Surface phosphophilicity of aluminum-containing adjuvants probed by their efficiency for catalyzing the P-O bond cleavage with chromogenic and fluorogenic substrates

Aluminum-containing adjuvants are widely used in a variety of vaccine produ cts, such as recombinant proteins, virus-like particles, conjugated polysac charides, and recently DNA vaccines. Aluminum-containing adjuvants are also known to have a high affinity to inorganic phosphate and its mono- or dies ters. Since phosphate groups are present in many antigens as well as the na tural physiological environment, a better understanding of the interactions between phosphate and phospho-containing species could help in the design of improved vaccines. This report describes a convenient and novel continuo us procedure to measure the avidity denoted by the new term "phosphophilici ty" of phosphate and phosphate esters to the surface of aluminum-containing adjuvants. The assay measures the rate of hydrolysis of a fluorogenic subs trate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)-with a microplat e reader. This method was based on the fundamental bioorganic phenomenon th at when a tight binding event occurs, the effective concentration of nucleo phile(s) will be significantly increased in the proximity of the P atom for a nucleophilic reaction (i.e., the cleavage of the P-O bond) to take place . A very good leaving group (pK(a) of DiFMU similar to 4.7) in the phosphat e monoester substrate makes the assay highly sensitive. Top reading of the nascent fluorescence makes the assay very convenient with no need to separa te the particulate adjuvants from the reaction mixtures. The results from t his assay are consistent with catalysis of the chromogenic phosphate mono- or diesters. (C) 2001 Academic Press.