Liposome immunoassay (LIA) for gentamicin using phospholipase C.

A simple and sensitive liposome immunoassay for gentamicin was developed using the cytolytic agent, phospholipase C, instead of complement. Liposomes entrapping a fluorescent marker, calcein, were prepared by the reverse-phase evaporation method from a mixture of dimyristoylphosphatidylcholine and cholesterol (molar ratio of 3:1). Gentamicin, a model analyte, was covalently coupled to phospholipase C by 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide and N-hydroxysuccinimide. Liposomes were lysed by gentamicin-phospholipase C conjugate and an entrapped fluorescent marker was released. The lytic activity of gentamicin-phospholipase C conjugate was inhibited in the presence of gentamicin antiserum. The standard calibration curve was constructed by plotting percentage of liposome lysis versus log concentration of free gentamicin. The standard calibration curve was linear over 2.5 pg/ml approximately 2.5 ng/ml of gentamicin concentrations. This newly developed immunoassay is simple, relatively rapid and potentially applicable to the determination of concentration of antigens, drugs and endogenous substances.