Isotype Determination of Antibodies

Peter Hornbeck1, Thomas A. Fleisher2, Nicholas M. Papadopoulos2

1 University of Maryland, Baltimore, Maryland, 2 Warren Grant Magnuson Clinical Center, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.3
DOI:  10.1002/0471142727.mb1103s18
Online Posting Date:  May, 2001
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Abstract

Frequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti‚Äźimmunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.

     
 
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Table of Contents

  • Basic Protocol 1: Sandwich ELISA for Isotype Detection
  • Basic Protocol 2: Detecting and Isotyping Antibodies by Electrophoresis and Immunofixation
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Sandwich ELISA for Isotype Detection

  Materials
  • Capture anti‐isotype antibodies: heavy‐chain class‐specific antibodies (anti‐µ, ‐α, ‐γ, ‐δ, ‐ɛ), heavy‐chain subclass‐specific antibodies (anti‐γ1, ‐γ2a, ‐γ2b, ‐γ3, ‐γ4), or light‐chain isotype‐specific antibodies (anti‐κ, ‐λ)
  • PBS ( appendix 22) containing 0.05% NaN 3 (PBSN)
  • Test antibodies: hybridoma supernatants, ascites fluid, or antisera
  • Blocking buffer (unit 11.2)
  • Standard isotype antibodies (i.e., purified antibodies of known isotypes)
  • Developing reagent: anti‐Ig antibody (specific for all heavy‐chain classes)–alkaline phosphatase conjugate (see unit 11.1; Southern Biotechnology or Linscott's Directory)
  • MUP or NPP substrate solution (unit 11.2)
  • Immulon 2 or 4 microtiter plates (or equivalent; unit 11.2)
  • Additional reagents and equipment for ELISA (unit 11.2)

Basic Protocol 2: Detecting and Isotyping Antibodies by Electrophoresis and Immunofixation

  Materials
  • Serum (or other biological fluid)
  • Normal saline
  • 95% methanol/5% acetic acid
  • 1% amido black (1 g in 100 ml of 2.5% acetic acid)
  • 2.5% (v/v) acetic acid
  • 2 × 2–cm cellulose acetate strip
  • Monospecific anti‐Ig, heavy‐chain‐specific (α, γ, µ, ɛ, or δ) or light‐chain‐specific (κ or λ)
  • 0.85% (w/v) NaCl
  • recipeAgarose gel–covered microscope slides
  • Plexiglas electrophoresis cell with two agarose bridges
  • Electrophoresis power supply (e.g., Pharmacia EPS 500/400)
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Figures

Videos

Literature Cited

Literature Cited
   Andrew, S.M. and Titus, J.A. 1991. Purification of immunoglobulin G. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.7.1‐2.7.12. Greene Publishing and Wiley‐Interscience, New York.
   Hornbeck, P. 1991. Double‐immunodiffusion assay for detecting specific antibodies. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeeck, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.3.1‐2.3.4. Greene Publishing and Wiley‐Interscience, New York.
   Johnson, M.A. 1982. Immunofixation electrophoresis. Clin. Chem. 28:1797‐1800.
   Johnson, M.A. 1986. Immunoprecipitation in gels. In Manual of Clinical Laboratory Immunology. (N.R. Rose, H. Friedman, and J.L. Fahey, eds.) pp. 14‐24. Am. Soc. Microbiol., Washington, D.C..
   Knapp, W., Stockinger, H., Majdic, O., and Shevach, E.M. 1991. The CD system of leukocyte surface molecules. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeeck, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.4.1‐A.4.28. Greene Publishing and Wiley‐Interscience, New York.
   Papadopoulos, N.M., Elin, R.J., and Wilson, D.M. 1982. Incidence of γ banding in a healthy population by high‐resolution immunofixation electrophoresis. Clin. Chem. 28:707‐708.
   Tiselius, A. 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 33:524‐526.
Key References
   Johnson, 1986. See above.
  A concise discussion of the principles of immunoprecipitation with specific reference to immunofixation electrophoresis.
   Maggio, E.T. 1981. Enzyme Immunoassay. CRC Press, Boca Raton, Fla.
  A valuable reference describing parameters of ELISA technology.
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