1. Field of the Invention
This invention relates to the field of specific binding assays, particularly immunoassays for determining substances of clinical interest. The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between a ligand, i.e., a bindable analyte under determination, and a binding partner therefor, i.e., receptor. The presence of the receptor can be used to effect a mechanical separation of bound and unbound labeled analyte or can affect the label in such a way as to modulate the detectable signal. The former situation is normally referred to as heterogeneous and the latter as homogeneous, in that the latter technique avoids a separation step. Where one of the ligand and its binding partner is a hapten or antigen and the other is a corresponding antibody, the assay is known as an immunoassay. See, generally, Odell and Daughaday (Eds.), Principles of Competitive Protein-Binding Assays, J. B. Lippincott Co., Philadelphia (1971).
In conventional label conjugate specific binding assay techniques, a sample of the liquid medium to be assayed is combined with various reagent compositions. Such compositions include a label conjugate comprising a binding component incorporated with a label. The binding component in the conjugate participates with other constituents, if any, of the reagent composition and the ligand in the medium under assay to form a binding reaction system producing two species or forms of the conjugate, e.g., a bound-species (conjugate complex) and a free-species. In the bound-species, the binding component, e.g., a hapten or antigen, of the conjugate is bound by a corresponding binding partner, e.g., an antibody, whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand to be detected in the test sample.
2. Brief Description of the Prior Art
Many properties of natural cell membranes can be duplicated in simple lipid bilayer systems, referred to as liposomes. One of these properties is lysis. Lysis can be achieved by conventional chemical agents, such as detergents, or by immunological reactions. When a vesicular, e.g., liposome, membrane contains an externally accessible antigen it will react with corresponding antibody, causing agglutination. When the antigen-sensitized liposome reacts with corresponding antibody in the presence of complement the membrane is irreversibly damaged and can no longer function as the intact selective permeability barrier. This is immunolysis. The extent of immunolysis has been monitored by using antigen-sensitized liposomes containing entrapped marker molecules which are released upon immunolysis.
The lysis of liposomes has been studied using a wide variety of marker systems. One type of marker system, which uses no marker-reactive reagents external to the liposome, has been disclosed in which the liposome encloses an electron paramagnetic resonance spin marker, e.g., stable free radical, such as tempocholine. Tempocholine is water soluble but membrane impermeable, therefore it can be entrapped within liposomes or vesicles and it does not leak across the lipid bilayers. Tempocholine enclosed within liposomes produces a characteristic broad paramagnetic resonance signal of small amplitude. This is because there is a high concentration of spin molecules which exchange signals due to their close proximity to one another. When the liposome membranes are ruptured, such as by activation of complement, the tempocholine molecules are released and diluted in the external medium. This results in a readily detectable, qualitative and quantitative, alteration in the paramagnetic resonance spectrum. Humphries, G. M. and McConnell, H. M. Proc. Nat. Acad. Sci. USA, 72: 2483-2487 (1975). See, also, Wei, et al., J. Immunol. Methods, 9: 165-170 (1975); Chan, et al., J. Immunol. Methods, 21: 185-195 (1978); and Hsia, et al., Ann. N.Y. Acad. Sci., 308: 139-148 (1978). Another marker system which uses no marker-reactive reagents external to the liposome employs a composition containing a fluor, such as 1-aminonaphthalene-3,6,8-trisulfonate, and a quencher, such as a,a'-dipyridinium p-xylene dibromide, entrapped within the liposome. Fluor and quencher escape upon immunolysis of the liposome and their subsequent dilution in the external volume abolishes the quenching, resulting in a high fluorescent signal. See Smolarsky, et al., J. Immunol. Methods, 15: 255-265 (1977) and Geiger, et al., J. Immunol. Methods, 17: 7-19 (1977).
Another type of marker system employs an electrode in the reaction environment external to the liposome. One example of this uses a potassium loaded liposome. Potassium ion escapes upon liposome immunolysis and reacts with an ion-selective electrode in the external environment. See Katsu, et al., Chem. Pharm. Bull, 30: 1504-1507 (1982). Tetrapentylammonium ions have also been enclosed in liposomes and detected upon immunolysis using an ion-selective electrode. See Shiba, et al., Anal. Chem., 52: 1610 (1980) and Chem. Lett. 155 (1980). Also, sheep erythrocyte ghosts (membranes) have been loaded with trimethylphenyl ammonium ion and lysis has been detected with an ion-selective electrode. D'Orazio, et al., Anal. Chem., 49: 2083 (1977) and D'Orazio, et al., Anal. Chem. Acta. 25: 109 (1979).
One of the first types of marker systems to be reported employs substrate entrapped within the liposome or erythrocyte membrane vesicle. The substrate escapes upon immunolysis of the liposome and reacts with an enzyme-containing composition in the external volume to produce a detectable response. The early examples of this use glucose entrapped within the liposome. The glucose escapes upon immunolysis and its release from the liposomes is measured by the increase in absorbance at 340 nanometers which occurs upon reduction of NADP.sup.+ in the presence of hexokinase, glucose-6-phosphate dehydrogenase and the requisite cofactors. See Hixby, et al., Proc. Nat. Acad. Sci., 64: 290-295 (1969); Kinsky, et al., Biochemistry, 8: 4149-4158 (1969); Kinsky, et al., Biochemistry, 9: 1048 (1970). In a more recent example of this type of system, a fluorogenic substrate (umbelliferone phosphate) or a chromogenic substrate (p-nitrophenyl phosphate) is enclosed in the liposome. The substrate escapes upon immunolysis of the liposome and reacts with an enzyme (alkaline phosphatase) in the external volume. Free substrate is produced, resulting in an increased signal. See Six, et al., Biochemistry, 13: 4050 (1974); Uemura, et al., J. Biochem, 87: 1221 (1980); and Uemura, et al., J. Immunol. Methods, 53: 221-232 (1982).
Lysis, including immunolysis, of liposomes having internally entrapped enzymes as markers has also been reported. The enzyme reacts with substrate in the external reaction medium resulting in a detectable response. For example, experiments have been performed using trapped enzyme markers including hexokinase, glucose-6-phosphate dehydrogenase and B-galactosidase. See Kataoka, et al., Biochem. Biophys. Acta, 298: 158-179 (1973). Also, horseradish peroxidase can be entrapped within the liposome. The peroxidase escapes upon immunolysis and catalyzes the following reaction: ##STR1## Oxygen is consumed by the oxidation of NADH and resultant production of water. The depletion of oxygen is detected by an oxygen electrode. See Haga, Biochem. Biophys. Res. Comm., 95: 187-192 (1980).
Several references report the enhancement of enzymic activity upon lysis of enzyme-containing liposomes. For example, Solomon, et al., Biochem. Biophys. Acta, 455: 332-342 (1976) report this observation upon lysis of glucose oxidase-containing liposomes by either sonication or exposure to detergent. The enzymic activity of the entrapped glucose oxidase served as a measure for the permeability of the bilayer membrane of the liposomes to glucose in a non-separation assay. The oxidation of glucose was followed in the same reaction mixture before and after detergent lysis of the enzyme-containing liposome by oxygen uptake using an O.sub.2 electrode. Observed enzymic activity was as high as 4.60 times greater after lysis than it was before lysis.
Tokunaga, et al., FEBS Letters, 106: 85-88 (1979) report an in vitro non-separation method for determining permeability of liposomes containing alkaline phosphatase, a-glucosidase and a-galactosidase. Enzymic activity was determined using substrate and a coupled enzyme reaction in the same reaction mixture before and after detergent lysis. Using alkaline phosphatase, observed enzymic activity was as high as 17.5 times greater after lysis than it was before lysis. These authors state that if the K.sub.m of the particular enzyme/substrate pair were the same both inside and outside of the liposome, the intravesicular enzyme activity could be related to the apparent rate of substrate permeability.
Magee, et al., J. Cell Biology, 63: 492-504 (1974) have used horseradish peroxidase-containing liposomes. The enzymic activity of horseradish peroxidase was determined spectrophotometrically in the same reaction mixture before and after detergent lysis. The ratios of the observed enzymic activity after lysis to the observed enzymic activity before lysis were as high as 8.3. They note that liposomes have been reported as useful models for membranes in permeability studies owing to their sensitivity to polyene antibiotics and susceptibility to immune lysis.
Several different homogeneous label conjugate specific binding assay systems are known in the art, one of which is disclosed by Ullman, et al., in U.S. Pat. No. 4,193,983. In this assay system, a label and a ligand or ligand analog are non-covalently bound to the external surface of a colloidal particle, such as a liposome, which is capable of maintaining its integrity in an aqueous environment. The discrete colloidal particle serves as a hub or nucleus which retains the ligand or its analog and the label in a substantially fixed average spatial relationship. By having the label and ligand in relatively close proximity on the surface of the particle, the proximity of the label and the receptor bound to the ligand adjacent the label can be used in accordance with the prior art techniques to modulate the signal from the label. Nowhere is immunolysis suggested. Indeed, the clear teaching of this reference is that lysis of the vesicle would adversely affect the spatial relationship (proximity) of label and ligand which is required for this assay procedure.
Specific binding assay systems have been proposed, using a multilayered lipid membrane vesicle which has been prepared or treated to have surface-bound ligand or ligand analog and a marker or reagent substance enclosed within the vesicle. The remaining reagents for the assay include: (1) a binding partner, e.g., antibody, for the ligand; and (2) complement to effect lysis of the vesicle upon binding of the binding partner to surface-bound ligand. Generally, see McConnell, U.S. Pat. No. 3,850,578 and McConnell, et al., U.S. Pat. No. 3,887,698 and Gregoriadis, et al., Liposomes in Biological Systems, John Wiley & Sons, N.Y. (1980), especially Chapter 12 entitled "Liposomes as Diagnostic Tools".
More particularly, immunoassay systems have been disclosed in which the use of enzyme-encapsulating liposomes is suggested. Hsia, et al., U.S. Pat. No. 4,235,792 describes a competitive homogeneous immunoassay method which employs immunolysis of an antigen-sensitized liposome containing a marker. Enzymes are among the markers disclosed (col. 6, lines 24-28).
Cole, U.S. Pat. No. 4,342,826 discloses a specific binding assay using antigen-sensitized, enzyme-containing liposomes. These liposomes are immunospecifically caused to release enzyme upon binding of corresponding antibody and fixing of active complement. Upon enzyme release, the presence or absence of enzymatic activity is detected. Cole emphasizes the advantage of providing a homogeneous system in which enzymic activity is substantially greater upon lysis, e.g., a "signal:noise" ratio of at least 5-10 and preferably above 60.
Each of the above approaches to vesicular marker systems has provided an advance of one sort or another in sequestering marker from the reaction medium and, thus, minimizing the generation of signal (latency) prior to immunolysis. That is, the signal observed from intact liposomes is considerably less than that from lysed liposomes. As demonstrated by the references, this end has been widely recognized as a major consideration in the improvement of liposome immunoassays. Further, the combined teaching of the literature in this area has been that the advantages to be achieved are enhanced when complete sequestration prior to lysis is combined with the production of a high intensity signal upon immunolysis.