Monitoring the Retention of a Protein Antigen in Complete Freund's Adjuvant, Alum, and Pluronic F-127 Gel Formulations by X-ray Fluorescence

Adjuvants function by protecting antigens from rapid degradation or dispersal. The effectiveness of experimental adjuvants can be assessed by measuring antibody titers to the antigen of interest or, less frequently, by evaluating the retention and distribution of antigen at the application site. In this study, we used X-ray fluorescence (XRF) to monitor the release of an iodinated protein (I-bovine serum albumin) from several adjuvant formulations after its subcutaneous injection in rats. The interaction of the tagged antigen with an external Am-241 source leads to the emission of iodine X-rays from the application site; the number of these X-rays is proportional to the concentration of the protein remaining at the injection site. The disappearance of the iodine X-rays, and hence the antigen, from the injection site followed first-order kinetics for all adjuvant formulations tested; mean half-life values were as follows: in 50% Freund's adjuvant, 17.1 ± 1.1 h; in 4-hour-old 25% Alum, 11.78 ± 0.08 h; in 4-h-old 50% Alum, 13.2 ± 2 h; in 3-day-old 50% Alum, 15.8 ± 1.5 h; and in 240 mg mL Pluronic F-127, 7.9 ± 0.7 h. We conclude that XRF is an easy, reliable, noninvasive method to monitor the retention of antigens in these adjuvant solutions.