WO1992007959A1

WO1992007959A1 - Homogeneous membrane lytic immunoassay method utilizing contact sensitive liposome formulations

Info

Publication number: WO1992007959A1
Application number: PCT/US1991/008293
Authority: WO
Inventors: Bruce P. Babbitt; Larry R. Hillis
Original assignee: Biowhittaker Inc.
Priority date: 1990-11-05
Filing date: 1991-11-05
Publication date: 1992-05-14

Abstract

A contact sensitive liposome formulation, comprising from about 0.1 to about 99.9 mole percent of one or more inverted hexagonal phase forming lipids, said liposome stabilized in the bilayer phase with from about 0.1 to about 99.9 mole percent of one or more analyte independent amphipathic stabilizer molecules or mixtures thereof, said liposome further comprising one or more targeting molecules on the surface thereof.

Description

HOMOGENEOUS MEMBRANE LYTIC IMMUNOASSAY METHOD UTILIZING CONTACT SENSITIVE LIPOSOME FORMULATIONS

BACKGROUND OF THE INVENTION

Liposomes are microscopic unilamellar or multilamellar phospholipid bilayer vesicles which enclose an aqueous compartment. They are uniquely suited as carriers for a variety of compounds, both biological and nonbiological. Such compounds can be encapsulated in the interior aqueous compartment of the liposome, or, they may be inserted in or attached to the lipid bilayers, which bound and limit the internal aqueous space. Due to their relatively simple composition, and their flexibility for chemical, physical, and immunological manipulations, liposomes are a favorite material for membrane lytic assays, especially immunoassays.

Currently used high volume clinical immunoassay methods include radioimmunoassay (RIA) , fluorescence immunoassay (FIA) , as well as a variety of enzyme - based technologies (ELISA) . Generally these assays require several separation steps (washes, centrifugations , filtrations, etc.) , and long incubations, making it essential to hire trained personnel and/or purchase and maintain highly automated equipment, often at substantially high cost.

Over the past several years, many groups have devised membrane lytic, liposome based, immunoassays, in the hope of utilizing the following presumed advantages of liposomal diagnostic reagents:

1. low cost and high stability of reagents,

2. increased sensitivities, due to the release of hundreds to thousands of entrapped reporter molecules upon detection of a small number of analyte molecules,

3. flexibility of the system, in that a large number of a wide variety of compounds are compatible with a liposome based immunoassay format,

4. potential for homogeneous assay format(s) , which allows for much simpler testing protocols, less trained personnel, and less cumbersome equipment design (i.e., lower costs) .

For example, McConnel et al., in U.S. Patent No. 3,887,698, described the use of liposomes containing stable free radicals in an EPR monitored immunoassay. However, the sensitivity of this assay, compared to other detection systems was poor. Similarly, Mandle et al., in U.S. Patent No. 4,372,745, described the use of- liposome encapsulated fluorescent dyes useful in an immunoassay. However, this method required detergent lysis of analyte captured liposomes to release the entrapped fluorescent signal, a cumbersome step which, if avoidable, should be avoided.

Kins y et al., Biochemistry. 8 (1969), page 4149, were the first to show that liposomes containing haptenated lipids could bind with an antibody and fix or activate complement, resulting in lysis of the hapten tagged liposomes.

Cole, in U.S. Patent No. 4,342,826, describes an immunoassay method which utilized antigen/analyte coated liposomes, containing entrapped enzymes, which were made to rupture immunospecifically, in the presence of the cognate antibody and active complement molecules. The assay utilized a homogeneous phase reaction between the antigen, antibody, and complement to release the enzyme, with a resultant increase in signal by optical density measurement.

Despite the efforts of Cole and many others since that time (for a recent review see Alving & Richards, in "LIPOSOMES¹*, Ostro, editor, pages 209 - 287, Marcel Dekker, New York, 1983) , this type of liposome based immunoassay has proven very difficult to scale up and commercialize, mostly due to the extreme difficulties in storing a functionally reproducible stock of complement proteins at reasonable cost and customer convenience. More recently, several liposome based membrane lytic immunoassays, that are independent of complement proteins, have been developed. For example, binding of antibody to haptens or drugs conjugated to another membrane lytic protein, melittin, blocked the liposome lytic activity of the molecule, proportional to the concentration of the free drug or analyte in solution. (See, Freytag et al., U.S. Patent No. 4,517,303, and Biophysical Journal, 45 (1984), page 360(a)).

Another example involves the binding of antibodies from lupus patient sera to liposomes containing cardiolipin (antigen) to inhibit the lysis of the liposomes by Mg⁺⁺ ions in solution (see, Janoff et al.. Clinical Chemistry. 29, (1983), page 1587). Although independent of complement proteins in particular, these approaches and many others similar to them, are still dependent upon the addition of membrane lytic molecules or ions as additional required reagents to the liposomes themselves. As a result, although the reaction are performed in a homogeneous manner, they still require multiple steps, and are sometimes difficult to reproduce, scale up and automate.

In order to circumvent the requirement for an exogenously added membrane lytic compound(s) , Huang & Ho constructed phosphatidylethanolamine (PE) - based liposomes, which typically under physiological conditions, are unable to form intact liposomes (lipid bilayer vesicles) but instead, form what is referred to as "hexagonal phases" in aqueous buffers. These so- called hexagonal phases are totally incapable of entrapping small or large molecules (i.e., "reporter molecules") , as normal bilayer vesicles do. An example of a hexagonal phase forming lipid is an unsaturated PE, such as egg PE or dioleoyl -PE. Unsaturated PE by itself does not form stable bilayer liposomes at room temperature and neutral pH. Lipids, in addition to PE which can form the hexagonal phase include monogalac osyl diglyceride, cardiolipin, and phosphatidic acid (see, U.S. Patent No. 4,708,933).

Cardiolipin and phosphatidic acid form the hexagonal phase in the presence of a divalent cation, such as Ca⁺⁺» No other neutral lipids have been reported to form the hexagonal phase under physiological conditions of temperature and salt concentration. It appears that the general requirement for the structure of a hexagonal phase forming lipid is that it possess a relatively poorly hydrated polar head group coupled to a bulky hydrophobic moiety; the overall shape of the molecule is therefore that of a cone. It has been postulated that molecules having a complementary, so called "inverted cone" shape, may form stable, bilayer liposomes when appropriately mixed with hexagonal phase forming lipids. Such stabilizer molecules should contain a bulky hydrophilic head group coupled to a small, hydrophobic moiety (see Cullis and DeKruijff, Biochem. Biophvs. Acta. 559 (1979), page 339.)

Huang et al. were able to stabilize the PE phospholipids in a bilayer form by the addition of 12 mole percent of a hapten-phospholipid conjugate, whose molecular shape approximated that of an inverted cone. Upon binding to solid surfaces containing multivalent anti-hapten antibodies, the hapten coated liposomes were disrupted, and released dye into the media. The proposed mechanism was one in which an antibody induce_d phase separation of the haptenated phospholipid molecules (served the dual role of stabilizer and targeting molecule) away from the bulk PE lipids at the area of contact between the liposomes and the solid surface, induced a rapid and leaky bilayer-to-hexagonal lipid phase transition. Significantly, agglutination of the liposomes by the same (bivalent) antibody in solution phase (instead of on a solid surface as a multivalent compound) did not result in any measurable destabilization/leakage of the liposomes.

More recently, Huang, in U.S. Patent No. 4,957,735, described an advancement of this technology, in which a stabilized and targeted PE-based immunoliposome was prepared by the incorporation of an acylated, anti-herpes monoclonal antibody into the vesicle bilayer. Upon binding to virus infected cells, or to partially purified virus particles devoid of cells, they were able to show multivalent, antigen specific destabilization and lysis of the liposomes, in a totally homogeneous immunoassay format.

Taken together, the inventions of the two Huang patents exhibit the following two major technological restrictions (and, key differences from the present invention) , in terms of their application to analyte detection by immunoassay in a commercially viable format:

1. the Huang liposomes are stabilized and targeted by the same molecule (a very restrictive parameter, in terms of broad applicability to a range of analytes and manufacturability of the reagents) and. 2. there is the requirement for a multivalent binding interaction between the target analyte and the liposome, in order to induce vesicle lysis.

Consequently, it follows that the underlying mechanism of liposome lysis in the Huang & Ho invention(s) , versus the present invention, is not the same; this invention requires "contact sensitive" liposomes to be brought into contact with each other, by any of a variety of pathways, dependent upon analyte concentration (independent of multivalent interaction of the liposome with the analyte and consequent contact capping of stabilizer molecules; bivalent antibodies can cause "contact sensitive" liposome lysis, without any direct binding to stabilizer molecules, which allows the use of the tremendous store of mouse monoclonal antibodies available commercially in the design of the immunoassays utilizing this invention) , while the Huang & Ho "target sensitive" liposomes require contact with a multivalent target surface (viral membrane, coated glass, etc.) to induce lysis.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that "contact sensitive" liposomes, composed of a hexagonal phase forming lipid as their major component, may be employed in a one or multistep, competitive or noncompetitive, homogeneous membrane lytic immunoassay format, with the following key performance characteristics: 1. liposome membrane lysis, and subsequent reporter molecule readout, is induced/inhibited either directly or indirectly by specific linker molecule - analyte interaction(s) , at the contact sensitive liposome surface,

2. utilization of separate and independent groups of compounds to stabilize and target the contact sensitive liposomes, imparting a large degree of flexibility in the formulation/design of the liposome reagents,

3. detection of analytes ranging from very small to very large molecular weight, including particulates, utilizing a variety of contact sensitive liposome formulations; multivalent interactions with analyte are not required; monovalent and bivalent binding are also able to induce/inhibit liposome lysis,

4. utilization of a "generic" contact sensitive liposome formulation to detect a wide variety of analytes; therefore, only one type of liposome formulation need be manufactured and stored,

5. dual readout capability of the immunoassay, in that agglutination of the liposomes and/or lysis of the liposomes, with resultant color generation, may be used as the signal for detection of analyte,

6. attainment of analyte specific membrane lysis, without the necessity to add any complement or lytic molecules/ions to the immunoassay mixture,

7. compatibility of the system with a very wide range of commercially available analyte specific linker molecules, such that all types of immunoassay reagents now in use are expected to work with the present liposome formulation,

8. achievement of a rapid (e.g., < 5 minutes), and sensitive (e.g., < ng/ l levels), analyte specific membrane lysis, at temperatures ranging from room temperature up to at least 70°C,

9. compatibility of the contact sensitive liposome formulation with a wide variety of commercially available reporter molecule systems, including absorbant, fluorescent, chemiluminescent and bioluminescent compounds coupled, if desired, to a variety of enzyme catalysts.

While not wishing to be bound by conjecture, the presumed mechanism is generally believed to involve an analyte mediated/dependent surface destabilization of a contact sensitive liposome resulting in the release of reporter molecules.

It is believed that the liposomal immunoassay system of the present invention is an excellent alternative for homogeneous phase detection of low molecular weight analytes (such as therapeutic drugs, drugs of abuse,etc.), which does not rely upon or mimic any systems such as, Syntex (EMIT ^R technology) , Abbott (TDx ^R technology) , and Microgenics (CEDIA™ technology) , to name but a few. More importantly, this invention may represent the only commercially viable method to perform homogeneous immunoassays on a wide variety of large molecular weight analytes, solution phase and particulate (tumor markers, hormones. lymphokines, antibodies, viruses, bacteria, mycoplasma, etc.) .

In describing the chemical composition of the liposomes and the accompanying immunoassays used in this invention, the following abbreviated terms will be used for the sake of simplicity and brevity :

ABBREVIATIONS

LUV : LARGE UNILAMELLAR VESICLES

PE : PHOSPHATIDYLETHANOLAMINE

DOPE : DIOLEOYL PHOSPHATIDYLETHANOLAMINE

DOPC : DIOLEOYL PHOSPHATIDYLCHOLINE

DOPA : DIOLEOYL PHOSPHATIDIC ACID

BIOTIN-CAP-DPPE: BIOTIN CAPROYL DIPALMITOYL

PHOSPHATIDYLETHANOLAMINE

DOPS : DIOLEOYL PHOSPHATIDYLSERINE MPB - PE : P - MALEIMIDOPHENYL - BUTYRYL - PE PBS : PHOSPHATE BUFFERED SALINE 3H - CE : HEXADECYL [³H] CHOLESTANYL ETHER DMPS : DIMYRISTOYL PHOSPHATIDYLSERINE N-GPE : N-GLUTARYL PHOSPHATIDYLETHANOLAMINE CHEMS : CHOLESTERYL HEMISUCCINATE DOSG: DIOLEOYL SUCCINYL GLYCEROL DMPG : DIMYRISTOYL PHOSPHATIDYLGLYCEROL DOPG : DIOLEOYL PHOSPHATIDYLGLYCEROL OA: OLEIC ACID

BRIJ ^R78 POLYOXYETHYLENE (20) STEARYL ETHER HSV : HERPES SIMPLEX VIRUS

The following definitions may also prove useful and helpful to the reader in comprehending the scope of the present invention: DEFINITION OF TERMS

1. As used herein, the term "inverted hexagonal phase forming lipids" (see Cullis, P.R. and de Kruijff, B., Biochi . Biophvs. Acta. 569. 399-420; (1979)) is use_d to refer to the so-called "cone" shaped lipids that in aqueous buffers under physiological conditions of temperature,salt, pH, etc., self aggregate into nonbilayer/nonlamellar, reverse/inverted micelles, consisting of hexagonally/closed packed extended lipid cylinders with a hydrophilic core.

2. As used herein, the term "targeting molecule(s) ", is used to refer to any compound, molecule, or composition on the surface of the liposome, that either directly or indirectly binds the analyte. These molecules can be either analyte specific or nonspecific. Examples of commercially available materials include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, and antibodies.

3. As used herein, the term "stabilizing molecule(s) ", is used to refer to any amphipathic compound, molecule, or composition, that upon addition to an inverted hexagonal phase forming lipid, allows the mixture to form a lipid bilayer phase (liposome) , enclosing an aqueous compartment. The stabilizer molecule is/can be independent of both the analyte(s), and the targeting molecule(s) ; it does not necessarily bind the analyte, either directly, or indirectly. Examples of commercially available materials include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, and antibodies. 4. As used herein, the term "amphipathic", is used to refer to any compound, molecule, or composition, that contains both a hydrophobic and a hydrophilic region.

5. As used herein, the term "contact sensitive", is used to refer to a liposome formulation which renders the phospholipid vesicles capable of a membrane lytic event, which is dependent upon analyte induced effects at the surface of the liposome(s) , and, independent of exogenously added lytic molecules or ions.

6. As used herein, the term "reporter molecule(s) ", is used to refer to any compound, molecule, or composition, which, upon release from the liposome, is itself directly capable, or able to generate another compound, molecule, or composition, capable of detection by eye or machine. Examples of commercially available materials include colorogenic or fluorogenic dyes, enzymes and their substrates, chemiluminescent species, and bioactive compounds, such as nucleic acids, hormones, lymphokines, etc., together with their appropriate cellular and noncellular targets.

7. As used herein, the term "analyte(s)", is used to refer to any compound, molecule, or composition to be qualitatively or quantitatively detected in the test sample media. The analyte can be any substance for which there exists a naturally occurring specific binding member, or, for which a specific binding member can be prepared. Examples include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, antibodies, viruses, and bacteria. 8. As used herein, the term "homogeneous immunoassay", is used to refer to an antibody - antigen binding mediated diagnostic assay, in which a single step or a sequence of steps, are performed without any intervening washing or separation steps required.

9. As used herein, the term "linker molecule(s)", is used to refer to an analyte specific or analyte nonspecific molecule(s) , present in solution (or in suspension, or on a solid support) together with the liposome reagent and the analyte, that binds directly or indirectly, to either the targeting molecule on the liposome surface, or the analyte, or both, or other linker molecules, allowing the analyte to induce a "contact sensitive" liposome lysis. Examples include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, antibodies, receptors and ligands.

It has been determined that a mathematical relationship can be.expressed between the targeting molecule(s), analyte(s) , and linker molecule(s) . In this expression, the targeting molecule(s) on the liposome surface are abbreviated "TM", the linker molecule(s) are abbreviated "LM", and the analyte(s) is abbreviated "AN". The mathematical relationship is:

TM + (LM-L + LM₂ + ... LM_n) + AN -

LIPOSOME MEMBRANE LYSIS

WHEREIN : n = 0, 1, 2 ... etc. Given the above expression, the following types of assays are possible herein:

1. WHEN n = 0, THERE ARE NO LINKER MOLECULES UTILIZED IN THE ASSAY, AND THE TARGETING MOLECULE ON THE LIPOSOME SURFACE BINDS DIRECTLY TO THE ANALYTE, CAUSING LIPOSOME MEMBRANE LYSIS.

2. WHEN ή = 1, THERE IS A SINGLE LINKER MOLECULE UTILIZED IN THE ASSAY, AND BOTH THE TARGETING MOLECULE ON THE LIPOSOME SURFACE AND THE ANALYTE BIND TO THE LINKER MOLECULE, CAUSING LIPOSOME MEMBRANE LYSIS.

3. WHEN n = 2, THERE ARE TWO LINKER MOLECULES UTILIZED IN THE ASSAY, ONE OF WHICH BINDS DIRECTLY TO THE TARGETING MOLECULE ON THE LIPOSOME SURFACE, WHILE THE OTHER BINDS DIRECTLY TO THE ANALYTE; LIPOSOME MEMBRANE LYSIS IS CAUSED BY THE BINDING OF THE LINKER MOLECULES TO EACH OTHER.

4. WHEN n = 3, THERE ARE THREE LINKER MOLECULES UTILIZED IN THE ASSAY, ONE OF WHICH BINDS DIRECTLY TO THE TARGETING MOLECULE ON THE LIPOSOME SURFACE, ANOTHER OF WHICH BINDS DIRECTLY TO THE ANALYTE, AND, ANOTHER OF WHICH BINDS THE OTHER TWO LINKER MOLECULES TO EACH OTHER RESULTING IN LIPOSOME MEMBRANE LYSIS.

5. WHEN n > 3, MORE LINKER MOLECULES ARE UTILIZED, WITH AN ACCOMPANYING INCREASE IN THE NUMBER OF LINKER MOLECULE INTERACTIONS REQUIRED TO CAUSE ANALYTE-DEPENDENT, LIPOSOME MEMBRANE LYSIS. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB illustrate the effect of analyte addition on the size of LUV composed of 90 mole percent DOPE, 10 mole percent MPB-PE as stabilizer, and 1.0 mole percent Biotin-PE as targeting molecule. Five μl (50 nmol) of LUV were added to 10 μl of PBS, in the presence (IB) or absence (1A) of 2.5 μg of polyclonal goat anti-biotin antibody. After 1 hour at 37°C, samples were analyzed by electron microscopy, and sized using photographically enlarged micrographs. The magnification shown is 20,OO0X and the bar insert in Fig. 1A represents a length of 500 nm.

Figure 2 depicts LUV containing 50 mM calcein and composed of 90 mole percent DOPE, 10 mole percent MPB-PE as stabilizer, and either 0.1 (ref. line 20) or 1.0 (ref. line 22) mole percent Biotin-PE as the targeting molecule. The LUV were incubated at 37°C for 1 hour with various amounts of goat anti-biotin antibody (analyte) .. Percent quenching was measured as described in the examples.

Figure 3 illustrates the effect of temperature on analyte induced lysis of LUV containing 50 mM calcein, composed of 85 mole percent of either DOPE (ref. line 30) or DOPC (ref. line 32) , stabilized with 15 mole percent DOPS, and containing 1 mole percent Biotin-PE as the targeting molecule. Ten μg of goat anti-biotin antibody (analyte) was added to the liposome suspension at a variety of temperatures. DOPE controls (ref. line 34) and DOPC controls (ref. line 36) were run in the absence of analyte. Percent quenching was measured after a 5 minute incubation as described in the examples. Figure 4 illustrates the effect of analyte concentration on lysis of LUV containing 50 mM calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1 mole percent Biotin-PE as targeting molecule. Incubation was at 70°C for 5 minutes. Percent quenching was measured as described in the examples.

Figure 5 illustrates the effect of free theophylline on the antibody mediated lysis of LUV containing 50 mM calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1 mole percent theophylline - DOPE as the targeting molecule/analyte. A mouse monoclonal anti-theophylline antibody was pre-incubated for 5 minutes at room temperature with a range of free theophylline in PBS (0-500 nmoles) , followed by addition of liposomes for 5 minutes at 70°C. Percent quenching was measured as described in the examples.

Figure 6 illustrates the detection of streptavidin in human serum samples, utilizing LUV containing 70 mM entrapped calcein, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent Biotin-Cap-DPPE as targeting molecule. LUV (146 nmol) were added to 7 different human sera that had been spiked with streptavidin, in the presence (ref. line 60) or absence (ref. line 62) of 1 μmol of SUV composed of 20 mole percent DOPA and 80 mole percent DOPE, added in order to inhibit nonspecific serum lysis of the LUV. Following incubation at 37°C, in a final volume of 200 μl at pH 6.5 in 200 mM HEPES, percent quenching was determined as previously described. Figure 7 illustrates the correlation of the Lipogen contact sensitive, liposome based competitive immunoassay for serum theophylline, as compared to the commercially available Dupont Petinia^R assay. Five hundred nmol of LUV containing 70 mM entrapped calcein, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent theophylline-DOPE as targeting molecule, were mixed with serum samples that had been briefly pre-incubated with mouse monoclonal anti-theophylline antibody and sonicated liposomes, composed of 75 mole percent DOPE and 25 mole percent DOPA (added to adsorb a nonspecific lytic serum component) . Incubation was in final volume of 100 μl at 37°C for 15 minutes. Percent quenching was measured as previously described in the examples.

Figure 8 illustrates the detection of HCG in human serum samples, utilizing LUV containing glucose oxidase, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent Biotin-Cap-DPPE as targeting molecule on the liposome surface. LUV (10.5 nmol) were added to sera that had been spiked with HCG, and preincubated briefly with 1 μmol of "empty bags", composed of 25 mole percent DOPA and 75 mole percent DOPE. Biotinylated mouse monoclonal anti-HCG (75 ng) together with streptavidin (9 ng) was added to the test samples as "linker molecules". Following incubation at 37°C for 5 minutes, in a final volume of 143.8 μl at pH 6.5 in 75 mM HEPES, 300 μl of a substrate mix containing glucose, HRP, and OPD, at pH 7.4, was added in order to allow released glucose oxidase to generate product. After a 3 minute incubation at room temperature, the reaction was quenched by the addition of 1.2 N HC1 and 1.2 % Triton-X 100. Reactions were carried out in microtiter plates and the final optical density was measured on a BioTek microplate reader at 490 n .

Figure 9 illustrates the detection of antibodies in human serum samples, utilizing LUV containing 70 mM calcein, and composed of 74.5 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.5 mole percent theophylline-DOPE conjugate as targeting molecule on the liposome surface. LUV (200 nmol) were added to sera that had been spiked with mouse monoclonal anti-theophylline antibody (1-5 μg) , and preincubated briefly with 1.5 μmol of "empty bags", composed of 25 mole percent DOPA and 75 mole percent DOPE. Following incubation at 37°C for 10 minutes, in a final volume of 250 μl at pH 6.5 in 75 mM MES buffer, percent quenching was measured as previously described.

Figure 10 depicts the virus induced lysis of LUV containing 50 mM calcein, composed of 80 mole percent DOPE, 20 mole percent DOPA as stabilizer, and 0.06 mole percent of anti-herpes mouse monoclonal antibody (conjugated to NGPE) as targeting molecule. Liposomes (500 μM) were incubated at room temperature with HSV (2 mg viral protein/ml) in the presence (ref. line 100) or absence (ref. line 110) of excess antibody, Sendai virus (ref. line 120) , or uninfected Vero cell supernatant (ref. line 130) . At various time intervals samples were measured for percent dye released as described in the examples. Control liposomes, containing DOPE/DOPA, but without targeting antibodies, were also tested for lysis by HSV (ref. line 140) . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Figure 1 (B) shows that the contact sensitive liposomes are extensively aggregated by the appropriate antibody, to form a heterogeneous mixture of multi-liposome particles, with an average diameter of between 1,000 - 2,500 nm. Leakage of entrapped calcein dye occurred subsequent to the agglutination step(s) , and the final fluorescent signal was proportional to the degree of liposome aggregation (data not shown) . Addition of a wide range of the monovalent. Fab fragment of specific antibody to the liposome suspension failed to either agglutinate the liposomes or induce calcein leakage (data not shown) . This data, taken together, emphasizes the "contact sensitivity" characteristic of the present liposome formulation. In all immunoassays performed thus far, liposome-liposome close contact appears to be a prerequisite for analyte induced, entrapped reporter molecule leakage. As a control. Figure 1 (A) shows an even dispersion of contact sensitive liposomes, incubated in the absence of antibody, with an average size (diameter) of 250 nm.

Figure 2 shows the results of an experiment described in Example 3, that shows that the degree of lysis of extruded liposomes containing DOPE as their bulk lipid is dependent upon both the concentration of analyte and the targeting molecule surface density, when temperature, liposome concentration, and time, are held constant. Specificity of the lysis reaction was demonstrated by the complete inhibition of dye leakage when an excess of free/soluble biotin was added to the mixture (data not shown) . The Figure 2 results lead to two important conclusions:

1. the DOPE liposome system can detect a 150,000 dalton, nonmultivalent analyte in a "contact sensitive" homogeneous format, and,

2. there is no need for the analyte to bind directly to the stabilizer molecule as in the Huang & Ho approach, so that a generic liposome design can be utilized to detect a wide variety of analytes, as long as a low/sufficient mole percent of the appropriate targeting molecule is present on the bilayer stabilized DOPE liposome surface.

Figure 3 shows that results of an experiment described in Example 4, that shows that a rapid and strongly temperature dependent analyte induced lysis occurred when liposomes composed of DOPE were utilized, while no such dye release was seen when conventional DOPC based liposomes, of otherwise identical composition and size were employed. At 70°C, even though DOPC based liposomes were aggregated by analyte, no lysis was seen for up to 30 minutes of incubation, further supporting the uniqueness of the PE lipid formulation in the present "contact sensitive" liposomal immunoassay. Just as significant, it was found, that even at temperatures as high as 70°C, DOPE liposomes incubated for up to 15 minutes in the absence of analyte, remained totally nonleaky, exhibiting surprising reagent stability.

Figure 4 shows the results of an experiment described in Example 5, which shows that across a wide range of analyte concentration, there exists a bell shaped liposome lysis curve, similar to that seen for classical antigen - antibody agglutination reactions. The significance of such a dose - response is the flexibility it allows in terms of immunodiagnostic assay design. One may design the system to start the reaction at the beginning, midpoint, or endpoint of the bell shaped curve, depending upon the particular needs of the assay. In subsequent experiments it was found that the sharpness/slope of the curve could be controllably fine - tuned by manipulation of the identity and mole percent of the liposome stabilizer molecule, and the mole percent of the targeting molecule (data not shown) .

Manipulations of divalent cation concentrations, salt concentrations (osmotic pressures), and pH levels, in the assay incubation buffer, were found to decrease both the required time, and incubation temperature, of the assay (data not shown) . For example, it is possible to perform the assay at room temperature and at 37°C, in less than 5 minutes.

Figure 5 shows the results of an experiment described in Example 6 in which an analyte of actual commercial and therapeutic interest, theophylline, is coupled to DOPE as a "targeting molecule" on the surface of the contact sensitive liposome. Increasing amounts of free theophylline pre-mixed with the liposome reagent were able to inhibit mouse monoclonal anti-theophylline antibody induced liposome agglutination and lysis in a dose dependent manner. Such a homogeneous two step competition assay for small drugs is similar to commercially available technologies such as the Syva EMIT ^R system and the ABBOTT TDx ^R system. Significantly, unlike the data shown in Figures 1A and IB for the biotin model system, a commercially available mouse monoclonal antibody was utilized (whereas previously goat polyclonal anti - biotin antibody was used) to effect liposome aggregation and lysis. To check the specificity of the immunoassay a variety of mouse monoclonal antibodies nonreactive toward theophylline as well as a variety of small therapeutic drugs unrelated to theophylline were tested and found to have no effect on the assay performance (data not shown) .

Figure 6 shows that the results of an experiment described in Example 7, that shows the use of SUV (and, in subsequent experiments, EV; data not shown) composed of DOPE and DOPA (containing no reporter molecule(s) , and no surface targeting molecule(s); referred to as "empty bags") , allows the detection of a high molecular weight, model "analyte", streptavidin, in human serum, in the ng/ml range of sensitivity, utilizing contact sensitive LUV (60) . In the absence of SUV (62) , there is no dose response proportional to analyte concentration, due to a large degree of nonspecific LUV lysis by the serum samples. The major significance of these data is the ability to broaden the use of the contact sensitive liposome reagent to detection of analytes in human serum, employing the empty bags to adsorb out any interfering substances.

The ability to quantitatively detect streptavidin in 5 minutes, in serum, or in a variety of conventional buffers, by a single step, homogeneous immunoassay format, allows the contact sensitive liposome formulation to be utilized in a large number of conventional ELISA protocols, currently utilizing biotin/streptavidin bridges, in- the diagnostics field. An interesting possibility is the use of the contact sensitive liposome to quantitate the amount of DNA synthesized by the enzymes used in the PCR gene amplification technology. Another intriguing possibility is the use of the liposomes of the present invention in conjunction with a variety of biosensor probe technologies.

Figure 7.shows the results of an experiment described in Example 8, that shows that the present contact sensitive liposome formulation compares quite favorably in performance to the commercially well established Dupont Petinia ^R theophylline immunoassay kit. The present inventors were able to achieve almost a 90% correlation with the Dupont assay, while evaluating 21 human patient sera obtained from a local hospital.

The present homogeneous, liposome based membrane lytic immunoassay was linear over the entire therapeutically relevant range of theophylline (0-40 μg/ml) tested. (Results of an experiment in which theophylline levels were quantitated in serum, which is the relevant biological fluid for the detection of this particular drug (and most other therapeutic drugs) . When switching from an aqueous buffer (PBS) to actual human serum samples it was discovered that it was necessary to include appropriate levels of liposomes containing neither entrapped dye nor any targeting molecule on their surface ("empty bags") and/or adjust the liposome formulation in order to circumvent a somewhat strong decrease in the reaction rate due to unidentified serum components) ."

Figure 8 shows the results of an experiment described in Example 9, that shows that the contact sensitive DOPE-based liposome is able to detect, by a totally homogeneous (one or two step) immunoassay format, a high molecular weight, solution phase hormone analyte, human chorionic gonadotropin (HCG: 33,000 daltons) , in human serum. Utilizing glucose oxidase entrapped in the liposome, coupled with HRP in the readout system, 30 ng of HCG was clearly detectable. A "generic" liposome formulation was used, employing a biotinylated phospholipid as the phospholipid vesicle surface handle ("targeting" molecule) , together with the appropriate analyte specific (biotinylated monoclonal antibodies) , and analyte nonspecific (streptavidin), "linker molecules", added in solution phase. This represents a significant decrease in the complexity and cost of assay manufacturing, since a panel of analytes is detectable by a single generic liposome formulation.

The present inventors have since been able to detect LH, FSH and TSH, utilizing the same generic liposome formulation, simply by changing to the appropriate linker molecules in each case (data not shown) .

Significantly, these results show that this contact sensitive liposome formulation is able to release latent enzyme activity proportional to analyte concentration, at 37° C in 5-10 minutes, (or at room temperature in 20 min. - data not shown) , without any washing or separation steps. Essentially, "antigen capture" is accomplished in the solution/suspension phase, with no need to manufacture any solid phase antibody coated microwells, for example. Consequently, these liposome reagents should be compatible with a wide variety of ELISA based immunoassays; ranging from microwell to dedicated equipment formats; at the same time performing in much shorter time frames, homogeneous immunodiagnostic assays for a wide variety of small/single epitope, and large/multiepitope analytes, requiring little or no training of laboratory personnel, and highly simplified clinical equipment.

In addition, the coupling of a homogeneous immunoassay format (for large or small analytes) , together with the utilization of an enzyme as a reporter molecule, allows a tremendous amount of assay design flexibility, in terms of the variety of kinetic measurements available to the end user (initial, midpoint, endpoint reaction time measurements) .

In a separate series of experiments, immunoliposomes were constructed with biotinylated anti-HCG antibodies attached through a streptavidin bridge to a biotinylated phospholipid on the surface of a contact sensitive liposome. Such a "nongeneric" liposome is presumably harder to manufacture, but may allow higher immunoassay sensitivities, since there should be much less unused linker molecule(s) - analyte complexes in the test sample. Incubation of the liposomes with either, 1.) streptavidin plus biotinylated anti - HCG antibodies, or, 2.) antigen (HCG) alone, did not lead to liposome lysis; only upon mixing of all components did an analyte dependent contact sensitive liposome entrapped enzyme leakage occur.

Figure 9 shows the results of an experiment described in Example 10, that shows the detection of specific antibodies in human serum, once again utilizing the contact sensitive liposome reagent(s), in a totally homogeneous immunoassay format. Although only μg quantities of serum antibody were homogeneously detected in this case, it is expected that one will be able to increase the detectability to the ng and pg levels by replacement of enzymes for fluorescent dyes as reporter molecules (see HCG results. Figure 8) . These results extend the use of the liposomes of the- present invention to the quantitative measurement of analytes as large as 150,000 daltons, as compared to HCG (and related hormones) at approximately 33,000 daltons. Furthermore, these data indicate the potential to utilize the contact sensitive liposomes as a screen for serum antibodies. For example, it is of interest to the present inventors to chemically link an appropriate mixture of AIDS virus specific peptides to the liposome surface, as targeting molecules, and devise a single step, homogeneous, quantitative immunoassay for antibodies to the AIDS virus (or detect virus itself, by a two step, homogeneous competition immunoassay utilizing the same liposomes) .

Figure 10 shows the results of an experiment described in Example 11, that shows an extension of the contact sensitive liposome immunoassay to the detection of multivalent, particulate analytes. In addition, these results were the first obtained utilizing as reagents true "immunoliposomes", in that the analyte specific linker molecules/antibodies were covalently attached to the surface of the liposome. When a cellular extract of HSV was added to the immunoliposomes at room temperature, a rapid lysis of the vesicles, leading to an increase in fluorescent signal, was observed. The lysis could be partially blocked by the addition of a 100-fold excess of solution phase antibody, and appeared to be specific in that Sendai virus at the same protein concentration gave little dye release.

Table I shows a list of stabilizers that have been utilized in the various immunoassays described in this text. This list is not meant to be exhaustive, but instead, is included to point out the broad range of stabilizers available to construct/formulate the "contact sensitive" liposome of this invention. In addition, the percent stabilizer shown in this table simply represents the most commonly used mole percent of each compound utilized in the present inventors attempt to most controllably and conveniently manufacture and store the liposomes, as well as to optimize, up to this point in time, analyte detection, in terms of assay time, temperature, sensitivity, sample fluid media compatibility, etc.

TABLE I

STABILIZERS UTILIZED IN THE CONSTRUCTION OF CONTACT

SENSITIVE LIPOSOMES

STABILIZER MOLE PERCENT

BIOTIN-PE 10

MPB-PE 10

DOPS 15

DPPG 15

DMPG 15

STERALYLAMINE 15

OLEIC ACID 15

DOPA 20

DOPG 20

CHEMS 25

DOSG 25

BRIJ^R78 10

The present invention will be further illustrated with reference to the following examples which aid in the understanding of the present invention, but which are not to be construed as limitations thereof. All percentages reported herein, unless otherwise specified, are percent by weight. All temperatures are expressed in degrees Celsius.

REAGENTS

All synthetic phospholipids were purchased as a chloroform solution from Avanti Polar Lipids, Inc. Goat anti - biotin antibody, and calcein were purchased from Sigma Chemical Co. The mouse monoclonal anti - theophylline antibody was purchased from Medix. Human chorionic gonadotropin and streptavidin were purchased from Scripps Laboratories. The ³H - hexadecyl cholestanyl ether was a gift from Dr. Leaf Huang at the University of Tennessee. Theophylline - DOPE was synthesized and purified as described below.

EXAMPLE 1

PREPARATION OF DOPE-BASED, CONTACT SENSITIVE LARGE UNILAMELLAR LIPOSOMES (LUV)

Lipid preparations were composed of either DOPC or DOPE as the bulk phospholipid (80-90 mole percent) , 10-20 mole percent of an amphipathic stabilizer compound, and 0.1-1.0 mole percent of a surface targeting molecule(s) . In some preparations 3H-CE was included in the lipid mixture as a tracer. Lipid films were generated by evaporation of solvent from a chloroform/phospholipid solution utilizing a N₂ gas stream, followed by desiccation of the lipids at reduced pressure for a minimum--of four hours. Lipids (10 mM) were hydrated at room temperature for at least 30 minutes in PBS containing 50 mM calcein dye. Calcein, 2^/,7'-[[bis(carboxymethyl)amino]methyl] - fluorescein, is a self - quenching dye that, at concentrations greater than 5 μM, undergoes progressive collissional fluorescence quenching. It has been widely used for analysis of liposome lysis/leakage , especially in the design of homogeneous phase systems. The calcein solution was prepared at 50 mM in distilled water and the pH adjusted to 7.4 with 10 N NaOH. The osmolarity of the solution was adjusted with 10X PBS to that of human serum, approximately 330 milliosmoles. The lipid suspensions were formed by rigorous vortexing and sized by repeated extrusion (10 times) through Nucleopore polycarbonate filters (0.2 μm pore size) at 200 psi utilizing a Lipex Extruder.

Separation of liposomes with entrapped, quenched fluorescent dye, from unentrapped, nonquenched soluble dye, was accomplished by column chromatography (BioGel A15M) . In all cases, the lipid yield off of the column was greater than 90% and the vesicles were quite homogeneous in size (162 +/- 33 nm diameter) . No changes in either percent quench (see below) or vesicle size was detectable following 6 months storage at 4°C.

The percent fluorescent quench of the entrapped calcein in the liposomes eluting in the BioGel column void volume was calculated using the following equation:

Percent quench = [(F(t) - F(i))/F(t) ] X 100 , where F(i) is the initial fluorescence, F(t) is the total fluorescence after the addition of detergent (which releases all of the entrapped calcein) . Initial calcein fluorescence was measured by adding a 5 μl lipid sample to 3 ml of PBS in a quartz cuvette, and reading light emission intensity on a Perkin Elmer LS - 5B Luminescence Spectrophotometer at 490 nm excitation/ 520 nm emission. Total calcein fluorescence was monitored after the addition of 10 μl of 10% Triton X - 100 to completely lyse the vesicles and release all entrapped dye.

The size of the liposomes was determined by laser light scattering using the Coulter N4MD submicron particle analyzer.

EXAMPLE 2

AGGLUTINATION OF ANTIGEN/HAPTEN TARGETED CONTACT SENSITIVE LIPOSOMES BY SPECIFIC ANTIBODY

Five μl (50 nmol) of LUV composed of 90 mole percent DOPE, 10 mole percent MPB-PE as stabilizer, and 1.0 mole percent Biotin-PE as targeting molecule on the liposome surface, were added to 10 μl of PBS, in the presence or absence of 2.5 μg of polyclonal goat anti-biotin antibody. After a 1 hour incubation at 37°C, the liposome samples were visualized using negative stain electron microscopy, and a Hitachi 600 electron microscope; the size of the liposomes was measured on photographically enlarged micrographs. EXAMPLE 3

EFFECT OF TARGETING MOLECULE SURFACE DENSITY AND ANALYTE CONCENTRATION ON CONTACT SENSITIVE LIPOSOME LYSIS

Ten μl (70 nmol) of LUV with 50 mM entrapped calcein, composed of 90 mole percent DOPE, 10 mole percent MPB - PE as stabilizer, and either 0.1 or 1.0 mole percent Biotin - PE as targeting molecule on the liposome surface, were added to 200 μl PBS. The goat anti - biotin polyclonal antibody, which represents a large, solution phase analyte in this case, was added (0 - 20 μg) to the liposome suspensions, and incubated for one hour at 37°C in a water bath, prior to the measurement of percent quench as described above.

EXAMPLE 4

EFFECT OF BULK PHOSPHOLIPID AND INCUBATION TEMPERATURE ON CONTACT SENSITIVE LIPOSOME LYSIS

Ten μl (70 nmol) of LUV with 50 mM entrapped calcein, composed of either 85 mole percent DOPE or DOPC, 10 mole percent DOPS as stabilizer, and 1.0 mole percent Biotin - PE as targeting molecule on the liposome surface, were added to 200 μl PBS. Approximately 10 μg of goat anti - biotin antibody was added as analyte to the suspension and incubated for 5 minutes in a Multi - Block heater (American Scientific Products) at various temperatures; controls contained no antibody. Percent quench was determined as previously described. EXAMPLE 5

EFFECT OF ANALYTE CONCENTRATION ON CONTACT SENSITIVE LIPOSOME LYSIS

Three μl (21 nmol) of LUV with 50 mM entrapped calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1.0 mole percent Biotin - PE as targeting molecule on the liposome surface, were added to 200 μl PBS. Goat anti - biotin antibody (0 - 200 μg) was added as analyte to the liposome suspensions, and then incubated for 5 minutes at 70°C. Percent quench was measured as previously described.

EXAMPLE 6

DETECTION OF THEOPHYLLINE BY COMPETITIVE INHIBITION OF CONTACT SENSITIVE LIPOSOME LYSIS

Four μl (32 nmol) of LUV with 50 mM entrapped calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1.0 mole percent theophylline - DOPE conjugate as targeting molecule / analyte on the liposome surface, were added to 200 μl PBS. Mouse monoclonal anti - theophylline antibody was premixed for 5 minutes at room temperature with a range of theophylline concentrations in PBS (0 - 500 nmoles) , followed by addition to the liposome suspension for a 5 minute incubation at 70°C. Percent quench was determined as described above.

The theophylline - DOPE conjugate was prepared by the condensation of 5,6-diamino-l,3 - dimethyluracil with succinic anhydride to form 8-(2-carboxyethyl)- theophylline. This theophylline derivative, in turn, generated the desired lipid conjugate upon condensation with DOPE. Following purification by preparative TLC the drug conjugate was mixed with the appropriate phospholipids and liposomes were formed by the extrusion method as previously described.

EXAMPLE 7

DETECTION OF STREPTAVIDIN IN SERUM UTILIZING CONTACT SENSITIVE LIPOSOMES AND "EMPTY BAGS"

One μmol of sonicated liposomes, composed of 20 mole percent DOPA and 80 mole percent DOPE (containing neither targeting molecules nor entrapped reporter molecules; henceforth referred to as "empty bags"), was added, in order to inhibit an unidentified, nonspecific serum lytic factor, to seven different human sera spiked with various amounts of streptavidin. After brief mixing, 146 nmol of LUV containing 70 mM of entrapped calcein, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent Biotin-Cap-DPPE as targeting molecule on the liposome surface, were added to bring the mixture to a final volume of 200 μl at pH 6.5 in 200 mM HEPES. Following incubation for 5 minutes at 37°C, percent quenching was measured as previously described. EXAMPLE 8

QUANTITATIVE DETERMINATION OF SERUM THEOPHYLLINE LEVELS CORRELATION OF CONTACT SENSITIVE LIPOSOMAL IMMUNOASSAY WITH COMMERCIAL DUPONT PETINIA ASSAY

Two μl of serum or standard(s) was added to 5 μl (10.8 μg) of a mouse monoclonal anti-theophylline antibody, followed by the addition of 20 μl (1 μmol) of sonicated liposomes, composed of 25 mole percent DOPA and 75 mole percent DOPE in 20 mM HEPES, pH 7.4 ("empty bags", added in order to inhibit an unidentified, nonspecific serum lytic factor) . After brief mixing, 60 μl of 290 mM HEPES, pH 6.5, and 10 μl (500 nmol) of LUV with 70 mM entrapped calcein, composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent theophylline-DOPE conjugate as targeting molecule/ nalyte on the liposome surface, were added sequentially, and incubated at 37°C for 15 minutes. Percent quenching was measured as previously described. The standard curve consisted of 0, 5, 10, 20, 30, and 40 μg/ml free theophylline in serum calibrators.

EXAMPLE 9

DETECTION OF SERUM HUMAN CHORIONIC GONADOTROPIN (HCG) UTILIZING CONTACT SENSITIVE LIPOSOMES CONTAINING ENTRAPPED ENZYMES

Ten μl (10.5 nmol) of LUV with 189 μmol of entrapped glucose oxidase, composed of 74.5 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.5 mole percent Biotin-Cap-DMPE as targeting molecule on the liposome surface, were added to 133.8 μl of a HEPES buffered mixture (pH 6.5)-, containing 1 μmol of "empty bags", and 75 ng of biotinylated mouse monoclonal anti-HCG together with 9 ng of streptavidin, as "linker molecules". To this mixture was added 2 μl of human serum spiked with increasing amounts of HCG, followed by incubation at 37°C for 5 minutes in microtiter plate polystyrene wells. At the end of the incubation, 300 μl of an enzyme substrate mix containing glucose, HRP, and OPD was added to react with released glucose oxidase. After a 3 minute incubation at room temperature, 1.2 N HC1 and 1.2% Triton-X 100 was added to stop the reaction, and the final optical density was measured at 490 nm utilizing a BioTek microwell reader.

EXAMPLE 10

DETECTION OF SERUM ANTIBODIES UTILIZING CONTACT SENSITIVE LIPOSOMES

Twenty μl (200 nmol) of LUV with 70 M entrapped calcein, composed of 74.5 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.5 mole percent theophylline-DOPE conjugate as targeting molecule on the liposome surface, was added to 230 μl of MES buffer, pH 6.5, containing 1.5 μmol of "empty bags", composed of 25 mole percent DOPA and 75 mole percent DOPE, and 3 μl of human serum spiked with increasing amounts (1-5 μg) of a mouse monoclonal anti-theophylline antibody (IgG, subclass 2b) . Following a 10 minute incubation at 37°C percent quenching was measured as previously described. EXAMPLE 11

DETECTION OF HERPES SIMPLEX VIRUS (HSV) UTILIZING CONTACT SENSITIVE IMMUNOLIPOSOMES

Twenty μl (100 nmol) of LUV with 50 mM entrapped calcein, composed of 80 mole percent DOPE, 20 mole percent DOPA as stabilizer, and 0.06 mole percent anti - herpes mouse monoclonal antibody as targeting molecule, was added to 200 μl PBS containing HSV (2 mg viral protein/ml) , in the presence or absence of excess antibody, Sendai (control) virus, or uninfected Vero cell supernatant. After incubation at room temperature for various times, percent quench was measured as described.

In order to incorporate the anti-HSV monoclonal antibody onto the surface of the liposome, it was necessary to conjugate the antibody to N-glutaryl-PE in the presence of carbodiimide and N-hydroxy sulfosuccinimide (S-NHS) . Following the coupling reaction, the NGPE-IgG mixture was dialyzed against PBS, and then co-sonicated with the appropriate phospholipid mixture, in order to insert the antibody-lipid complex into the liposome bilayer.

The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention and still be within the scope and spirit of this invention as set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A contact sensitive liposome formulation, comprising from about 0.1 to about 99.9 mole percent o_f one or more inverted hexagonal phase forming lipids, said liposome stabilized in the bilayer phase with from about 0.1 to about 99.9 mole percent of one or more analyte independent amphipathic stabilizer molecules or mixtures thereof, said liposome further comprising one or more targeting molecules on the surface thereof.

2. A contact sensitive liposome formulation, comprising from about 0.1 to about 99.9 mole percent of one or more inverted hexagonal phase forming lipids, said liposome stabilized in the bilayer phase with from about 0.1 to about 99.9 mole percent of one or more analyte independent amphipathic stabilizer molecules or mixtures thereof, said liposome further comprising one or more targeting molecules on the surface thereof, and said liposome further containing one or more entrapped reporter molecules.

3. A contact sensitive liposome formulation, comprising from about 0.1 to about 99.9 mole percent of one or more inverted hexagonal phase forming lipids, said liposome stabilized in the bilayer phase with from about 0.1 to about 99.9 mole percent of one or more analyte independent amphipathic stabilizer molecules or mixtures thereof, said liposome further comprising one or more analyte molecules, or analyte fragments, binding domains, or subunits on the surface thereof.

4. A contact sensitive liposome formulation. comprising from about 0.1 to about 99.9 mole percent of one or more inverted hexagonal hase forming lipids, said liposome stabilized in the bilayer phase with from about 0.1 to about 99.9 mole percent of one or more analyte independent amphipathic stabilizer molecules or mixtures thereof, said liposome further comprising one or more analyte molecules, or analyte fragments, binding domains, or subunits on the surface thereof, and said liposome further containing one or more entrapped reporter molecules.

5. The contact sensitive liposome formulation of claim 1, 2, 3, or 4, wherein the inverted hexagonal phase forming lipid, is selected from the group of phosphatidylethanolamine (PE) lipids, and PE-like lipids, consisting of saturated PE lipids, unsaturated PE lipids, and cardiolipin.

6. The contact sensitive liposome formulation of claim 1, 2, 3, or 4, wherein the analyte independent amphipathic stabilizer molecule is selected from the group consisting of carbohydrates, proteins, nucleic acids, lipids, drugs, steroids, hormones, lectins, enzymes, metabolites, antigens, haptens, and antibodies.

7. ^" The contact sensitive liposome formulation of claim 1 or 2, wherein the targeting molecule is selected from the group consisting of carbohydrates, proteins, nucleic acids, lipids, drugs, steroids, hormones, lectins, enzymes, metabolites, antigens, haptens, and antibodies.

8. The contact sensitive liposome formulation of claim 3 or 4, wherein the analyte molecule on the liposome surface is selected from the group consisting of carbohydrates, proteins, nucleic acids, lipids, drugs, steroids, hormones, lectins, enzymes, metabolites, antigens, haptens, and antibodies.

9. The contact sensitive liposome formulation of claim 2 or 4, wherein the reporter molecule is selected from the group consisting of colorogenic dyes, fluorogenic dyes, phosphorescent dyes, enzymes or enzyme substrates, cofactors and inhibitors, chemiluminescent compounds, spin labels, heavy metal cations, chelators, and bioactive compounds.

10. A direct,noncompetitive, homogeneous, liposome based , membrane lytic immunoassay for multi-epitope, solution phase or particulate, analytes in a test sample, said assay utilizing the contact sensitive liposome reagent of claim 2, including an analyte specific targeting molecule(s) on the liposome surface, said assay including the following steps:

(a) adding the liposome reagent to a buffered solution;

(b) adding the analyte suspect test sample to the buffered liposome reagent in step (a) ; and,

(c) monitoring the solution in step (b) for analyte dependent release of liposome entrapped reporter molecules;

(d) using analyte standards, generating a standard curve; and,

(e) determining the level of analyte present in the test sample by comparison of the level of reporter molecule release measured in step (c) , with the standard values generated in step (d) .

11. A direct, noncompetitive, homogeneous, liposome based, membrane lytic ύmmunoassay for multi-epitope, solution phase or particulate, analytes in a test sample, said assay utilizing the contact sensitive liposome reagent of claim 2, including an analyte-nonspecific targeting molecul (s) on the liposome surface, said assay including the following steps:

(a) adding the appropriate mixture of analyte specific and analyte nonspecific linker molecules to the analyte suspect test sample;

(b) adding the liposome reagent in a buffered solution to solution (a) ; and,

(c) monitoring the solution of step (b) for analyte dependent release of the liposome entrapped reporter molecules;

(d) using analyte standards, generating a standard curv ; and,

12. An indirect, competitive, homogeneous, liposome based, membrane lytic immunoassay for single or multi-epitope, solution phase or particulate, analytes in a test sample, said assay utilizing the contact sensitive liposome reagent of claim 4, including an analyte molecule(s), or fragment thereof, on the liposome surface, said assay including the following steps:

(a) adding analyte specific linker molecules to the analyte suspect test sample;

(b) adding the liposome reagent in a buffered solution to said test sample;

(c) monitoring the solution in step (b) for analyte dependent inhibition of release of the liposome entrapped reporter molecules;

(d) using analyte standards, generating a standard curve; and,