Quantitative analysis of a synthetic adjuvant by an immunoassay

Antibodies against a sulpholipo-derivative of synthetic polysucrose (SL-Ficoll) were prepared with the purpose of developing a detection system for this adjuvant component. SL-Ficolls with polysucrose backbones of 400 kDa (SL-Ficoll400) or 22 kDa (SL-Ficoll22) were not immunogenic. However, SL-Ficoll22 conjugated to bovine serum albumin (SL-Ficoll22-BSA) induced high levels of specific antibodies against SL-Ficoll in mice as determined by an indirect ELISA with either SL-Ficoll400 or SL-Ficoll22 as coating antigen. The specificity of the antibodies was analysed further in a blocking ELISA with SL-Ficoll400 as coat. Dose-dependent inhibition of the ELISA titre was obtained with SL-Ficoll22 and SL-Ficoll400 and the concentration causing 50% inhibition (IC50) was < 1 microg/ml. Non-derivatised polysucrose (Ficoll400) and compounds lacking either lipid or sulphate were not recognised (IC50 > 1000 microg/ml). The reaction of SL-Ficoll400-derivatives with antibodies increased with increasing sulphate and lipid content and maximal inhibition was produced by compounds with a composition similar to the SL-Ficoll22 used for immunisation. Quantitative analysis of SL-Ficoll400 in adjuvant formulations comprising SL-Ficoll400 incorporated in a squalane-in-water emulsion revealed high recovery and sufficient precision. From these data, we conclude that specific antibodies are generated against the synthetic SL-Ficoll400 and that this component of a novel adjuvant formulation can be quantified by an immunoassay.