Induction and detection of antibodies to squalene

An enzyme-linked immunosorbent assay (ELISA) utilizing antigen coated on hy drophobic polyvinyldiene fluoride (PVDF) membranes is described for detecti ng antibodies that bind to squalene (SQE). Because of the prior lack of ava ilability of validated antibodies to SQE, positive controls for the assay w ere made by immunization with formulations containing SQE to create monoclo nal antibodies (mAbs) that reacted with SQE. Among eight immunogens tested, only two induced detectable murine antibodies to SQE: liposomes containing dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, 71% SQE , and lipid A [L(71% SQE+LA)], and, to a much lesser extent, an oil-in-wate r emulsion containing SQE, Tween 80, Span 85, and lipid A. In each case, li pid A served as an adjuvant, but neither SQE alone, SQE mixed with lipid A, liposomes containing 43% SQE and lipid A, nor several other emulsions cont aining both SQE and lipid A, induced antibodies that reacted with SQE. Mono clonal antibodies produced after immunizing mice with [L(71% SQE+LA)] serve d as positive controls for developing the ELISA. Monoclonal antibodies were produced that either recognized SQE alone but did not recognize squalane ( SQA, the hydrogenated form of SQE), or that recognized both SQE and SQA. As found previously with other liposomal lipid antigens, liposomes containing lipid A also induced antibodies that reacted with the liposomal phospholip ids. However, mAbs were also identified that reacted with SQE on PVDF membr anes, but did not recognize either SQA or liposomal phospholipid. The polyc lonal antiserum produced by immunizing mice with [L(71% SQE+LA)] therefore contained a mixed population of antibody specificities and, as expected, th e ELISA of polyclonal antiserum with PVDF membranes detected antibodies bot h to SQE and SQA. We conclude that SQE is a weak antigen, but that antibodi es that specifically bind to SQE can be readily induced by immunization wit h [L(71% SQE+LA)] and detected by ELISA with PVDF membranes coated with SQE . (C) 2000 Elsevier Science BN. All rights reserved.