EP0258388A1 - Immunoliposome assay - methods and products - Google Patents

Abstract

New membranolytic immunological analysis. In one embodiment of this analysis, in particular in a solution phase system, an antibody is first covalently linked to a lipid which must serve as an anchor for the introduction into the membrane of the liposome. This antibody-anchor complex is used in combination with a lipid or a mixture of lipids which do not otherwise form a bilayer, to obtain vesicles of stable bilayer liposomes further containing a self-extinguishing fluorescent dye. When this liposome containing an antibody and a dye is brought into contact with a solution containing antigen molecules, a rapid bond occurs between the antibody-anchor complex and the antigen, which causes the liposome to rupture and release dye. Dye release can be quantified by spectrophotometric or spectrofluorometric methods using standard solutions.

Description

IMMUNOLIPOSOME ASSAY - METHODS AND PRODUCTS

FIELD OF THE INVENTION

This invention is directed to a membrane lytic immunoliposome assay and to products useful therein, especially in kit form. The assay utilizes the lateral phase separation of an antigenic or antibody liposome resulting in the destabilization and lysis of the liposome which may be quantified and employed in determining the presence and/or concentration of antigens, antibodies and like agents in biological fluids and other samples.

BACKGROUND OF THE INVENTION

High volume screening assays are commonly employed for detecting the presence of, and quantitatively measuring antigenic materials, antibodies and analytes in biological samples. For example, radioimmuno?ssay (RIA) techniques are commonly employed for clinical diagnostics. However, RIA procedures are often incompatible with large scale screening program. Radiotracers, by their very nature, are of .limited stability and they require special handling during use. special disposal techniques and sophisticated instrumentation.

Other immunoassay methods currently available include fluorescent and enzymatic techniques. Generally, these assays require a separation step, either by filtration or centrifugation in order to be interpreted. These separation requirements make the assay methods slow and difficult to automate.

Lipo-jcmes have previously been reported as useful components for immunoassays. For example, McConnell et al., U.S. Patent No. 3,887,698, describe the use of liposomes containing stable free radicals in an electron paramagnetic resonance (EPR) monitored immunoassay. Mandle et al. , U.S. Patent No. 4,372,745, describe the use of liposomes as fluorescer containing microcapsules, useful in an immunoassay. This assay requires the use of a detergent such as, Triton X-100 to break the liposomes and release the fluorescent compound. Liposomes have also been employed as a marker carrier in an immunoassay described by Ull an et al., U.S. Patent No. 4,193,983. Markers used in this assay included fluorescers, enzymes and chemiluminescent compounds.

Kinsky and his colleagues were the first to show that liposomes containing haptenated lipids could bind with an antibody and fix the complement thereof (Haxby et al., Biochem. - 8. 1582 (1969); Kinsky et al., Biochem. , 8_ 4149 (1969) ) . The result was the lysis of the liposomes by the activated complement components. Cole, U.S. Patent No. 4,342,826, describes an immunoassay method which utilizes antigen-tagged, enzyme-encapsulated liposomes which are immunospecifically ruptured in the presence of the cognate antibody and an active complement. The assay utilizes the homogeneous phase reaction between the antibody and complement to release the enzyme marker. This complement mediated event has been the focal point for a large amount of literature (for a recent review, see Alving & Richards, Liposomes, Ostro, ed. ■ 209-287 (Marcel Dekker, New York, 1983)).

Recently several noncomplement mediated liposome lytic assays have been developed. For example, binding of an antibody to haptens conjugated to a membrane lytic protein, melittin, blocks the liposome lytic activity of the melittin (Freytag et al. ,

Biophvs. CL_., 4_5_. 360(a) (1984)). Binding of the antibody in the Lupus serum to liposomes containing cardiolipin prevents the lysis the liposome by Mg⁺2 ions (Janoff et al. , Clin. Chem. , 29 1587 (1983)). While no complement is required each of these assays requires either a membrane lytic molecule or ion.

Although the previously described assays may be quite sensitive, they often involve many steps, and are sometimes difficult to reproduce and/or automate. Thus, new and more efficient assays are desirable.

SUMMARY OF THE INVENTION

The present invention is directed to an immunoassay wherein the lysis of the liposomes is a direct consequence of the immune complex formation. The assay of this invention is as sensitive as RIA, providing rapid determinations, yet it does not require the presence of membrane lytic molecules, ions, or active complements.

This invention is directed to a new membrane lytic immunoassay. Accordingly, said membrane lytic immunoassay comprises the steps of:

(a) forming liposomes with the analyte of interest in the liposome membrane, said liposomes containing a marker compound;

(b) mixing a test fluid containing a receptor for the analyte of interest with said liposomes of step (a) for sufficient time to saturate the liposomes with the receptor present in said test fluid;

(c) determining the presence of marker compound released by the liposomes in step (b) .

In one embodiment of this assay, namely a solution phase system, an antibody is first covalently coupled to a lipid which is to serve as an anchor for insertion into the liposome membrane. This antibody-anchor complex is used in conjunction with an otherwise non-bilayer forming lipid or mixture of lipids to form stable bilayer liposome vesicles which additionally contain a self-quenching fluorescent dye. When this antibody and dye containing liposome is brought into contact with a solution containing antigen molecules, rapid binding occurs between the antibody-anchor complex and the antigen, disrupting the liposome and releasing the dye. Release of the dye can be quantified by spectrophotometric or spectrofluorometric methods using standardized solutions. The amount of antigen in an unknown sample is then determined by comparison with the amount in known standards.

In another embodiment of this assay, namely, a solid-phase supported system, an antigen is first covalently coupled to a lipid which is to serve as an anchor for insertion into liposome membranes and this antigen-anchor complex is used in conjunction with an otherwise non-bilayer forming lipid or mixture of lipids. to form stable bilayer liposome vesicles which additionally contain a self-quenching fluorescent dye. When this antigen and dye containing liposome is brought into contact with an inert solid surface having attached thereto, antibody molecules, rapid binding occurs between the antigen-lipid complex and the antibody, disrupting the liposome and releasing the dye. Release of the dye can be quantified using standardized solutions. The amount of analyte in an unknown sample is then determined by comparison with the amount in known standards. The invention is also directed to products useful in said assay, especially in kit form.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates the stabilization of DOPE and DOPC liposomes with DNP-cap-PE. 90° light scattering of the sonicated lipid were measured for DOPE (a) and DOPC (b) liposomes;

Fig. 2 is a negative stain electron microscopic photograph of DOPE-DNP-cap-PE (88:12) liposomes at a magnification of 1.12 x 106;

Fig. 3 illustrates the immunospecificity of DOPE liposome lysis by antibody attached to a glass slide; Fig. 4 illustrates that DOPC liposomes are not lysed by antibody attached to a glass slide.;

Fig 5 illustrates the effect of the attached antibody concentration on DOPE liposome lysis.

Anti-DNP IgG (c) or normal IgG (d) at indicated concentrations was used to coat a glass slide, and the DOPE liposome lysis was measured by calcein release;

Fig. 6 illustrates the inhibition of DOPE liposome lysis by free hapten. DNP-Gly (e) or Gly (f) of indicated concentration was added to attached anti-DNP IgG on a glass slide before liposome addition. Fig. 7 illustrates the inhibition of DOPE liposome lysis by free antibody. Liposomes were preincubated with free anti-DNP IgG (g) or normal IgG (h) at indicated concentrations before adding to the attached antibody on a glass slide.

Fig. 8 is a schematic representation of a solid phase immunoliposome assay.

Fig. 9 illustrates the optimal palmitic acid to IgG coupling ratio for the preparation of a ligand-anchor (A) , and the optimal palmitoyl IgG to DOPE ratio for the preparation of the stable immunoliposome (B) . Stability of the liposomes were measured by both the calcein encapsulation and the degree of fluorescence quenching.

Fig. 10 illustrates the Herpes Simplex Virus-induced liposome lysis as measured by the release of the entrapped calcein. Other indicator viruses do not induced lysis.

Fig. 11 illustrates the color change of the liposomes when they are lysed by the Herpes Simplex

Virus. The tube on the right contains the intact liposomes and the one on the left contains lysed liposomes.

Fig. 12 illustrates the inhibition of liposome lysis by preincubation of the Herpes Simplex Virus with free antibody (anti-HSV-gD) but not by an unrelated antibody (anti-HSV-gB) or BSA.

Fig. 13 is a schematic representation of the solution phase immunoliposome assay embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Liposomes are microscopic vesicles composed of closed lipid bilayers. See: Papahadjopoulos, Ann. N.Y. Acad. Sci. , 301 1 (1978). Due to their relatively simple composition and their flexibility for chemical, physical and immunological manipulations, liposomes are a favorite material for membrane lytic assays.

When compared to other immunoassay techniques, there are several advantages in using a membrane lytic immunoassay: (1) a single lytic event can lead to the release of many signal molecules and hence there is a high degree of signal amplification, (2) it is rarely necessary to separate the immune complex from the free antibody or antigen and hence it is generally a homogeneous assay; and (3) optical measurements such as colorimetric and fluorometric techniques can be used and hence it avoids any requirement for the use of radioisotopes. For these reasons, membrane lytic assays have received increasing attention in the recent development of immunoassays.

The immunoliposome assay of the present invention will be illustrated by referring to the assay for one particular entity, i.e., an antigen. The general principles and techniques described herein for assaying an antigen can then be applied to assay for other species such as, for- instance antibodies, haptens, etc.

In order to aid in the understanding of the present invention, the following terms as used herein and in the claims have the following meanings:

Analyte - the compound or composition to be measured, which may be a ligand, such as an antigen, hapten or an antibody. For example, in a preferred embodiment, the analyte may be either an antigen or an antibody.

Ligand - any compound for which an immunological receptor naturally exists or can be made. When the ligand is an antibody , the immunological receptor can be an antigen or an anti-antibody.

Ligand-anchor complex - a covalently bonded species comprising the analyte of interest and a hydrophobic composition compatible with the lipid or lipids used to form the liposomes for the assay herein. If the ligand of interest cannot be directly used to stabilize the lipid bilayer for the formation of vesicles, the ligand must first be coupled to a suitable lipid, hydrophobic peptide or protein using conventional coupling chemistry. Similar coupling chemistry is described herein below for coupling between the anti-ligand and the inert solid support in the embodiment utilizing a solid-phase assay.

Lipids useful for the anchor complex of this invention include OCIQ-¹ fatty acids, phospholipids, OCIQ) hydrocarbons, large cyclic hydrocarbons, polycyclic hydrocarbons and others readily selectable by those skilled in the art. Hydrophobic peptides and proteins prepared synthetically or by recombinant DNA techniques can also be used as the anchor. The ligand-anchor complex is employed to stabilize an otherwise unstable liposome composition. In a preferred embodiment, i.e., a solution phase assay, an antibody covalently bound to a fatty acid was used to stabilize liposome formed predominantly (99.95 mole percent) of phosphatidylethanolamine.

Marker compound - any compound capable of ready detection other than a radiotracer. Especially useful herein are markers such as enzymes, chemiluminescent species, colorigenic agents and fluorogenic agents. The most preferred marker compounds are self-quenching fluorescent dyes. These compounds include water soluble derivatives of fluorescein such as carboxyfluorescein and calcein. Another suitable marker is a combination of water soluble fluorophore, e.g., 8-amino-naphthalene-l,3 ,6-trisufonic acid and a water soluble quencher, such as p-xylene bis (pyridinium) bromide. As the dye/quencher combination is released from the liposome at lysis, dilution allows for dequenching of and thus detection of the fluorophore. See for example, Ellens et al. , Biochem. 2! 1532 (1984).

Receptor - any compound or composition capable of recognizing a particular spatial and polar organization of another molecule. Natural receptors include antibodies, enzymes, lectins, and the like. For any specific ligand, the receptor may be generally termed an anti-ligand. Depending upon the circumstances the terms may be interchangeable, i.e., receptors in one case can be ligands in another. For example, in a preferred embodiment, the receptor for an antigen is an antibody, while the receptor for an antibody is either an anti-antibody or, preferably, that antibody's cognate antigen.

System - a combination of analyte, ligand and/or receptor reagents, usually formulated with ancillary reagents such as buffers, salts, stabilizers and the like, and supplied in individual containers, generally in the form of an assay kit. For example, a system for detecting the presence and/or the concentration of an antigen using the assay of the present invention would include appropriate containers with (1) antibody or a chemical derivative thereof in the membrane of liposomes and (2) a fluorescer or other suitable marker encapsulated in the liposomes of (1); (3) antigen standards of known concentration for preparing a curve for comparison of known dye release with the unknown dye release, or a predetermined comparison curve and one standard for a control.

In the preferred embodiment of this invention, the assay for an antigen is believed to involve the lateral phase separation of a liposome formed from an antigen-lipid complex or an antibody-lipid complex resulting in the destabilization of the liposome and release of a marker compound such as a fluorescent dye.

Any lipid, natural or synthetic, that is capable of forming the reverse hexagonal phase- at mild, e.g., physiological conditions can be used to form liposomes useful in the practice of this invention. One such suitable lipid is an unsaturated phosphatidylethanolamine (PE) such as egg PE or dioleyl PE. Unsaturated PE by, itself does not form stable liposomes at room temperature and neutral pH. Stable bilayer liposomes can be formed by the addition of a ligand-anchor complex. Lipids in addition to PE which can be stabilized by addition of a ligand-anchor complex include cardiolipid and phosphatidic acid. These lipids form the hexagonal (HJJ) phase in the presence of a divalent cation, such as Ca²⁺. Glucosyldiglyceride can also form the hexagonal phase at physiological conditions. No other natural lipids have been reported to form the H j phase under physiological conditions of temperature and salt concentration. However, the general requirement for the structure of an Hji-forming lipid is a relatively small hydrophilic group coupled to a relatively bulky hydrophobic moiety (the overall molecular shape being cone-like) will also exhibit the H.j phase, and be useful herein to form the liposomes together with ligand-anchor complex.

Furthermore, it has been postulated that lipids, proteins, glycoproteins, and other molecules having a complementary shape, that is, an inverted cone, may be employed as the ligand-anchor complex of the present invention. These molecules would comprise a bulky hydrophilic group coupled to a small hydrophobic moiety. It will be apparent to those skilled in the art that the conjugated complex of a water-soluble ligand, such as an antigen or antibody, and a lipid having sufficient hydrophobic character, will have the molecular configuration of an inverted cone. Similarly, a water-soluble ligand, such as an antigen or antibody, which is coupled to a hydrophobic peptide or protein will also have a molecular configuration of an inverted cone. Thus, this type of ligand-anchor complex will be use,ful to stabilize an otherwise unstable liposome bilayer. See Cullis and DeDruijff, Biochem. Biophvs. Acta, 559 399 (1979) , the disclosure of which is incorporated herein by reference.

In the solution-phase assay, in the presence of a ligand-anchor complex, such as an antibody-fatty acid complex, and a hexagonal phase-forming lipid, such as unsaturated PE, stable immunoliposomes can be prepared by sonication, dialysis, or by other conventional techniques and a marker compound such as a self-quenching fluorescent dye can be entrapped within the liposomes. When the antibody-containing liposomes come into contact with an multivalent antigen in a test fluid, such as a virus, bacterium, or other pathogen, the antibody-fatty acid complexes laterally migrate to bind with the antigen. This lateral phase separation of the liposomes results in a rapid bilayer to hexagonal phase transition, leading to the release of the entrapped marker. Thus, for example, a fluorescent signal is released which can be readily measured without the need for separation of any reagents or other material from the multivalent angiten. By comparing the amount of marker release in the test fluid wirh those released in the standardized solutions of known antigen concentration, the amount of antigen in the test fluid can be quantified. The solution-phase assay, is thus a direct assay for multivalent antigens.

The liposomes containing antigen-anchor complex described in the solid-phase assay can also be used in a direct assay for multivalent antibody. For example, liposomes containing autoimmune antigens coupled to a hydrophobic anchor will rupture when they come into contact with a test serum from an autoimmune patient which has been previously adsorbed to a solid support such as a plastic or a glass surface.

The liposomes containing antibody-anchor complex described in the solution-phase assay can also be used in an inhibition assay for free antibody titer. For example, antibody in patient's serum, or a suitable dilution of it, when added to and incubated with a standardized multivalent antigen, can saturate the binding sites of the antigen. The lysis of the subsequently added liposomes will thus be inhibited.

The assay of this invention is preferably carried out in an aqueous medium at a moderate pH, such as neutral pH, generally close to optimum assay sensitivity, without the need f- r separation of the assay components or products. The assay zone for the determination of analyte is prepared by employing an appropriate aqueous solution., normally buffers containing the unknown sample, which may have been subject to prior treatment, the liposome-analyte-fluorescer reagent, any auxiliary materials associated with production of the detectable signal, as well as when appropriate, a modified or unmodified receptor.

The concentration of a ligand or anti-ligand as the analyte in the biological sample will affect the degree of binding between the immunoliposomes and antigens in the solution-phase assay, and influence the production of the detectable signal.

In carrying out the assay an aqueous medium will normally be employed. Other polar solvents may also be employed, usually oxygenated organic solvents of from 1 to 6, more usually from 1 to 4 carbon atoms, including alcohols, ethers and the like. Usually these cosolvents will be present in less than about 40 volume percent, more usually in less than about 20 volume percent.

The pH for the medium will usually be in the range of about 4 to 10, more usually in the range of about 5 to 9, and preferably in the range of about 5.5 to 8.5. The pH is chosen so as to maintain a significant level of specific binding by the receptor while optimizing signal producing proficiency. In some instances, a compromise will be made between these two considerations. Various buffers may be used to achieve the desired pH and to maintain the pH during the determination. Illustrative buffers include borate, phosphate, carbonate, Tris, Tris HC1, barbital and the like. The particular buffer employed is not critical to this invention but in individual assays, one buffer may be preferred over another.

Moderate temperatures are normally employed for carrying out the assays and usually a constant temperature is maintained during the period of assay. The temperatures for the determination will generally range from about 10° to 50°C. , preferably from about 15° to 40°C, and more preferably be about 22°C, where applicable.

The concentration of analyte which may be assayed will generally vary from about 10~4 to 10^~l*-> molar, more usually from about 10~6 to 10~13 molar. Considerations such as whether the assays are qualitative, semi-quantitative or quantitative, the particular detection technique and the expected concentration of the analyte of interest will normally determine the concentrations of the other reagents.

Although the concentrations of the various reagents will generally be determined by the expected concentration range of interest of the analyte, the final concentration of each of the reagents will normally be determined empirically to optimize the sensitivity of the assay over the range of interest. There are many methods available for the preparation of liposomes. Some of them are used to prepare small vesicles (d<0.05 micrometer) , some for large vesicles (d>0.05 micrometer). Some are used to prepare multilamellar vesicles, some to unilamellar ones. For the present invention, unilamellar vesicles are preferred because a lytic event on the membrane means the lysis of the entire vesicle. However, multilamellar vesicles could also be used, perhaps with reduced efficiency. Methods for liposome preparation are exhaustively described in several review articles such as Szoka and Papahadjopoulos, Ann. Rev. Biophys. Bioenq. , 9_ 467 (1980), Deamer and Uster, in Liposomes, ed. M. J. Ostro, Marcel Dekker, New York, 1983, p. 27-51. The recently published monographs Liposome Technology, ed. G. Gregoriadis, CRC Press, Boca Raton, also contain up-to-date information, especially in Volume I.

The assay and products of the present invention will be further illustrated with reference to the following examples, which aid in understanding the invention, but are not to be construed as a limitation on the scope of the invention, which is set forth in the appended " claims. All percentages reported herein are, unless otherwise specified, mole percent. All ratios, are unless otherwise specified, mole ratios. All temperatures are expressed in degrees Celsius and are uncorrected.

Liposome Preparation

DOPE or DOPC (8.8 micromole) DNP-cap-PE (1.2 micromole) and trace amount of hexadecyl [^H. cholestanyl .ether (final specific activity 5.7 x 10-^ cpm/mol) were mixed and evaporated free of solvent with a stream of N2 gas. The dry lipid was vacuum dessicated for at least 30 minutes. One hundred microliters of PBS containing 4 micromoles calcein, pH 7.4, was added. The mixture was sonicated for 20 minutes at room temperature in a bath sonicator (Laboratory supplies, Inc. , Hicksville, NY) until a uniform translucent liposome suspension was obtained. The liposome suspension was then chromatographed on a biogel A50M column to remove any untrapped calcein. The liposome eluted with PBS in the void volume fractions and was detected by counting ---Η radioactivity, pooled and stored at 4°C.

Liposome-Antibody Interactions Liposome suspension (0.09 to 1.9 nanomoles lipid in 5 to 45 microliters) was added to the spot on the glass slide which had previously coated with IgG. After 20 minutes incubation in a moist chamber at room temperature, the glass slide was rinsed with 2 ml PBS to quantitatively transfer the liposome into a quartz cuvette. The fluorescence was measured with a Perkin Elmer LS5 spectrofluorometer vith lambda _ex = 490 nm and lambda _em = 520 nm. The total calcein fluorescence in the liposome was measured after the addition of sodium deoxychol be to a final concentration of 0.12%. The percent of dye release is defined as:

F - F₀

% release = X 100

Ft " Fo where F₀ and F are the calcein fluorescence of the liposome sample before and after the interactions with the immobilized antibody, respectively. Ft is the total calcein fluorescence after releasing with the deoxycholate.

For the inhibition of dye release, free hapten in 40 microliters was added to the immobilized antibody on the glass slide and incubated for 20 minutes at room temperature before the addition of the liposome and antibody were mixed and preincubated for 20 minutes at room temperature before being added to the σiass slide.

90°C Light Scattering of Liposomes

In order to test the liposome formation, sonicated lipids were diluted 100 fold in PBS. 90° light scattering was measured in a Perkin Elmer LS5 spectrofluorometer at lambda_ex = Lambda_em = 660 nm with a slit width of 3 nm.

Electron Microscopy

Liposome (0.75 micromole/ml) were negatively stained with 0.5% aqueous uranyl acetate and viewed in a Hitachi 600 electron microscope operating at 75 KV. The size of the liposomes was measured on photographically enlarged micrographs.

EXAMPLE 1

Stabilization of DOPE Liposome Bilayer by DNP-cap-PE

Formation of a stable liposome was monitored by 90° light scattering at 660 nm. To determine the minimum amount of DNP-cap-PE, a ligand-anchor complex, which would stabilize the DOPE bilayer, various amounts of DNP-cap-PE were mixed with DOPE or DOPC and the solvent-free lipid mixtures were sonicated in phosphate-buffered saline and measured for light scattering. When stable sonicated liposomes were formed, the turbidity of the suspension was low and hence low light scattering was detected.

As shown in Fig. 1, at concentrations above 12% DNP-cap-PE, stable DOPE liposomes were generated. Between 6 to 11%, liposome suspensions were quite turbid and hence exhibited a high level of light scattering. Below 6%, large aggregates of lipid were seen and the light scattering was again low. Pure DOPE (without the ligand-anchor complex) only forms large aggregates even after prolonged sonication. In contrast, DOPC formed stable, low light scattering liposomes at all concentrations of DNP-cap-PE. It was concluded that a minimum of 12% DNP-cap-PE was required for stable DOPE liposome formation. This composition was used for the experiments of Examples 2 - 7.

EXAMPLE 2

Size of Liposomes

Sonicated liposomes composed of DOPE:DNP-cap-PE (88:12) (hereafter called DOPE liposomes) were unilamellar and relatively homogeneous in size as examined by negative stain electron microscopy (Fig. 2) . The average diameter of the liposomes was 908 + 134 angstroms.

EXAMPLE 3

Trapped Volume of Liposomes

From the average diameter of the DOPE liposomes, the trapped or encased volume of the liposome can be calculated according to Enoch et al. , Proc. Natl. Acad. Sci., USA, 76_. 145 ll979) to be 2.66 microliter/micromole lipid. The trapped volume was also directly estimated by measuring the amount of calcein trapped in the liposomes after the removal of the untrapped calcein by gei filtration. This was done by constructing a standard curve of fluorescence intensity vs. calcein concentration (0 to 0.5 micromolar) ; and by measuring the ^H-cpm in a liposome suspension additi^onally containing a trace amount of hexadecyl [^H] cholestanyl ether to determine the lipid mass. Assuming the calcein concentration inside the liposomes was 40 millimolar, the trapped volume was determined to be 2.08 microliter/micromole lipid. This is in agreement with the value calculated from the size of the liposomes. The trapped volume of the DOPC:DNP-cap-PE (88:12) liposomes (hereafter called DOPC liposomes) was 0.54 microliter/micromole, indicating that these liposomes were much smaller in size. DOPE or DOPC liposomes containing calcein could be stably stored at 4°C for at least one month without significant dye leakage.

EXAMPLE 4

Dye Release upon Liposo e-Antibody Interaction

In order to measure liposome lysis, calcein was trapped at a 40 millimolar concentration inside the liposome. At this concentration calcein fluorescence is self-quenched. Fluorescence is greatly enhanced when the dye leaks out of the liposomes (Allen et al. , Biochim. Biophys. Acta, 597 418 (1980)). Using a glass surface coated with various types of protein, the ability to induce the calcein release from the liposomes was tested. As can be seen from Fig. 3, bare glass surface and glass surface coated with BSA could not induce liposome leakage. However, when DOPE liposomes came in contact with the glass surface coated with anti-DNP IgG, all of the entrapped calcein was released. Glass surface coated with normal IgG did have some effect but the magnitude was much less than that of the anti-DNP IgG. Dye release was blocked by a pretreatment of the glass surface with free hapten, DNP-Gly, or by a preincubation of the liposomes with free anti-DNP IgG, but not by free normal IgG or BSA. These results strongly indicate that the dye release from the DOPE liposomes is a direct result of the antibody-hapten binding at the glass surface. DOPC liposomes were very stable; none of the glass surface types tested could induce the dye release (Fig. 4)

EXAMPLE 5

Effect of Immobilized Antibody Concentration on Dye Release

The concentrations of the IgG solution used in the coating of the glass surface was also varied. Fig. 5 shows that the dye release was dependent on the antibody concentration on the glass surface. Nearly total release was observed for glass surface coated with anti-DNP IgG solution of a concentration greater than 1 icrogram/ml. Below this concentration, progressively lower release was seen. At high concentrations (above 10 microgram/ml) , normal IgG also showed a non-specific effect on liposome lysis, however, the magnitude was much lower than those caused by the anti-DNP IgG. For the subsequent experiment, Example 6, below, and IgG concentration of 10 microgram/ml to coat the glass surface was used.

EXAMPLE 6

Inhibition of Dye Release by Free Hapten

The inhibitory effect of the free hapten to liposome lysis was also examined. As can be seen in Fig. 6, free hapten, DNP-Gly could effectively inhibit the dye release from the DOPE liposomes. The concentration of the free hapten which caused 50% inhibition was calculated to be 0.35 micromolar, which was equal to 14 picomoles in 40 microliters in the preincubation medium. A non-hapten analog, Gly had no effect on the dye release even at 1 millimolar concentration.

EXAMPLE 7

Inhibition of Dye Release by Antibody

Free anti-DNP IgG did not cause dye release even at 10 mg/ml. However, visible aggregation of the liposomes of DOPE or DOPC type was observed when free anti-DNP IgG was preincubated with the liposomes. Preincubation of the DOPE liposomes with free anti-DNP IgG, but not normal IgG, caused inhibition of the dye release (Fig. 7) . Fifty percent inhibition took place at the free antibody concentration of 0.5 mg/ml, which is equal to 2.5 micrograms in a 5 microliters preincubation volume.

DOPE was purchased from Avanti Polar Lipids, Inc. (Birmingham, AL) . Calcein and BSA were purchased from Sigma Chemical Co. (St. Louis, MO). Other reagents were analytical grade.

Antibody

Mouse monoclonal antibody against Herpes Simplex Virus antigen gD was isolated from ascities fluid and was kindly provided by Dr. Steve Norley. IgG from the ascities fluid were purified by protein A-sepharose affinity chromatography followed by DEAE sephadex G25 ion-exchange chromatography. The purified antibody was stored in PBS at -20°C, When required, IgG were radio-labeled with 125j_ -using chloramine T to a specific activity of 1 X 10*5 to 5 X 10^ cpm/ug.

Derivatization of IgG with NHSP

Coupling of N-hydroxysuccinimide ester of palmitic acid (NHSP) to IgG was done following the procedure of Huang et al._r J. Biol. Chem. , 255, 8015 (1980). Briefly 125i-labeled IgG was added to the [³H]NHSP or NHSP in PBS such that the final deoxycholate (DOC) concentration was 2%. The coupling was performed at 37°C for 12 hr. Palmitic acid, the hydrolyzed product of NHSP, in the reaction mixture was separated from derivatized IgG using Sephadex G75 column and eluted with PBS containing 0.16% DOC as described previously (Huang et al., 1980). The purified and derivatized IgG was concentrated with a Centricion 30 microconcentrator (Amicon Co. , MA) followed by dialyzing against PBS containing 0.15% DOC. Binding activity of palmitoyl-IgG (plgG) was tested by a radioimmunoassay method using the -*-25ι-pigG. Briefly 4 X 10^β pfu of HSV in 50 ul of PBS pH 7.6 was incubated at 4°C in an Immulon Removawell (Dynatech Lab, Inc.) overnight. After washing away the unadsorbed HSV, the Removawells were incubated with 50 ul of 5% goat serum in PBS for 1 hour to block the nonspecific binding sites. Then, after washing various amounts (0.1 to 10 ug/ml) of ^{1 5}I-pIgG in 40 ul were added. The binding of plgG was carried out at 4°C for 1 hour. After washing with PBS, pH 7.4, the Removawells were counted for -*-25ι_pigG bound to the HSV adsorbed on the well. All of the measurements were done in duplicate. The dissociation constant K(3 were determined by Scatchard analysis (Schatchard, Ann. N.Y. Acad. Sci., 51, 660 (1949)).

Liposome Preparation

DOPE (1 - 4 umole) and a trace amount of hexadecyl [³H] cholestanyl ether (CE) (Pool et al., Lipids, 17, 448 (1982)) (final specific activity 4 - 11 x 10*^- cpm/mole total lipid) were mixed and evaporated free of solvent with a gentle stream of 2 gas. The dry lipid was vacuum desiccated for a minimum of 30 min. 200 ul PBS containing varying amounts of derivatized IgG, 50 mM calcein, 0.09% DOC, pH 8.0 was added to hydrate the lipid. The mixture was sonicated in a bath type sonicator (Laboratory Supplies, Inc. , Hicksville, NY) for two five-min cycles with 30 min. rest period at room temperature. Liposome suspension was then chromatographed on a Biogel A-0.5 M column to remove the untrapped calcein as well as the detergent DOC. The liposomes, eluted with PBS pH 8.0 in the void volume, were detected by counting ^H-radioactivity. These fractions were pooled and stored at 4°C.

Lysis of Immunoliposomes by Virus

DOPE immunoliposomes bearing pal itoyl anti-HSV-gD-IgG (0.81 nmoles in 2 ul) was added to 5 ul of purified virus in a V-bottom microtiter plate and incubated at room temperature for 20 min. For the inhibition assay using the free antibody, the virus was preincubated with the free antibody for 20 min at room temperature before the addition of i munoliposomes. After dilution to 2 ml with PBS, pH 8.0, liposome lysis was determined fluorometrically by the release of the entrapped calcein as described above.

Solution-Phase Assay Theory

Native IgG does not bind to liposomes under physiologic conditions. However, when the monoclonal IgG against Herpes Simplex Virus (HSV) surface antigen glycoprotein D (gD) was derivatized with N-hydroxysuccinimide ester of palmitic acid, the increased hydrophobitity of the antibody enabled the antibody to incorporate into the lipid bilayer. Therefore, the palmitoyl anti-HSV-gD is a ligand-anchor complex. In addition, it has been found that the palmitoyl antibody is capable of stabilizing the bilayer of dioleoyl phosphatidylethanolamine (DOPE) which does not form bilayer by itself at neutral pH. Mixing of palmitoyl anti-HSV-gD IgG with DOPE at 1:4000 molar ratio was sufficient to form stable liposomes by sonication, as judged by the entrapment of 50mM self quenching dye, calcein. When the antibody stabilized DOPE immunoliposomes were incubated with intact HSV, a multivalent antigen lysis of liposomes occurred with t.e release of calcein into the medium as detected by the enhancement of calcein fluorescence.

Since no lysis activity was observed with other viruses such as Sendai, Semliki ^"Forest ^"and Sindbis, this direct solution-phase assay is specific for HSV. While not wishing to be bound by theory, a likely mechanism for liposome lysis is the aggregation (contact capping) of antibody at the contact area between the virus and liposome due to multiple immune complex formation (Fig. 13) .. As a result of lateral phase separation in the immunoliposome membrane, DOPE is believed to undergo the bilayer-to-hexagonal phase transition, causing the release of the entrapped dye. Furthermore, the HSV specific liposome lysis was inhibited by free antibody, but not by other IgG's. Using this principle, it has been shown that this novel solution-phase immunoliposome assay is sensitive to 50,000 plaque forming units of HSV and the inhibition assay is sensitive to nanogram quantities of the free antibody in 5 microliters of incubation medium.

EXAMPLE 8

Derivatization of anti-HSV-gD-IgG with N-hydroxy succinimide ester of plamitic acid (NHSP)

The degree of coupling palmitic acid to the IgG was examined with varying input molar ratio of [³H.NHSP to [¹ 5i]_IgG from 0 to 44. The final molar ratio of palmitic acid to IgG was determined from the pooled IgG fractions (void volume) of sephadex G75 column (Huang et al. , Biσohim. Biophys. Acta, 716, 140 (1982)). These values represent the average ratio since the pooled samples contain both derivatized and underivatized antibodies.

Table 1 shows the data for the preparation of a ligand-anchor complex, namely the palmitoyl anti-HSV-gD-IgG, with covalently coupled palmitic acid. The dissociation constants of the coupled antibodies are also shown.

TABLE 1 Derivatization of anti-HSV-gD-IgG with NHSP

10

Input molar ratio Coupling stoichiometry Kd NGSP/IgG Palmitic Acid/IgG (xl0⁸M)

0 0 0.75 ^a

15 11 1.4 ND ^b 14 2.1 ND 20 2.2 1.17 25 5.14 ND

20 30 6 . 66 ND 44 14.6 1.90

r- -. (a) K_fj for native anti-HSV-gD-IgG was ⁵ 0.48 x 10^~8 M.

(b) Not determined

30 As shown in Table 1, increasing input ratio of NHSP/IgG resulted in more palmitic acid coupled per IgG without significant change in the antigen binding affinity as measured by the dissociation constant K^j of the antibody. Therefore the coupling condition was sufficiently mild and the coupling stoichiometry can be controlled by varying the input NHSP/IgG ratio.

EXAMPLE 9

Preparation of Palmitoyl anti-HSV-gD-IgG as Liqand-Anchor

Formation of stable liposomes was monitored by their ability to encapsulate self-quenching fluorescence dye, calcein. At 50 millimolar concentration, calcein fluorescence was self-quenched to greater than 70%. Florescence was greatly enhanced as the . dye was diluted upon released from liposomes. In order to determine the optimal palmitic acid to IgG coupling stoichiometry for DOPE liposome formation, the palmitoyl IgG to (plgG) to DOPE ratio of each plgG preparation was varied (coupling stoichiometry, palmitic acid/IgG = 0 - 14.6). The protein and lipid mixture was sonicated in the presence of 50 mM calcein. After chromatographed to remove the untrapped dye, liposome formation was detected by analyzing the total amount of calcein encapsulated per unit mass of DOPE (Fig. 9). In order to demonstrate the encapsulation of calcein, quenching of calcein fluorescence was also presented in Figure 9. Furthermore, a series of linear gradient (5 - 20% sucrose) centrifugations were performed to quantitate the degree of plgG incorporation into the DOPE liposomes. With the palmitic acid to IgG coupling stoichiometry of 2.2 to 5.1, it was found that within this range practically all plgG's were incorporated into liposomes prepared with a plgG/DOPE ratio of 1:4000 (Fig. 9A) . Outside this range, the plgG had no stabilization activity for DOPE liposomes. This result clearly indicates that only plgG of certain coupling stoichiometries can be used as a ligand-anchor for the stabilisation of DOPE liposomes.

EXAMPLE 10

Optimal Ligand-Anchor to Lipid Ratio for

Immunoliposome Formation

In order to determine the optimal plgG to DOPE ratio, the calcein encapsulation and the percent quenching of the DOPE immunoliposomes with varying ratio of plgG/DOPE were analyzed. In this experiment, coupling stoichiometry, palmitic acid to IgG was 2.2. As shown in Figure 9B, the optimal plgG to DOPE ratio was found to be 2.5 - 4.2 X 10^-4. Within this range, the highest amount of quenched (up to 70%) calcein fluorescence per unit mass of DOPE was detected; increasing or decreasing this ratio outside the range resulted in negligible amounts of total calcein encapsulation. With a plgG to DOPE ratio less than 2.5 X 10~⁴, a sharp decrease was detected in the calcein encapsulation without significant decrease in the degree quenching. This was attributed to the heterogeneity of the liposome population. A small number of plgG stabilized liposomes in the population could show a high degree of clacein quenching. At a plgG to DOPE ratio greater than 4.2 x 10^~4, the excess plgG were self aggregated.

A minimum coupling stoichiometry of 2.2 palmitic acid per IgG was required in order for the plgG to stabilize the DOPE liposomes (Figure 9A) . In addition,, minimal plgG to DOPE ratio was found to be 2.3 x 10^~4. This combination was used to prepare the DOPE immunoliposomes for both of the subsequent experiments (Examples 11 - 12) . Under this condition, knowing the average diameter of the liposomes to be 500A, that there were an estimated five plgG molecules per DOPE immunoliposome.

EXAMPLE 11

Virus-Mediated Lysis of DOPE Immunoliposomes

With varying concentrations of purified virus, lysis of DOPE immunoliposomes bearing palmitoyl anti-HSV-gD-IgG was tested by detecting the release of calcein into the medium. As shown in Figure 10, only HSV caused total release of calcein at viral protein concentration of 158 ug/ml. No other viruses (Sendai, Semliki Forest and Sinbis) could cause significant amount of lysis over the wide concentration range tested. Therefore, this assay for HSV is viral specific. Fifty percent release took place at the HSV concentration of 6.3 ug/ml, which corresponds to 32 ng of viral protein or approximately 50,000 pfu in 5 ul incubation volume. Fig. 11 shows the visual change of color when liposomes are lysed by virus. In.tact liposomes with high concentration of entrapped calcein are not fluorescent, only a dark orange color can be seen. When liposomes are lysed by virus, calcein is released and the fluorescence is greatly enhanced. The color becomes a bright yellow-g eenish under regular fluorescence room lighting. The liposomes could also be lysed by cells infected with HSV but not by normal cells.

EXAMPLE 12

Inhibition of Immunoliposome Lysis by Free Antibody

Preincubation of the free anti-HSV-gD-IgG with HSV blocked the binding of HSV of the DOPE immunoliposomes. As a result, the lysis of the immunoliposomes was inhibited. As shown in Figure 12, inhibition caused by preincubation of HSV with antibody was specific for anti-HSV-gD-IgG, neither a closely related antibody anti-HSV-gB-IgG nor BSA could inhibit. Therefore, this assay could be used to analyze the free anti-HSV titer in a test solution. The concentration of antibody that caused 50% inhibition was 2 ug/ml or 10 ng in a 5 ul preincubation medium.

The method of the present invention can be applied to a wide variety of analytes. Antibodies, both polyclonal and monoclonal, can be raised using standard immunological techniques to numerous analytes. Other membrane-lytic techniques are also contemplated herein, for example, detection of enzymes or enzyme substrates using the solid-phase assay of the present invention can be accomplished in a manner analogous to the detection of antigens or antibodies described supra.

In general, an enzyme substrate, which has been coupled to a suitable hydrophobic anchor (if necessary) is used to form stable liposomes containing a marker, such as the fluorescent dye and interaction between these liposomes and the appropriate enzyme bound to a solid support causes lysis of the liposomes, releasing the fluorescent dye. Calibration of dye release is accomplished using standard enzyme or substrate concentrations and inhibition of dye release by unknown quantities of enzyme or substrate in a biological test sample may readily be determined and the concentration calculated from the standard plots.

Enzymes detectable by the assays of the present invention include, but are not limited to; oxidoreductases such as alcohol dehydrogenase, glycerol dehydrogenase, glyoxylate reductase, L-lactate reductase, malate reductase, glucose 6-phosphate dehydrogenase, mannitol 1-phosphate dehydrogenase, L-lactate dehydrogenase, glucose oxidase, galactose oxidase, L-amino acid oxidase, D-amino acid oxidase, polyphenol oxidase, ascorbate oxidase, catalase, peroxidase; hydrolases such as carboxylic ester hydrolases, cholinesterase, phosphoric monester hydrolase, alkaline phosphatase, phosphoric diester hydrolase, phospholipase C (when the lipid is not a phospholipid) ; glycoside hydrolases including alpha-amylase, cellulase, lysozyme, beta-galactosidase, amyloglucosidase, beta- glucuronidase; peptidyl-amino acid hydrolase, carboxypeptidase A. peptidy1-peptlde hydrolase, alpha-chromotrypsin, papain, urease, inorganic pyrophosphataseg; lyases such as carbon-carbon lyases, e.g. , aldehyde lyases, such as aldola-.e; carbon-oxygen lyases, e.g., hydrolases, such as carbonic anhydrase; carbon-nitrogen lyases, e.g., ammonia lyases, such as histidase. These enzymes have been previously encapsulated in liposomes and employed as a marker to tag for an immunoassay, see Cole, U.S. Patent No. 4,342,825, the disclosure of which is incorporated herein by reference.

The assay of the present invention can be employed in the detection and concentration calculation of circulating hormones in biological samples. Antibodies to these hormones may be raised using standard immunological techniques. These hormones include thyroid hormones such as thyroxine, triiodothyronine, parathyroid hormone and calcitonin; pancreatic hormoens such as insulin, proinsulin, and glucagon; pituitary hormones including prolactin, adrencorticotropic hormone, tyrotropin, oxytocin, and vasopressin; uterine and placental hormones such as chorionic gonadotripin, placental lactogens, chorionic thyrotropin and relaxin; steroid hormones including estradiol, estrone, estriol, testosterone, and dihydrotestosterone; growth factors such as urogastrone, nerve growth factors and the somatomedins.

Similarly, the method may be usefully applied to the intracellular messengers, the cyclic nucleotides inositol polyphosphate and prostaglandins.

The present invention may likewise be applied to the screening of circulating levels of therapeutic drugs, e.g. , the cardiac glycosides; digoxin, digitoxin, anticonvulsants, diphenylhydantoin, mesantoin, phenobarbital, and mephobarbital. Of particular interest are those drugs with narrow thereapeutic index, i.e., a certain minimal circulating level is required for thereapeuti-" efficacy while a moderately higher level elicits toxic or harmful reactions.

The procedure may also be adapted to screening for antibodies raised against antibiotics, or to the antibiotics themselves, such as penicillins, cephalosporins, thienamycins, clavulanic acids, monobactams, streptomycin, and tetracyclines, chloretracycline, oxtetracycline, and tetracycline, chloramphenicol, erythromycin, caromycin, polymyxin B. The aminoglycoside antibiotics gentamycin, amikacin, tobramycin, kanamycin and neomycin employed in the management of aerobic Gram negative bacillary infections can be conveniently assayed by the present invention. Likewise, the method may be applied to the detection and estimation of drugs of abuse such as opiates - morphine, heroin, meperidine and methadone; ergot alkaloids such as lysergic acid diethylamide; marijuana; barbiturates and cocaine and its derivatives.

The method is not restricted to small molecules. Marcromolecular species including DNA, and large antigens such as egg albumin, can be directly or after conjugation with suitable hydrophobic anchors incorporated into stable bilayer liposome vesicles. Thus, the present invention can also be applied to detection of macromolecular species such as large antigens, plasma proteins, hepatitis associated antigens, hiεtocompatibility markers, and the like. The solution-phase assay is especially suitable for detecting pathogens in the biological fluids, such as serum. Assays for viruses, such as AIDS, Hepatitis, Herpes, Influenza, Rabies, and many others including those infecting human animals and plants, can be constructed. Bacteria, ycobacteria, fungi, protozoas and other pathogenic organisms of humans, animals, and plants can also be assayed with this invention. Similarly, nonpathogenic organisms may be detected using the assay of the present invention.

Since the present invention is very simple in performance and does not employ unstable or hazardous reagents, the assay method is applicable in environments which are less well-equipped and less sophisticated than typical diagnostic laboratories. For example, the assay method can be applied to screening food and environmental toxins. In food screening, important antigens would be mycotoxins and natural toxicants. This area involves such major toxins as aflatoxins, ochratoxin, patulin, penicillic acid, zearelonone; and tricothecene toxins, as well as toxic metabolites such- as ipomeamerone that occur naturally in foods. In addition to the natural toxicants there are a wide variety of environmental contaminants, the presence of which in foods even in trace amounts, poses a significant threat to mankind. These may be industrial byproducts or pesticides, e.g. , polychlorinated biphenyls, chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, heptachlorepoxide, dieldrin, and DDT, l,l'-(2,2,2- trichloro-ethylidene)-bis(4-chlorobenzene) .

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 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 membrane lytic immunoassay method comprising the steps of:

(a) forming liposomes with a stabilizing amount of the analyte of interest in the liposome membrane, said liposomes containing a marker compound;

(b) contacting the liposomes of step (a) with a multivalent receptor for the analyte of interest, for a sufficient time to saturate the liposomes with the receptor; and

(c) determining the presence of marker compound released by the liposomes of step (b).

2. The immunoassay of claim 1, further comprising step (al) , which follows step (a) and preceeds step (b); said step comprising mixing a test fluid containing the analyte of interest with the multivalent receptor used in step (b) , for a sufficient time to saturate the multivalent receptor.

3. The immunoassay of claim 2, wherein the multivalent receptor is attached to an inert support.

4. The immunoassay of claim 2, wherein the decrease in the amount of marker compound released by the liposomes is caused by the presence of the analyte of interest in the test fluid of step (al) .

5. The immunoassay of claim 1, wherein the multivalent receptor is prepared by chemical means.

o. The immunoassay of claim 1, wherein the multivalent receptor is prepared by biological means.

7. The immunoassay of claim 1, wherein the multivalent receptor of step (b) is in the test fluid.

8. ^" The immunoassay of claim 1, wherein step (c) further comprises quantifying the amount of marker compound released.

9. The immunoassay of claim 8, wherein the increase in the amount of marker compound released by the liposomes is caused by the presence of the multivalent receptor for the analyte of interest in the test fluid.

10. The immunoassay of claim 1, wherein the liposomes further comprise at least one lipid in the liposome membrane, said lipid forming the reverse hexagonal phase in excess water under mild conditions of pH, temperature, and salt concentration.

11. The immunoassay of claim 10, wherein the lipid is selected from naturally occurring lipids, synthetically prepared lipids, or mixtures thereof.

12. The immunoassay of claim 2, wherein said analyte of interest is a ligand-anchor complex.

13. The immunoassay of claim 12, wherein the ligand of the ligand-anchor complex is an antigen.

14. The immunoassay of claim 13, wherein multivalent receptor of said antigen is an antibody.

15. The immunoassay of claim 14, wherein the antibody is polyclonal or monoclonal.

16. The immunoassay of claim 13, wherein said antigen is selected from the group consistinig of proteins, peptides, glycoproteins, lipoproteins, haptens, nucleic acids, polysaccharides, and lipids.

17. The immunoassay of claim 12, wherein the anchor of the ligand-anchor complex is a hydrophobic molecule.

18. The immunoassay of claim 17, wherein the hydrophobic molecule is selected from the group consisting of lipids, fatty acids, steriods, hydrophobic peptides, and hydrophobic proteins.

19. The immunoassay of claim 12, wherein the ligand-anchor complex is a chemically coupled entity.

20. The immunoassay of claim 12, wherein the ligand-anchor complex is prepared biologically in a cell.

21. The immunoassay of claim 20, wherein the ligand-anchor complex is prepared using recombinant DNA technology.

22. The immunoassay of claim 12, wherein the ligand of the ligand-anchor complex is selected from the group consisting of antibodies, nucleic acids, and enzymes.

23. The immunoassay of claim 22, wherein the receptor for the ligand of the ligand-anchor complex is selected from the group consisting of antigens, complementary strands of nucleic acids, and enzyme receptors.

24. The immunoassay of claim 1, wherein the analyte of interest is a ligand-anchor complex.

25. The immunoassay of claim 24, wherein the ligand of the ligand-anchor complex is an antibody.

26. The immunoassay of claim 25, wherein the antibody is monoclonal.

27. The immunoassay of claim 26, wherein the monoclonal antibody is substantially pure.

28. The immunoassay of claim 27, wherein the antibody is IgG antibody or fragments thereof.

29. The immunoassay of claim 24, wherein the anchor of the ligand-anchor complex is a hydrophobic molecule.

30. The immunoassay of claim 29, wherein the hydrophobic molecule is selected from the group consisting of lipids, fatty acids, steriods, hydrophobic peptides, and hydrophobic proteins.

31. The immunoassay of claim 24, wherein the ligand-anchor complex is a chemically coupled entity.

32. The immunoassay of claim 24, wherein the ligand-anchor complex is prepared biologically _iτ. a cell ,

33. The immunoassay of claim 32, wherein the ligand-anchor complex is prepared using recombinant DNA technology.

34. The immunoassay of claim 24, wherein the ligand of the ligand-anchor complex is selected from the group consisting of antibodies, nucleic acids, and enzymes.

35. The immunoassay of claim 34, wherein the receptor for the ligand of the ligand-anchor complex is selected from the group consisting of antigens, complementary strands of nucleic acids, and enzyme receptors.

36. The immunoassay of claim 7, wherein the multivalent receptor is an antigen.

37. The immunoassay of claim 36, wherein the antigen selected from an organism or a fragment of an organism.

38. The immunoassay of claim 37, wherein said organism or fragment thereof, is selected from live, dead, or processed material.

39. The immunoassay of claim 38, wherein the organism os selected from the group consisting of viruses, bactreia, fungi, protazoa, parasites, and cells of higher organisms.

40. The immunoassay of claim 39, wherein the cells of higher organisms are selected from the group consisting of plant cells, animal cells, human cells.

41. The immunoassay of claim 40, wherein the cells are selected from cancer cells or normal cells.

42. The immunoassay of claim 38, wherein the organism or fragment thereof is pathogenic.

43. The immunoassay of claim 38, wherein the organism or fragment thereof is non-pathogenic.