ISOLATION AND QUANTIFICATION OF QUILLAJA-SAPONARIA MOLINA SAPONINS AND LIPIDS IN ISCOM-MATRIX AND ISCOMS

In the iscom, multiple copies of antigen are attached by hydrophobic i nteraction to a matrix which is built up by Quillaja triterpenoid sapo nins and lipids, Thus, the iscom presents antigen in multimeric from i n a small particle with a built-in adjuvant resulting in a highly immu nogenic antigen formulation. We have designed a chloroform-methanol-wa ter extraction procedure to isolate the triterpenoid saponins and lipi ds incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detec ting their carbohydrate chains and by HPLC, The cholesterol and phosph atidylcholine in the lipid phase were quantitated by HPLC and a commer cial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and l ipids in their respective phases, while protein was detected in all ph ases after extraction, The protein content was determined by the metho d of Lowry and by amino acid analysis. Amino acid analysis was shown t o be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and conclusion, the triterpenoids and lipids adde d for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospectiv e vaccines.