Isotype Determination of Antibodies

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

1 Cell Signaling Technologies, Danvers, 2 Warren Grant Magnuson Clinical Center, Bethesda
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.2
DOI:  10.1002/cpim.19
Online Posting Date:  February, 2017
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On identifying a new monoclonal antibody, or in characterizing antibodies in sera evoked by disease or immunization, it is particularly informative to determine the serological class of the antibodies. The serological class of the protein is determined by the structure of the antibody constant region. Several methods of class or isotype determination are outlined in this unit: sandwich ELISA, electrophoresis, and immunofixation, or use of a variety of commercially available kits. Different purification schemes and approaches for enzymatic fragmentation of the antibodies depend on the class or isotype of the antibody, so this information streamlines these processes. © 2017 by John Wiley & Sons, Inc.

Keywords: antibody characterization; isotype; antibody class; antibody purification; antibody structure

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Table of Contents

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

  • Capture anti‐isotype antibodies for human: heavy‐chain class‐specific antibodies (anti‐µ, ‐α, ‐γ, ‐δ, ‐ε), heavy‐chain subclass‐specific antibodies (anti‐γ1, ‐γ2a, ‐γ2b, ‐γ3, ‐γ4), or light‐chain isotype‐specific antibodies (anti‐κ, ‐λ)
  • Capture anti‐isotype antibodies for mouse: anti‐γ1, anti‐γ2a, anti‐γ2b, anti‐γ3
  • PBS ( appendix 22) containing 0.05% NaN 3 (PBSN)
  • Test antibodies: hybridoma supernatants, ascites fluid, or antisera
  • Blocking buffer (unit 2.2; Hornbeck, )
  • 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 2.2 (Hornbeck, ) or available commercially]
  • MUP or NPP substrate solution (unit 2.2; Hornbeck, )
  • Immulon 2 or 4 microtiter plates (or equivalent; unit 2.2; Hornbeck, )
  • Additional reagents and equipment for ELISA (unit 2.2; Hornbeck, )

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

  • 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, ε, or δ) or light chain–specific (κ or λ)
  • 0.85% (w/v) NaCl
  • Agarose gelcovered microscope slides
  • Plexiglas electrophoresis cell with two agarose bridges
  • Electrophoresis power supply (e.g., Pharmacia EPS 500/400)
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Literature Cited

Literature Cited
  Alaverdi, N. 2002. Monoclonal antibodies to mouse cell‐surface antigens. Curr. Protoc. Immunol. 62:A.4B.1‐A.4B.25.
  Andrew, S.M. and Titus, J.A. 1997. Purification of immunoglobulin G. Curr. Protoc. Immunol. 21:2.7.1‐2.7.12.
  Hornbeck, P. 2015. Enzyme‐linked immunosorbent assays. Curr. Protoc. Immunol. 110:2.1.1‐2.1.23. doi: 10.1002/0471142735.im0201s110.
  Hornbeck, P. 2017. Double‐immunodiffusion assay for detecting specific antibodies. Curr. Protoc. Immunol. 00:2.3.1‐2.3.4.
  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.
  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.
  Yokoyama, W.M, Christensen, M, Santos, G.D, Miller, D, Ho, J, Wu, T, Dziegelewski, M, and Neethling, F.A. 2013. Production of monoclonal antibodies. Curr. Protoc. Immunol. 102:2.5.1‐2.5.29.
Key References
  Johnson, 1986. See above.
  A concise discussion of the principles of immunoprecipitation with specific reference to immunofixation electrophoresis.
  Lequin, R.M. 2005. Enzyme immunoassay (EIA)/enzyme‐linked immunosorbent assay (ELISA). Clin. Chem. 51:2415‐2418.
  A recent review that covers history and recent developments.
  Maggio, E.T. 1981. Enzyme Immunoassay. CRC Press, Boca Raton, Fla.
  A valuable reference describing parameters of ELISA technology.
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