242 Biotechnol. zyxwvutsrq Prog. zyxwvutsr 1993, 9, 242-258 Preparation and Characterization of Bifunctional Unilamellar Vesicles ?or Enhanced Immunosorbent Assays Matthew A. Jones, Peter K. Kilpatrick, and Ruben G. Carboriell* Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7904 Small unilamellar phospholipid vesicles with covalently attached biotin and horseradish peroxidase (HRP) were prepared and characterized in tetms of hydrodynamic diameter, amount and activity of immobilized enzyme, and number of biotin molecules on the outer vesicle surface. In addition, the specific adsorption of these bifunctional vesicles and commercially available biotin-labeled horseradish peroxidase (B-HRP) to anti- biotin antibody (ABA) coated polystyrene microtiter plate welfs was examined. At low antibody surface densities, the signal (AAlmin) generated by the vesicles adsorbed to the surface was approximately 100 times higher than the signal generated by B-HRP. It was also found that the biotin-conjugated vesicles were able to compete effectively with free biotin in solution for surface ABA sites. ThBse results indicate that this type of vesicle may be used in competitive and sandwich-type enzyme-linked immunoassays to improve the detection limits, increase the signal, and decrease the reaction time necessary to detkct a given analyte concentration in solution. Introduction Immunodiagnostic assays rely on the specific molecular recognition of antigens by their antibodies for the detection of a given analyte in solution. In medical applications, the analyte could be a viral protein, a drug, a hormone, or an antibody generated as a result of infection (Bluestein et al., 1990). Immunodiagnostic assays are becoming increasingly important in other fields, including the analysis of the levels of toxins, antibiotics, steroids, vitamins, and amino acids in food (Allen and Smith, 1987), as well as in the detection of pesticides, heavy metals, and other toxic chemicals (polychlorinated biphenyls, dioxin, phenols) in aqueous streams (Luong et al., 1988; Krull, 1990). Antibody-antigen recognition is the basis for most of the work currently being done on the development of biosensors for the detection of analytes at low concen- tration (Lowe, 1984; Turner et al., 1987). The medical applications of immunodiagnostic kits alone resulted in approximately 2 billion zyxwvuts U.S. dollars in sales worldwide in 1990,and there is a significant and increasing demand for assays that are more accurate, more reliable, easier, or more sensitive (Bluestein et al., 1990). Most of the medical applications require that the analyte be detected as quickly as possible after the onset of the disease. This often requires measurements of analytes in the nanomolar (10-9 M) range, but in many cases it would be advantageous to have a detection limit in the picomolar M) range. One of the most widely used immunodiagnostic tech- niques is the enzyme-linked immunosorbent assay (ELI- SA),in which one of the reactants is adsorbed on the surface of a solid support and the analyte is detected using an enzyme-labeled reactant. It is clear that in ELISAs, the larger the number of enzyme molecules conjugated to the labeled reactant, the larger the resulting signal for a given concentration of labeled reactant adsorbed on the solid surface. Additional label could potentially result in either shorter reaction times or lower detection limits for enzyme- linked assays. Bates (1987)and Kricka and Thorpe (1986) have summarized some of the approaches that have been * Author to whom correspondence should be addressed. used to enhance ELISA signals. These include the use of several enzyme molecules covalently linked together with an antibody and three biotin-conjugated enzymes bound to three of the binding sites of avidin while the fourth site is bound to a biotin-conjugated antibody (Bates, 1987). However, the direct attachment of several enzyme mol- ecules to a single antibody molecule or to a single antigen molecule can lead to loss of specificityand binding strength of the antigen-antibody interaction. This can come about as a result of steric hindrance upon binding to the surface as well as denaturation of the molecule during the coupling procedure. It is desirable to have alternative methods to increase the number of signal amplification labels without sacrificing specificity or the strength of the association constant. Recently, Janssen Biotech & Biochimica Prod- ucts (Flanders, NJ) introduced poly(HRP)-streptavidin, which is a chain of 18-20 horseradish peroxidase molecules conjugated to streptavidin. This reagent can be used to enhance signal generation in ELISAs by 4-20 times as compared to the conventional assay using the standard horseradish peroxidase-streptavidin conjugate (Janssen, 1991). In this article, we explore the properties of bifunctional vesicles which are conjugated with both a small ligand for molecular recognition as well as multiple HRP molecules for signal generation. These vesicles are a subset of the broad category of lipid vesicles known as liposomes; being approximately 1000 zyxw 8, in diameter and having a single lipid bilayer structure puts them in the category of small unilamellar vesicles (Szoka and Papahadjopoulos, 1980). Given that many enzyme molecules can be attached to the outer vesicle surface, the bifunctional vesicles have the potential of further increases in speed and sensitivity of these assays. Figure 1 illustrates how a bifunctional vesicle of this type could be used in a competitive immunoassay for a small analyte. With suitable modifications, it is also possible to develop noncompetitive or sandwich-type assays based on enzyme-conjugated vesicles. In principle, any small particle could be used to attach enzymes and ligands in an immunodiagnostic scheme such as that shown in Figure 1. For example, latex spheres are 8756-7938/93/3009-0242$04.00/0 0 1993 American Chemical Society and American Institute of Chemical Engineers