The present invention pertains to homogeneous immunoassay systems involving complement-mediated lysis of liposomes containing markers.
Liposomes are micron-sized spherical shells of amphipatic molecules which isolate an interior aqueous space from the bulk exterior aqueous environment. They can be made to contain hydrophobic molecules within their membrane, or hydrophilic markers within their internal aqueous space, or both. Because of this versatility, liposomes are of interest both as potential vehicles for the delivery of drugs in vivo and as the basis for immunoassay systems in vitro.
Various formats for liposome immunoassay systems have been developed including heterogeneous and homogeneous systems. Heterogeneous systems, which typically require an initial separation of bound and unbound forms of tracer, are described in O'Connell, et al., Clin. Chem., 31:1424-1426 (1985) and MacCrindle, et al , Clin. Chem., 31:1487-1490 (1985). Homogeneous methods, such as those based on (i) complement-mediated lysis, (ii) melittin-mediated lysis, (iii) color changes induced by cation-responsive dyes in perturbed membranes, and (iv) enhanced agglutination, have also been described. Some of these methods rely on liposomes to generally amplify immunological reactions whereas others rely on the utility of liposomes to encapsulate marker substances within the liposome and to subsequently release them in proportion to the amount of analyte present in a sample.
An immunoassay system of particular interest to the background of the invention is the Liposome Immuno-Lytic Assay (LILA) which involves the antibody-triggered complement-mediated lysis of liposomes. In an exemplary assay format, a liposome encapsulating a marker is first made immunoreactive by association of a first immunological binding pair member (e.g., an antigen) with its surface. The liposome is then incubated with a fluid sample to be analyzed for the presence of the corresponding binding pair member (e.g., an antibody). Typically, the binding of antibody to antigen (pre-bound to the liposome surface) generates a liposome immune complex and, upon the addition of serum, complement activation is initiated leading to lysis of the liposome and release of the internal marker substance. The amount of analyte present in the sample is proportional to the amount of marker substance released.
Liposome lysis can be detected in a variety of ways and depends upon the nature of the marker initially encapsulated within the liposome. Kataoka, et al., Eur. J. Biochem., 24:123 (1971), for example, describe Lipid A sensitized liposomes which release a spectrophotometrically detectable glucose marker when incubated with an anti-Lipid A anti-serum and complement source. Yet another means for detecting lysis involves initially encapsulating within the liposome a fluorophore at self-quenching concentrations. Upon liposome lysis, an extreme dilution of the fluorophore occurs and this dilution re-establishes fluorescence. The increase in fluorescence is proportional to the amount of analyte present in the sample. Ishimori, et al., J. Immuno. Methods, 75:351-360 (1984) describe an immunoassay technique using immunolysis of liposomes to measure antibody against protein antigens such as human IgG. The marker used was carboxyfluorescein and the technique was reported to be effective at detecting 10.sup.-15 mole of anti-human IgG antibody or human IgG. Similarly, Yasuda et al., J. Immun. Methods, 44:153-158 (1981), describe the utilization of complement-mediated immune lysis of liposomes entrapping carboxyfluorescein at self-quenching concentrations to measure anti-glycolipid antibody.
The use of antibody sensitized liposomes in Liposome Immuno-Lytic Assays presents a number of system design problems not present in assays employing antigen coupled liposomes. Of interest to the background of the present invention are references describing developments in the art relating to procedures for coupling antibodies to liposome surfaces (Heath, T. D. and Martin, F. J., Chemistry & Physics of Lipids, 40:347-358 (1986); Martin, F. J. and Kung, V. T., Annals New York Academy of Sciences, 446:443-456 (1985)) which describe binding characteristics of antibody-bearing liposomes; and especially those references which relate to avoidance of liposome aggregation--a phenomenon which can seriously limit the sensitivity of LILA's. As one example, Jou, et al., Fed. Proc., Fed. Am. Soc. E. Biol., 43:1971 (1984) disclose coupling procedures designed for avoidance of aggregation of antibody-sensitized liposomes through a series of steps including: (1) limiting the average number of reactive functional groups per antibody molecule to less than one; (2) providing for early "quenching" during the coupling reaction; and, (3) employing dialysis to remove uncoupled antibodies. In this reference however, only liposome aggregation as a result of antibody-liposome coupling was addressed and no information was provided regarding the use of antibody-coupled liposomes for immunoassays.
Umeda, et al., J. Immun. Meth., 95:15-21 (1986) (and Umeda, et al., Jap. patent appln. No. Sho 59 [1984-261806]) describe a series of studies regarding a complement-mediated liposome immune lysis assay using carboxyfluorescein-entrapped liposomes sensitized with antibody to C-reactive protein (CRP) antigen. Whole antibodies, derived from different animal sources, were modified and coupled to liposomes utilizing a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2 pyridyldithio)-propionate (SPDP) and dithiothreitol (DTT), a reducing agent. However, upon coupling certain antibodies, e.g., rabbit antibodies, to dithiopyridyl-substituted dipalmitoylphosphatidylethanolamine (DTP-DPPE) liposomes, complement activation and liposome lysis occurred even in the absence of sample containing analyte. The low level of complement reagent required to minimize this non-specific lysis necessarily lowered the overall sensitivity of the assay. In addition, only certain animal sources of complement, i.e., guinea pig serum, proved to be effective reagents and this effectiveness also depended upon the animal source of the antibody coupled to the liposome. For example, goat antibody was suitable as the antibody bound to the liposome, but was not suitable as the secondary antibody. Although the assay sensitivity was in proportion to the amount of antibody bound to the liposome, when more than 400 .mu.g of IgG/.mu.mol of lipid was bound to the liposomes, liposomes became fragile and their spontaneous release of carboxyfluorescein increased irrespective of the liposome lipid composition. The sensitivity of the assay was improved by purification of whole antibody (by passage through an affinity chromatography column) prior to binding to liposomes. However, sensitivity was not increased when Fab' antibody fragments (which were expected to be coupled to liposomes more efficiently than IgG) were bound to liposomes. The Fab' antibody fragments of the reference were prepared by reducing F(ab').sub.2 antibody fragments with mercaptoethylamine and were then coupled via thiol residues to derivatized liposomes containing DTP-DPPE. In the attempt to explain the lack of improved sensitivity over that obtained using liposomes bearing whole goat antibody, it was speculated that the affinity of Fab' antibody fragments for antigen may be reduced during the drastic pepsin digestion at pH 4.5. It was thus suggested that the use of "high affinity" Fab' fragments would result in a much higher sensitivity than use of whole IgG.
The coupling of Fab' antibody fragments to liposomes via a disulfide exchange reaction requires either a sulfhydryl reactive derivative on the liposome or a derivatization of the sulfhydryl group on the Fab' antibody fragment. For example, Martin, et al., Biochemistry, 20:4229 (1981), describes the use of N-[3-2-pyridyldithio)propionyl]phosphatidylethanolamine (PDP-PE) liposomes, in a disulfide exchange reaction. The sulfhydryl reactive derivative is a pyridyldithio derivative. Martin, et al., J. Biol. Chem. 257:286 (1982), describes the use of N-[4-(p-maleimidophenyl)butryl]phosphatidylethanolamine (MPB-PE) liposomes having reactive maleimide moieties for forming an essentially irreversible antibody-vesicle linkage which did not involve the usual disulfide linkage but rather involved the more stable thioether linkage. The liposomes of the two Martin references did not contain an encapsulated fluorophore as those liposomes were intended for "targetting" use rather than for use in an immunoassay. Bredehorst, et al., Biochemistry, 25:5693-5698 (1986) describes the coupling of Fab' fragments to MPB-PE liposomes. These liposomes did contain encapsulated fluorophore but the liposomes were noted to release up to 95% of the entrapped fluorophore. To overcome this leakage problem, a decrease in the molar concentration of the MPB-PE anchor in the liposomes was required which caused a corresponding decrease in the number of Fab' molecules bound per liposome. No evidence was given as to whether such coupled liposomes would be functional in an immunoassay.
A number of references have described derivatization of Fab' antibody fragments for use in the preparation of bispecific antibodies--hybrid immunoglobulins provided with two different antigen-binding sites through a chemical re-association of monovalent fragments derived. See, e.g., Brennan, et al., Science, 229:81 (1985) and Paulus, H.P., PCT patent application WO 85/04811. Both references show the preparation of Fab' thionitrobenzoate derivatives in which arsenite is used as a complexing agent to stabilize vicinal dithiols and to impede intramolecular disulfide formation.
In sum, several immunoassay systems involving complement mediated lysis of marker-encapsulating, antibody bound liposomes have been described. However, none of these are homogeneous systems totally responsive to the need in the art for highly sensitive assays for antigens in fluid samples.
BRIEF SUMMARY OF THE INVENTION
The present invention provides for novel homogeneous immunoassay systems involving complement-mediated lysis of marker-encapsulating lipid vesicles (liposomes) for detection of analyte in a fluid sample. These systems do not require the separation of unbound antigens and/or antibody conjugates yet provide highly sensitive procedures for analyte detection.
Liposomes containing a marker, e.g., a fluorescent marker, are coupled to antibody fragments (variously referred to as liposome-antibody conjugates or antibody sensitized liposomes). The antibody fragments of the invention confer the liposomes with immunological specificity yet avoid sensitizing the liposomes to complement mediated lysis in the absence of analyte. Antibody sensitized liposomes (the first reagent) are sequentially incubated with an analyte-containing sample, a second antibody (the second reagent), and finally with a complement source such as plasma. Complement is activated by the liposome-antibody-antigen-second antibody complex, and causes liposome lysis, a concomitant release of fluorophore and an increase in observable fluorescence.
Antibody of the first reagent may be an anti-analyte F(ab').sub.2 antibody fragment, or an anti-analyte Fab' antibody fragment. Antibody of the second reagent may be provided in either soluble form, or in insoluble form e.g., bound onto carboxylated polystyrene particles or coupled to a third antibody in the form of a "double antibody" immune precipitate. Where the antibody of the second reagent is provided in an insoluble form, the analyte-containing sample is preferably incubated with the second reagent, to form an analyte-second antibody complex, prior to incubation with the first reagent. Where the second reagent consists of soluble antibodies, the first reagent is incubated with the analyte containing sample prior to incubation with the second reagent.
In a presently preferred system, designed for detection of human chorionic gonadotropin (HCG) in a fluid sample, liposomes containing a fluorophore marker, such as calcein, are coupled to Fab'antibody fragments, e.g., goat anti-HCG Fab' antibody fragments, and incubated with an HCG-containing sample in the presence of dummy liposomes. This is followed by incubation with a second soluble antibody, e.g., goat anti-HCG IgG, and finally with complement, e.g., goat serum, leading to liposome lysis and an increase in observable fluorescence.
In another presently preferred system liposomes containing a fluorescent marker, e.g., calcein, are coupled to F(ab').sub.2 antibody fragments, e.g., goat anti-HCG F(ab').sub.2 antibody fragments, and the antibody sensitized liposomes are sequentially incubated with an HCG-containing sample and then with a second antibody, e.g., goat anti-HCG antibody, in solution form to form liposome-antibody-HCG-second antibody complexes, which are then lysed with complement, e.g., either human plasma or rabbit serum. In another preferred system, the second antibody, e.g., goat anti-HCG antibody, is coated onto carboxylated polystyrene particles or coupled to a third antibody, e.g., rabbit anti-goat IgG antibody, as a double antibody precipitate.
In another aspect of the invention designed to obtain improved sensitivity of the liposome immunolytic assay, antibody sensitized liposomes are mixed with "dummy" lipid vesicles, liposomes which do not contain encapsulated marker, prior to incubation with the analyte containing sample and with a second antibody.
In another of its aspects, the present invention provides methods for preparing antibody sensitized liposomes in a manner which minimizes liposome aggregation both during preparation and storage. In a preferred method for reducing lipid vesicle aggregation during antibody lipid vesicle conjugation, lipid vesicles having thiol groups are conjugated with modified antibody, i.e., Fab' and F(ab').sub.2 antibody fragments with thiol-reactive groups, in the presence of a polysaccharide capable of forming a reversible gel. The gel reaction mixture is cooled to form a gelate, unreacted thiol groups on the liposomes are alkylated, and the gelate, containing liposome-antibody conjugates, is washed and may be placed in a buffer for storage. By minimizing liposome aggregation, the accessible surface area of each liposome is increased as is the total reactivity of liposome preparations, thereby enhancing the efficacy of their diagnostic and therapeutic applications.
In another of its aspects, the present invention provides methods for preparing Fab' antibody fragments whereby antibody affinity for its complementary antigen is retained resulting in antibody sensitized liposomes with greatly improved sensitivity in a LILA. According to another aspect of the invention, antibody fragments are chemically modified to bear functional group(s) which are reactive toward certain moieties on the liposome surface and therefore are able to form antibody-liposome covalent linkages upon their mixing with liposomes. Illustratively, F(ab').sub.2 antibody fragments, prepared from whole antibody, e.g., goat anti-HCG antibody, are treated with a dithiol complexing agent, such as sodium arsenite, to stabilize vicinal dithiols and impede intramolecular disulfide formation, in the presence of a reductive cleavage agent such as cysteine-HCl. The resulting Fab' antibody fragments are then treated with a thiol activating agent, such as 5,5'-dithiobis(2-nitrobenzoic acid), to form thionitrobenzoate derivatized Fab' antibody fragments (TNB-Fab'). These derivatized Fab' antibody fragments may then be coupled to lipid vesicles to form antibody sensitized liposomes.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof which includes numerous illustrative examples of practice of the invention.