Measurement of TGF‐β in Biological Fluids

Hollie Hale‐Donze1, Sharon M. Wahl1, Raymond J. Jackson2, Jerry R. McGhee2

1 National Institute of Dental and Craniofacial Research, Bethesda, Maryland, 2 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.11
DOI:  10.1002/0471142735.im0611s41
Online Posting Date:  May, 2001
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Abstract

Transforming growth factor (TGF‐β) is an immunoregulatory cytokine produced by many different cell types, including lymphocytes and monocytes‐macrophages. Two assays are described for the quantitation of TGF‐β activity in supernatant fluids. The bioassay for TGF‐β is based on the potent ability of TGF‐β to suppress lymphocyte proliferation. The radioreceptor assay is based on the ability of TGF‐β‐containing supernatant fluids to inhibit the binding of radiolabeled TGF‐β to its cellular receptor. Two support protocols describe acid‐activation and neutralization of TGF‐β.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of TGF‐β1 Using a Flow Cytometry–Based Assay
  • Support Protocol 1: Acid‐Activation of TGF‐β
  • Support Protocol 2: Coupling Microspheres to TGF‐β1 Capture Antibody
  • Support Protocol 3: Removing Amine Groups from Capture Antibody Preparation
  • Support Protocol 4: Determining the Levels of TGF‐β1 in Samples Using the Luminex Multiplexed Analysis
  • Basic Protocol 2: Thymocyte Proliferation Assay for TGF‐β
  • Support Protocol 5: Neutralization of TGF‐β in Acid Activated Supernatant
  • Alternate Protocol 1: Luminescent ELISA Assay to Measure TGF‐β
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of TGF‐β1 Using a Flow Cytometry–Based Assay

  Materials
  • Sample buffer or medium
  • 1 µg/µl recombinant TGF‐β1 standard (rTGF‐β1; R&D Systems), untreated or acid‐activated (see protocol 2)
  • recipeAssay buffer (see recipe)
  • Sample: serum or supernatant, untreated and acid‐activated (see protocol 2)
  • 4 × 104 TGF‐β1 capture antibody‐coupled microspheres/ml (see protocol 3)
  • recipeWash buffer (see recipe)
  • 3 µg/ml biotin‐conjugated TGF‐β1 detection antibody (R&D Systems)
  • 4 µg/ml streptavidin‐FITC (SA‐FITC; Southern Biotech)
  • Fixative: 1% (v/v) formaldehyde in recipephosphate‐buffered saline (PBS; appendix 2A)
  • recipePBS ( appendix 2A)
  • Sonicator (Elma Ultrasonic)
  • 12 × 75–mm polystyrene round‐bottom tubes
  • 200‐µl pipet tips, sterile
  • FACScan flow cytometry system (Becton Dickinson) or equivalent
  • Luminex software (see protocol 5)

Support Protocol 1: Acid‐Activation of TGF‐β

  • recipe1 N HCl ( appendix 2A)
  • 1 N NaOH
  • Litmus paper
IMPORTANT NOTE: Nonactivated samples and standards should be treated in the same manner, substituting recipePBS ( appendix 2A) for both 1 N HCl and 1 N NaOH to control for dilution effects.

Support Protocol 2: Coupling Microspheres to TGF‐β1 Capture Antibody

  • 5 × 107 microsphere/ml (set 90/51; Luminex)
  • recipe100 mM monobasic sodium phosphate, pH 6.0 (see recipe)
  • recipe50 mg/ml N‐hydroxysulfosuccinimide (sulfo‐NHS; see recipe)
  • recipe50 mg/ml 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide⋅HCl (EDC; see recipe)
  • recipePBS, pH 7.0, sterile, azide‐free ( appendix 2A)
  • 250 µg/ml TGF‐β1 capture antibody, free of amine groups (see protocol 4)
  • 3 µg/ml FITC‐conjugated goat anti‐mouse detection antibody (Southern Biotech)
  • Orbital shaker
  • Additional reagents and equipment for determining concentration on a hemocytometer ( appendix 3A).
NOTE: Sterile conditions are required since there can be no azide in the buffers.

Support Protocol 3: Removing Amine Groups from Capture Antibody Preparation

  Additional Materials
  • recipePBS, pH 7.4, sterile, azide‐free ( appendix 2A)
  • PD‐10 columns (Pharmacia)
  • 1 mg/ml TGF‐β1 capture antibody (R & D Systems)
  • Ring stand with clasp
  • 50‐ml polypropylene conical tube
  • Scissors
IMPORTANT NOTE: Sterile conditions are required since there can be no azide in the buffers.

Support Protocol 4: Determining the Levels of TGF‐β1 in Samples Using the Luminex Multiplexed Analysis

  • Calibration microspheres (Luminex)
  • TGF‐β1 standards and samples (see protocol 1)
  • FACscan Luminex R/0 analysis software (Luminex)

Basic Protocol 2: Thymocyte Proliferation Assay for TGF‐β

  Materials
  • Complete RPMI‐5 medium ( appendix 2A) with 1.5 mM HEPES
  • recipe1 µg/ml TGF‐β stock (see recipe)
  • Acid‐activated samples (e.g., culture supernatants, plasma; see protocol 2)
  • 100 U/ml recombinant IL‐1β (Genzyme)
  • 3×107 cells/ml mouse thymocyte preparation (unit 3.1)
  • 4 µg/ml phytohemagglutinin (PHA, Difco)
  • [3H]thymidine
  • 96‐well flat‐bottom microtiter plate (Costar)
  • Additional reagents and equipment for determining [3H]thymidine incorporation ( appendix 3D).

Support Protocol 5: Neutralization of TGF‐β in Acid Activated Supernatant

  • 1 µg/µl anti‐TGF‐β1, ‐TGF‐β2, or ‐TGF‐β3 neutralizing antibody (R & D Systems)
  • Sample, acid‐activated (see protocol 2)
  • Phosphate‐buffered saline ( recipePBS; appendix 2A) or normal sera
  • Corresponding pre‐immune sera (Jackson Immunoresearch Laboratories)

Alternate Protocol 1: Luminescent ELISA Assay to Measure TGF‐β

  • Water‐soluble biotin‐x‐NHS (Calbiochem)
  • Monoclonal mouse‐anti‐TGF‐β1, ‐β2, ‐β3 (Genzyme Diagnostics)
  • Chicken anti‐TGF‐β neutralizing antibody or anti‐TGF‐β2 (R & D Systems) in recipe0.1 M sodium bicarbonate buffer, pH 8.1 (see recipe)
  • Superblock blocking buffer in PBS (Pierce)
  • recipePBS/0.05% Tween‐20 (PBS‐T; see recipe)
  • 2 ng/ml working dilution acid‐activated rTGF‐β (see protocol 2) in recipePBS‐BSA (see recipe)
  • 10 ng/ml working dilution latent and acid‐activated TGF‐β in recipePBS‐BSA (see recipe)
  • recipe3% BSA‐PBS (see recipe)
  • recipePBS‐T (see recipe)
  • recipePBS‐T/2 mM EGTA (see recipe)
  • recipeAquaLite Streptavidin (see recipe)
  • AquaLite Trigger Buffer (SeaLite Sciences) or recipe50 mM Tris/20 mM calcium acetate, pH 7.5 (see recipe)
  • Microlite 2 plate (Dynex Technologies)
  • Luminometer (Dynex)
  • Multichannel pipettor
  • Additional reagents and equipment for dialysis ( appendix 3H).
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Figures

Videos

Literature Cited

Literature Cited
   Abe, M., Harpel, J.G., Metz, C.N., Nunes, I., Loskutoff, D.J., and Rifkin, D.B. 1994. An assay for transforming growth factor‐beta using cells transfected with a plasminogen activator inhibitor‐1 promoter‐luciferase construct. Anal. Biochem. 216:276‐284.
   Brandes, M.E., Allen, J.B., Ogawa, Y., and Wahl, S.M. 1991. Transforming Growth Factor β1 suppresses acute and chronic arthritis in experimental animals. J. Clin. Invest. 87:1108‐1113.
   Chen, W., Jin, W., Cook, M., Weiner, H.L., and Wahl, S.M. 1998. Oral delivery of group A streptococcal cell wall augments circulating TGF‐β and suppresses streptococcal cell wall arthritis. J. Immunol. 161:6297‐6304.
   Derynck, R. and Choy, L. 1998. Transforming growth factor‐β and its receptors. In The Cytokine Handbook (A.W. Thomsom, ed.) pp. 953. Academic Press, Boston.
   Jackson, R.J., Fujuhashi, K., Kiyono, H., and McGhee, J.R. 1996. Luminometry: A novel bioluminescent assay enhances the quantitation of mucosal and systemic antibody responses. J. Immunol. Methods. 190:189‐198.
   Kulkarni, A.B., Huh, C.‐H., Becker, D., Geiser, A., Lyght, M., Flanders, K.C., Roberts, A.B., Sporn, M.B., Ward, J.M., and Karlsson, S. 1993. Transforming growth factor‐β1 null mutation in mice causes excessive inflammatory response and early death. Proc. Natl. Acad. Sci. U.S.A. 90:770‐774.
   Letterio, J.J. and Roberts, A.B. 1998. Regulation of immune responses by TGF‐beta. Annu. Rev. Immunol. 16:137‐161.
   Massague, J. 1998. TGF‐beta signal transduction. Annu. Rev. Biochem. 67:753‐791.
   McCartney‐Francis, N.L. and Wahl, S.M. 1994. Transforming growth factor β: A matter of life and death. J. Leuk. Biol. 55:401‐409.
   McCartney‐Francis, N.L., Frazier‐Jessen, M., and Wahl, S.M. 1998. TGF‐β: A balancing act. Intern. Rev. Immunol. 16:553‐580.
   Miyazono, K., Yuki, K., Takaku, F., Wernstedt, C., Kanzaki, T., Olofsson, A., Hellman, U., and Heldin, C.H. 1990. Latent forms of TGF‐β: Structure and biology. Ann. N.Y. Acad. Sci. 593:51‐58.
   Proetzel, G., Pawlowski, S.A., Wiles, M.V., Yin, M., Boivin, G.P., Howles, P.N., Ding, J., Fergurson, M.W.J., and Doetschman, T.D. 1995. Transforming growth factor TGF‐β3 is required for secondary palate fusion. Nature Genet. 11:409‐414.
   Sanderson, N., Factor, V., Nagy, P., Kopp, J., Kondaiah, P., Wakefield, L., Roberts, A.B., Sporn, M.B., and Thorgeirsson, S.S. 1995. Hepatic expression of mature transforming growth factor‐β1 in transgenic mice results in multiple tissue lesions. Proc. Natl. Acad. Sci. U.S.A. 92:2572‐2576.
   Sanford, L.P., Ormsby, I., Gittenberger‐de Groot, A.C., Sariola, H., Friedman, R., Boivin, G.P., Cardell, E.L., and Doetschman, T. 1997. TGF‐β2 knockout mice have multiple developmental defects that are nonlapping with other TGF‐β phenotypes. Development. 124:2659‐2670.
   Song, X.‐y., Gu, M., Jin, W.‐w., Klinman, D.M., and Wahl, S.M. 1998. Plasmid DNA Encoding Transforming Growth Factor‐β1 Suppresses Chronic Disease in a Streptococcal Cell Wall‐induced Arthritis Model. J. Clin. Invest. 101:2615‐2621.
   Wahl, S.M. 1994. Transforming Growth Factor‐β: The good, the bad, and the ugly. J. Exp. Med. 180:1587‐1590.
   Wahl, S.M. 1999. Transforming Growth Factor‐β (TGF‐β) in the Resolution and Repair of Inflammation. In Inflammation: Basic Principles and Clinical Correlates (J.I. Gallin and R. Snyderman, eds.) pp. 883‐892. Lippincott Williams & Wilkins, Philadelphia.
   Wakefield, L.M., Smith, D.M., Masui, T., Harris, C.C., and Sporn, M.B. 1987. Distribution and modulation of the cellular receptor for transforming growth factor‐beta. J. Cell Biol. 105:965‐975.
   Weiner, H.L. 1997. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol. Today. 18:335‐343.
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