Compendium of Factors for Optimization of the Ouchterlony Test

The Ouchterlony test or Ouchterlony Double Immunodifusion, also known as Passive Double Immunodiffusion, is an immunological technique used in the detection, identification and quantification of antibodies and antigens, such as immunoglobulins and extractable nuclear antigens.Five key aspects for optimization of the Passive Double Immunodiffusion technique are reviewed in this study: (a) advantages and disadvantages of agar versus agarose as diffusion matrix, (b) comparison of glass and plastic petri dishes in terms of clarity, (c) clarity of diffusion gels, (d) possibility to use effectively the least expensive gel densitometers and software for reading densities of protein in gels and the density of antibody-antigen complexes, and (e) the quantitative analysis potentials of the Ouchterlony method. Literature survey reveals that Agar and agarose are structurally different but their gelling properties are comparable. Pore sizes of Agar and agarose are much larger than the mean diameters and sizes of diffusing antigens and antibodies. The concentrations of 1.4 to 1.5% agar and 1% to 1.05% agarose are reported as optimal. Glass petri dishes offer a much better optical clarity than plastic petri dishes. Agar in its highly purified state, known as Noble Agar, exhibits excellent optical clarity but is very expensive. Gel densitometers and inexpensive image processing software exist for electrophoresis, but not yet for Ouchterlony. These densitometer-based methods can be adapted for use in Ouchterlony, and Radiolabelling and usage of fluorography are suggested as some of the means for rendering such densitometric methods more efficient. The quantitative analysis potentials of the Ouchterlony technique can be strengthened using existing models proposed by researchers in the past. These models are mainly based on linear relationships between position of precipitin line and log concentration of antigen or antibody.