Measurement of Polyclonal Immunoglobulin Synthesis Using ELISA

Thomas B. Nutman1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.12
DOI:  10.1002/0471142735.im0712s00
Online Posting Date:  May, 2001
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Abstract

The enzyme‐linked immunosorbent assay (ELISA) is a simple, specific, highly reproducible, and nonradioactive method for measuring immunoglobulin levels in cell culture supernatants and other biologic fluids. It can be used to measure the common immunoglobulin classes (IgG, IgM, and IgA) as described in the basic protocol, as well as classes and subclasses found in low levels in these fluids (IgG4 and IgE), as detailed in the alternate protocols.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of IgG, IgM, and IgA Responses by ELISA
  • Basic Protocol 2: Measurement of IgG Subclass Responses Using ELISA
  • Alternate Protocol 1: Measurement of IgE Responses Using ELISA
  • Support Protocol 1: Biotinylation of Immunoglobulin
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of IgG, IgM, and IgA Responses by ELISA

  Materials
  • First (capture) antibody: 10 µg/ml goat anti‐human IgM, IgG, or IgA (IgG fraction) in recipecoating buffer
  • Wash buffer: 0.05% (v/v) Tween 20 in PBS (unit 2.NaN)
  • Blocking buffer: 5% (w/v) BSA in wash buffer (filter sterilize; store at 4°C)
  • recipeImmunoglobulin standards (see recipe) or supernatant from mononuclear cell cultures (unit 7.13)
  • Diluent buffer: 1% (w/v) BSA in wash buffer (filter sterilize and store at 4°C)
  • Second antibody: affinity‐purified, Fc‐specific, alkaline phosphatase–conjugated goat anti‐human IgM, IgG, or IgA antibody ( Sigma or Jackson Immunoresearch)
  • 1 mg/ml p‐nitrophenyl phosphate (Sigma) in recipesubstrate buffer
  • 3 M NaOH
  • 96‐well ELISA plates (e.g., Immulon 4, Dynatech)
  • Plate sealers (Dynatech #001‐010‐350) or plastic wrap
  • Multiwell scanning spectrophotometer

Basic Protocol 2: Measurement of IgG Subclass Responses Using ELISA

  Additional Materials
  • Anti‐subclass antibody (first antibody):10 µg/ml IgG fraction of monoclonal antibodies directed against human IgG1, IgG2, IgG3, and IgG4, in PBS ( appendix 2A)
  • Biotinylated, Fc‐specific, affinity‐purified goat anti‐human IgG (e.g., GIBCO/BRL, or Jackson Immunoresearch)
  • Alkaline phosphatase–conjugated streptavidin

Alternate Protocol 1: Measurement of IgE Responses Using ELISA

  Additional Materials
  • 1 µg/ml monoclonal anti‐IgE antibody (Novo Biolabs) in recipecoating buffer
  • Biotinylated, Fc‐specific, affinity‐purified goat anti‐human IgE–F(ab′) 2 fragment or biotinylated, purified polyclonal or monoclonal anti‐IgE in diluent buffer
  • Alkaline phosphatase–streptavidin in diluent buffer

Support Protocol 1: Biotinylation of Immunoglobulin

  Additional Materials
  • Affinity‐purified polyclonal antibodies or monoclonal antibodies, precipitated twice with ammonium sulfate (unit 2.7)
  • 0.1 M NaHCO 3, pH 8.0
  • 1 mg/ml biotin N‐hydroxysuccinimide ester (Pierce) in dimethylsulfoxide (DMSO; store at −70°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Additional reagents and equipment for protein dialysis ( appendix 3H)
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Figures

Videos

Literature Cited

Literature Cited
   Engvall, E. and Perlmann, P. 1971. Enzyme‐linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G Immunochemistry 18:871‐874
   Engvall, E. and Perlmann, P. 1972. Enzyme‐linked immunosorbent assay (ELISA). 3. Quantitation of specific antibodies by enzyme‐labeled anti‐immunoglobulin in antigen‐coated tubes J. Immunol. 109:129‐135
   Fuller, S.A., Miyoko, R., and Hurrell, J.G.R. 1989. Direct enzyme linked immunosorbent assay (ELISA) for detection of antibodies in serum and hybridoma supernatants inCurrent Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 11.4.1‐11.4.4. Greene Publishing and Wiley‐Interscience, New York
   Helm, R.M., Buckley, R.H., Adkinson, N.F., Squillace, D.L., Gleich, G.J., and Yunginger, J.W. 1986. Variability of IgE protein measurement in cell‐culture supernatants: Results from a multicenter collaborative study J. Allergy Clin. Immunol. 77:880‐90
   Hussain, R., Poindexter, R.W., Wistar, R., and Reimer, C.B. 1986. Use of monoclonal antibodies to quantify subclasses of human IgG. I. Development of two‐site immunoenzymometric assays for total IgG subclass determinations J. Immunol. Methods 93:89‐96
   Rodbard, D., Feldman, Y. and 1978. Kinetics of two‐site immunoradiometric (‘sandwich') assays. I. Mathematical models for simulation, optimization, and curve fitting Immunochemistry 15(2):71‐76
   Voller, A., Bidwell, D. and 1987. Enzyme‐linked immunosorbent assay inManual of Clinical Immunology (N, Rose, W, Friedman, and J.L, Fahey, eds.) pp. w 99‐109. American Society for Microbiology, Washington, D.C
   Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 1989. Enzyme‐linked immunosorbent assays (ELISA) for detection of antigen inCurrent Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K, Struhl, eds.) pp. 11.2.1‐11.2.8. Greene Publishing and Wiley‐Interscience, New York
Key References
  Chan, D. and Perlstein, M.T. Immunoassays: A Practical Guide. Academic Press, San Diego.
  A useful guide to the theory behind using ELISAs for the measurement of antibodies with a particularly good discussion of the analysis of data generated by the immunoassays.
   Kemeny, D.M. and Challacombe, S.J., 1988. ELISA and Other Solid Phase Immunoassays: Theoretical and Practical Aspects. Wiley, New York.
  A comprehensive discussion of the theoretical aspects of solid phase antibody‐antibody interaction as it applies to immunoassays. There is particular reference to the selection of appropriate antibodies for use in two‐site (sandwich) immunoassays.
   Tijssen, P., 1985. Practice and theory of enzyme immunoassays. In Laboratory Techniques in Biochemistry and Molecular Biology (R.H. Burdon and P.H. Knippernberg, eds.) pp. 1‐349. Elsevier, New York.
  A good discussion of the theoretical considerations involved in antibody detection by ELISA.
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