Double‐Immunodiffusion Assay for Detecting Specific Antibodies (Ouchterlony)

Peter Hornbeck1

1 Cell Signaling Technologies, Danvers, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.3
DOI:  10.1002/cpim.18
Online Posting Date:  February, 2017
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Abstract

The method first described by Ouchterlony in 1948 is a classic and simple technique that permits evaluation and comparison of antibodies in animal or human sera directed against protein or complex carbohydrate antigens. It is a low‐tech procedure that may provide information on the relative quantity of antibody activity and the nature of the antigenic epitopes in different preparations. © 2017 by John Wiley & Sons, Inc.

Keywords: antibody assay; antigen; double‐diffusion; agar; gel

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Noble agar (Difco)
  • PBS ( appendix 2A) containing 0.05% NaN 3 (PBSN)
  • 4% PEG 6000 (J.T. Baker) in PBSN, prewarmed to 56°C (store at room temperature)
  • 1 mg/ml antigen
  • Antisera
  • Staining solution
  • Destaining solution
  • 2 × 3–in. microscope slides, precleaned
  • Boiling and 56°C water baths
  • 50°C oven
  • Template (see Fig.  )
  • 15‐G stainless steel needle (blunt‐ended and beveled) or immunodiffusion punch set (EC Apparatus)
  • 10‐µl Hamilton syringe
  • Humidified chamber (enclosed plastic container with moistened tissues
  • Staining rack and dish
  • Whatmann 3MM filter paper
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Figures

Videos

Literature Cited

Literature Cited
   Herren, T., Middendorp, M. A., and Zbinden, R. 2016. Quantification of the antibody response to Propionibacterium acnes in a patient with prosthetic valve endocarditis: a case report. BMC Infect. Dis. 16:185.
   Ouchterlony, O. 1948. In vitro method for testing the toxin‐producing capacity of diphtheria bacteria. Acta Pathol. Microbiol. Scand. 25:186‐191.
   Ouchterlony, O. and Nilsson, L‐A. 1986. Immunodiffusion and immunoelectrophoresis. In Handbook of Experimental Immunology, Vol. 1: Immunochemistry (D.M. Weir, L.A. Herzenberg, C. Blackwell, and L.A. Herzenberg, eds.) pp. 32.1‐32.50. Blackwell, Oxford.
   Rochu, D., Crespeau, H., Fine, A., and Fine, J‐M. 1989. A sensitive double‐diffusion microassay suitable for the detection of idiotype–anti‐idiotype precipitates. J. Immunol. Methods 118:67‐71.
Key References
  Ouchterlony and Nilsson, 1986. See above.
  Contains a detailed description of immunodiffusion techniques and provides detailed interpretations of various patterns of immunoprecipitation observed in double‐immunodiffusion experiments.
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