Liposome-Based Flow Injection Immunoassay System

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 then be monitored through the fluorescence of the encapsulated marker molecule. In this assay, each binding event is amplified approximately 1000 times. Liposomes containing 3 mmol/L carboxyfluorescein are about 0.1 ,um in diameter, and have a polydispersity of 0.1 which corresponds to a size distribution of 0.5% as determined by photon correlation spectroscopy with data analysis using the cumulants method [2]. The diffusion coefficient of these liposomes in water was determined to be 5 X 10' cm2 /s, and the liposomes contained 1-5 lamellae per liposome. These liposomes have been determined to be stable for greater than 3 months in TRIS buffer at room temperature. The flow injection system contains a glass reaction column (2.0 mm i. d. X 99.5 mm; total volume of 155 FtL) packed with nonporous glass beads 250±40 ,tm in diameter (60-80 mesh) which provide the solid phase for the immuno-specific reaction. The antibody is covalently bound to the solid support in a manner which ensures the proper orientation of the antibody binding site such that there is very little loss in activity. The antibody is prepared for binding by initially cleaving the Fc portion of the antibody with pepsin leaving an F(ab')2 fragment attached in the hinge region through a disulfide bond. Reducing this disulfide bond yields two Fab' fragments with equal affinity for antigen binding and with a sulfhydryl group at the base of the fragment available for attachment. Covalent immobilization of this Fab' fragment is achieved through silanization of the beads with an aminosilane reagent. An amide bond is formed between the aminosilane and a difunctional succinimidyl-maleimido linking agent. The Fab' fragment is then easily attached to the maleimido group through the sulfhydryl group at the base of the antibody fragment (fig. 1). Several preparations of nonporous soda lime glass beads derivatized with a monofunctional silane have proven to be stable in a packed column for 7 d when subjected to a flow rate of 1 mL/min in slightly basic TRIS buffer. Quantitation of silanized sites is accomplished through acetylation of the amino group using 1 4 C-