Prevention of nonspecific lysis in liposomal and erythrocyte immunoassay systems by small lipid vesicles and erythrocyte ghosts

Large unilamellar liposomes prepared by the reverse-phase evaporation method (REVs) were made immunoreactive by incorporating dinitrophenylaminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) or 8-(3-carboxypropyl)-theophylline-dipalmitoylphosphatidylethanolamine (Th-DPPE) into the phospholipid bilayer. Specific lysis in the presence of anti-DNP-BSA and goat anti-theophylline serum respectively, was induced by adding guinea pig serum as source for complement to these liposomes. However, specific lysis was found to be compromised by high levels of nonspecific lysis as monitored by the release of the fluorescent aqueous-space marker 6-carboxyfluorescein. Nonspecific lysis could be prevented without affecting specific lysis by pretreatment of complement or incubation of the reaction mixture with small unilamellar liposomes (SUVs). SUVs of various lipid compositions produced the desired effect; however, when the fraction of negative charge in the SUVs was increased to 30 mol%, specific lysis was inhibited as well. In a similar assay system consisting of hemolysin-sensitized sheep red blood cells it was also found that nonspecific lysis could be inhibited by addition of erythrocyte ghosts to the incubation medium, although specific lysis was somewhat depressed. However, SUVs or REVs of a composition similar to sheep erythrocytes were ineffective indicating a more selective nature of complement-mediated immunoreaction with erythrocyte membranes than with synthetic bilayer membranes.