Improved Technique for the Preparation of Waterin-Oil Emulsions Containing Protein Antigens

Water-in-oil emulsions are commonly used to provide persistent release of antigens necessary for sustained stimulation of the immune system. Optimal response of an animal’s immune system is dependent upon repeated, maintained exposure to the antigen. The sustained release of antigen, which delays catabolism, is thought to be one of the major methods by which adjuvants enhance the immune response. Water-in-oil emulsions are also used to provide sustained release of pharmaceutical agents and hormones. The most frequently used method for preparing a water-in-oil emulsion employs the “two-syringe” technique (1,3). The two syringes are connected with a double-ended Luer-lok® (Becton Dickinson, Lincoln, NJ, USA) connector (or three-way valve). The emulsion is generated by forcing a mixture of oil and water back and forth until the material becomes opaque white, homogeneous and viscous. The mixing is continued until emulsion thickens and it becomes difficult to extrude the material through the connector. Although the two-syringe method is conceptually very simple and requires only standard laboratory equipment, we have experienced difficulty producing durable water-in-oil emulsions using this technique. The emulsion produced by this technique is very sensitive to temperature of the antigen and adjuvant solutions, presence of detergent and many other factors including physical properties of the antigen. Many times it does not result in a stable water-in-oil emulsion, and it may produce an oil-in-water emulsion, which will disperse when tested. Our laboratory uses a different technique to produce a water-in-oil emulsion that is a modification of the method suggested by Crowle (2). This technique yields a durable emulsion with excellent stability when tested in water or physiological saline. The emulsion can be produced in samples that contain small amounts of nonionic detergents. In addition, most of the sample is recovered during preparation, an advantage when dealing with small amounts of valuable protein antigen. The only equipment required, in addition to standard laboratory equipment, is that of a variable-speed Dremel Moto tool (or equivalent), which is available at most hardware stores for about $50