Simple method for the preparation of antigen emulsions for immunization.

We have developed a rapid, safe, and reliable method to prepare emulsions of water-soluble antigens in an adjuvant oil phase for immunization purposes. The method, based on well established emulsification principles, employs a three-way 'T' connector to which three disposable syringes are attached. The system allows the stepwise addition of small volumes of the water phase, into the oil phase. We have compared the time required for emulsification, the rate of antigen release from the emulsion into a physiological phase, and the immunogenic properties of bovine serum albumin and transferrin contained in emulsions made by the new stepwise addition method, with those made by the widely used double-hubbed needle method. We report a significantly shorter (P < 0.001) and a more reproducible emulsification time for the stepwise addition method (6.1 +/- 2.1 min; mean +/- SD) than for the double-hubbed needle method (41.1 +/- 28.0 min). The stepwise addition method always yielded water-in-oil emulsions, while the double-hubbed needle method failed, about 20% of the time, to produce a water-in-oil emulsion after 120 min of mixing. Since the stepwise addition method employs a connector with a larger inner diameter (1.75 mm) than the one required for the double-hubbed needle method (0.84 mm); the pressure required for the former is markedly reduced compared with that required for the latter, thus making the new method safer and less labor-intensive. The rate of antigen release from the emulsions was significantly slower when the stepwise addition method was employed (P < 0.01). There were no differences in viscosity and stability in the emulsions prepared by the two methods. The ability of antigen-containing emulsions to elicit an immune response was found to be identical by the two methods; no significant differences were found in antibody titers as determined by enzyme-linked immunosorbent assays. These characteristics make the stepwise addition system the method of choice.