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Double‐Immunodiffusion Assay for Detecting Specific Antibodies

Peter Hornbeck¹

¹University of Maryland, Baltimore, Maryland

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 2.3

DOI:

10.1002/0471142735.im0203s00

Online Posting Date:

May, 2001

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Abstract

Double immunodiffusion owes its success to the unique nature of antibody-antigen interactions. When polyvalent antibodies with moderate-to-high intrinsic affinities are mixed with antigen at the right ratio (called the zone of equivalence) lattices of antibody-antigen complexes form and precipitate out of solution. When, as described in this unit, gradients of antigen and antibody are established by diffusion from adjacent wells in a bed of agar, a line of practically insoluble precipitation forms at the equivalence zone (precipitin lines).

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Basic Protocol
Reagents and Solutions
Commentary
Bibliography
Figures

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Materials

Basic Protocol

Materials

Noble agar (Difco)
PBS (appendix 2A) containing 0.05% NaN₃ (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. 2.3.1)
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; Fig. 3.8.1)
Staining rack and dish
Whatmann 3MM filter paper

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Figures

Figure 2.3.1

Double-diffusion template consisting of eight partially overlapping hexagonal arrays distributed around eight central wells. Central wells are represented by dark circles. A numbered set of wells arranged hexagonally around a central well is shown.

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Figure 2.3.2

Typical results of a double-immunodiffusion assay. Wells 1, 4, and 5 are positive for reactive antisera, while wells 2, 3, and 6 are negative.

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Literature Cited

Literature Cited
	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 Reference
	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.