Liposome-Based Flow Injection Immunoassay System

Abstract

The development of many biochemical assays is dependent upon the specific interaction of an antigen with its antibody. This interaction is usually monitored using secondary labels with the most popular immunoassays employing fluorescent or radioactive tracers for detection of a single binding event. We are developing a novel flow injection analysis (FIA) system which contains an immunospecific reactor column and utilizes liposomes for detection. The fluorophore-loaded liposomes used in this assay can be made to provide signal enhancement in the range of one thousand to one million times per binding event making fluorescent assays competitive in sensitivity with radioimmunoassays. Liposomes are spherical structures composed of phospholipid bilayers that form spontaneously when phospholipid molecules are dispersed in water. The interior and exterior environments of liposomes are aqueous and, therefore, liposomes can be prepared with large numbers of water-soluble marker molecules trapped in their internal aqueous space. Liposomes are prepared by the injection method [1] from a mixture of dimyristoylphosphatidylcholine: cholesterol: dicetyl phosphate with a molar ratio of 5:4:1. Approximately 1 X 103 carboxyfluorescein molecules are encapsulated inside each liposome when the liposomes are formed in 3 mmol/L carboxyfluorescein solution. The liposomes may be "sensitized" to a particular antigen through covalent binding of that antigen to the polar head group of a phospholipid molecule which is incorporated into the lipid mixture at about 1 mol % prior to liposome formation. When the liposome is formed in water, approximately half of the antigen will be exposed to the external solution, and, therefore, will be available to interact with antibody binding sites. The combining of antigen on these "sensitized" liposomes with antibody can