immediate and late allergic airway response of allergic rhinitis patients to segmental antigen challenge: Characterization of eosinophil and mast cell mediators. Amer. Rev. Respir. Dis. 144:1274‐1281.
   Slade, S.J. and Langhorne, J. 1989. Production of interferon‐gamma during infection of mice with Plasmodium chabaudi chabaudi. Immunobiology 179:353‐365.
   Spitalny, J.L. and Havell, E.A. 1984. Monoclonal antibody to murine γ‐interferon inhibits lymphokine‐induced antiviral and macrophage tumoricidal activation. J. Exp. Med. 159:1560‐1565.
   van Haelst Pisani, C., Kovach, J.S., Kita, H., Leiferman, K.M., Gleich, G.J., Silver, J.E., and Abrams, J.S. 1991. Administration of interleukin‐2 (IL‐2) results in increased plasma concentrations of IL‐5 and eosinophilia in patients with cancer. Blood 78:1538‐1544.
   Wilson, M.B. and Nakane, P.K. 1978. Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofluorescence and Related Staining Techniques (W. Knapp, K. Holubar, and G. Wick, eds.) pp. 215‐224. Elsevier/North Holland Biomedical Press, Amsterdam.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library