Fig. 5. Nature of the interaction of DCFHP antigen with Alhydrogel (AH) adjuvant as a function of storage time, temperature, and sodium phosphate concentration.

(A) ACE2 competitive ELISA results for the DCFHP formulations (three AH-adsorbed and one without alum as indicated) used for mouse immunization studies at time zero (first dose) and after 1 month storage at 4°C (second dose) as well as additional samples stored at 25°C for 1 month. Data are the mean of two independent samples, each measured twice with the error bars represent the standard deviation. (*) indicates a statistically significant difference by a two-tailed t-test and (ns) indicates the results were not significantly different. (B) Box plot analysis of ACE2 competitive ELISA data from 21 independent experiments of DCFHP antigen 100% adsorbed to Alhydrogel (no phosphate buffer) at time zero and after incubation at 4°C for an average of 10 days (range 3–21 days). *Results from a two tailed student’s t-test indicate the two data sets are significantly different (*p <0.0001). The dashed line represents the target concentration of 50 mcg/mL DCFHP. (C, D) Binding strength of DCFHP to AH increases as a function of storage time and temperature as measured by comparing “strong” vs “mild” desorption results; see text. (C) The percent of DCFHP bound to AH at time zero and after storage (4 and 25°C, 1 month) as determined by strong desorption (0.4 M sodium phosphate, LDS, 95°C incubation for 5–10 min), and (D) the percent of DCFHP desorbed from same samples using mild desorption conditions (0.4 M sodium phosphate, 37°C incubation for 30 min). ND- none detected. Data are the mean of two replicates with the error bars representing the data range.