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Highly sensitive immunoliposome assay of theophylline.

A highly sensitive and reproducible immunoassy method for determination of theophylline was developed by using large unilamellar liposomes. Vesicles incorporating theophylline-phosphatidylethanolamine conjugate on the membrane surface as a sensitizer and including a fluorescent marker, calcein, were prepared by the reverse-phase evaporation method from mixtures of phosphatidylcholines containing various fatty acids and cholesterol. Competitive binding of specific antibody to the analyte drug and the sensitizer on the liposomal membrane resulted in lysis of liposomes, and consequently the amount of entrapped calcein that leaked out was inversely proportional to the concentration of the analyte. From studies of various parameters influencing liposome lysis, it was concluded that the chain length of fatty acid most strongly affected the calcein release. The proposed system is simple, rapid, precise, and sensitive to nanomolar concentrations of theophylline. Furthermore, the sensitized liposomes were stable and gave reproducible results. The principle of this assay should be applicable to routine analysis of a wide variety of drugs in biological samples for the purpose of clinical diagnosis or monitoring.