EP0258388A1 - Immunoliposome assay - methods and products - Google Patents
Immunoliposome assay - methods and productsInfo
- Publication number
- EP0258388A1 EP0258388A1 EP87901814A EP87901814A EP0258388A1 EP 0258388 A1 EP0258388 A1 EP 0258388A1 EP 87901814 A EP87901814 A EP 87901814A EP 87901814 A EP87901814 A EP 87901814A EP 0258388 A1 EP0258388 A1 EP 0258388A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- immunoassay
- ligand
- liposomes
- antibody
- anchor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/586—Liposomes, microcapsules or cells
Definitions
- 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.
- Radioimmuno?ssay RIA
- Radiotracers by their very nature, are of .limited stability and they require special handling during use. special disposal techniques and sophisticated instrumentation.
- 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.
- McConnell et al. U.S. Patent No. 3,887,698
- EPR electron paramagnetic resonance
- Mandle et al. U.S. Patent No. 4,372,745
- 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.
- 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:
- step (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;
- step (c) determining the presence of marker compound released by the liposomes in step (b) .
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- the immunoliposome assay of the present invention will be illustrated by referring to the assay for one particular entity, i.e., an antigen.
- 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.
- Analyte - the compound or composition to be measured which may be a ligand, such as an antigen, hapten or an antibody.
- the analyte may be either an antigen or an antibody.
- Ligand any compound for which an immunological receptor naturally exists or can be made.
- 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.
- 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.
- 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.
- 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.
- the receptor may be generally termed an anti-ligand.
- the terms may be interchangeable, i.e., receptors in one case can be ligands in another.
- 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.
- ancillary reagents such as buffers, salts, stabilizers and the like
- 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.
- 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.
- 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.
- PE unsaturated phosphatidylethanolamine
- 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.
- HJJ hexagonal phase
- 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.
- 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.
- lipids, proteins, glycoproteins, and other molecules having a complementary shape 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.
- 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.
- 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.
- 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.
- stable immunoliposomes 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.
- a marker compound such as a self-quenching fluorescent dye can be entrapped within the liposomes.
- This lateral phase separation of the liposomes results in a rapid bilayer to hexagonal phase transition, leading to the release of the entrapped marker.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- oxygenated organic solvents usually from 1 to 6, more usually from 1 to 4 carbon atoms, including alcohols, ethers and the like.
- 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.
- 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.
- 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.
- liposomes 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.
- 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.
- 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 --- ⁇ radioactivity, pooled and stored at 4°C.
- Ft Fo where F 0 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.
- 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 ⁇ iass slide.
- 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.
- Formation of a stable liposome was monitored by 90° light scattering at 660 nm.
- 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.
- the turbidity of the suspension was low and hence low light scattering was detected.
- DOPE 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.
- 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.
- DOPC liposomes calcein concentration (0 to 0.5 micromolar) ; and by measuring the ⁇ H-cpm in a liposome suspension additi o nally 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.
- 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.
- 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.
- N-hydroxysuccinimide ester of palmitic acid was done following the procedure of Huang et al. r J. Biol. Chem. , 255, 8015 (1980). Briefly 125i-labeled IgG was added to the [ 3 H]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 -*-25 ⁇ -pigG. Briefly 4 X 10 ⁇ pfu of HSV in 50 ul of PBS pH 7.6 was incubated at 4°C in an Immulon Removawell (Dynatech Lab, Inc.) overnight.
- 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 -*-25 ⁇ _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)).
- DOPE (1 - 4 umole) and a trace amount of hexadecyl [ 3 H] 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.
- 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.
- 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.
- the palmitoyl anti-HSV-gD is a ligand-anchor complex.
- the palmitoyl antibody is capable of stabilizing the bilayer of dioleoyl phosphatidylethanolamine (DOPE) which does not form bilayer by itself at neutral pH.
- DOPE dioleoyl phosphatidylethanolamine
- 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.
- the degree of coupling palmitic acid to the IgG was examined with varying input molar ratio of [ 3 H.NHSP to [ 1 5i] I gG 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. , Bi ⁇ ohim. 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.
- K f j for native anti-HSV-gD-IgG was 5 0.48 x 10 ⁇ 8 M.
- Palmitoyl anti-HSV-gD-IgG as Liqand-Anchor
- 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.
- the optimal plgG to DOPE ratio 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.
- 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.
- 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)
- 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.
- 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.
- 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.
- thyroid hormones such as thyroxine, triiod
- 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.
- therapeutic drugs e.g. , the cardiac glycosides; digoxin, digitoxin, anticonvulsants, diphenylhydantoin, mesantoin, phenobarbital, and mephobarbital.
- 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.
- 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.
- 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.
- the present invention can also be applied to detection of macromolecular species such as large antigens, plasma proteins, hepatitis associated antigens, hi ⁇ tocompatibility 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.
- the assay method is applicable in environments which are less well-equipped and less sophisticated than typical diagnostic laboratories.
- the assay method can be applied to screening food and environmental toxins.
- 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.
- toxic metabolites such- as ipomeamerone that occur naturally in foods.
- 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 centuries.
- 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) .
- 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) .
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Abstract
Nouvelle analyse immunologique membranolytique. Dans une forme d'exécution de cette analyse, notamment dans un système à phase en solution, un anticorps est d'abord lié de manière covalente à un lipide devant servir d'ancre pour l'introduction dans la membrane du liposome. Ce complexe anticorps-ancre est utilisé en combinaison avec un lipide ou un mélange de lipides ne formant pas autrement de bi-couches, pour obtenir des vésicules de liposomes bi-couches stables contenant en outre un colorant fluorescent à auto-extinction. Lorsque ce liposome contenant un anticorps et un colorant est mis en contact avec une solution contenant des molécules d'antigènes, il se produit une liaison rapide entre le complexe anticorps-ancre et l'antigène, ce qui provoque la rupture du liposome et la libération du colorant. La libération de colorant peut être quantifiée par des procédés spectrophotométriques ou spectrofluorométriques employant des solutions normalisées.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
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP19850903151 EP0185738A4 (en) | 1984-06-12 | 1985-06-11 | Immunoliposome assay - methods and products. |
PCT/US1987/000222 WO1987004795A1 (en) | 1985-06-11 | 1987-01-23 | Immunoliposome assay - methods and products |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0258388A1 true EP0258388A1 (en) | 1988-03-09 |
EP0258388A4 EP0258388A4 (en) | 1990-01-26 |
Family
ID=8194785
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19870901814 Withdrawn EP0258388A4 (en) | 1985-06-11 | 1987-01-23 | Immunoliposome assay - methods and products. |
Country Status (3)
Country | Link |
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EP (1) | EP0258388A4 (en) |
JP (1) | JPH01500848A (en) |
WO (1) | WO1987004795A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0194261A (en) * | 1987-07-13 | 1989-04-12 | Univ Tennessee Res Corp | Amplification of signal utilizing liposome in homogeneous system for assay involving enzyme |
JPH04303770A (en) * | 1991-03-30 | 1992-10-27 | Toyo Ink Mfg Co Ltd | Analyzing method for immunity of antigen |
CN113406335A (en) * | 2021-06-15 | 2021-09-17 | 安徽大千生物工程有限公司 | Complement valence liposome immunoassay kit and preparation and use method thereof |
CN114088675A (en) * | 2021-11-17 | 2022-02-25 | 浙江科技学院 | Immunoliposome wrapping fluorescent dye and application |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4483929A (en) * | 1982-05-03 | 1984-11-20 | Liposome Technology Incorporated | Liposomes with glycolipid-linked antibodies |
US4517303A (en) * | 1982-10-20 | 1985-05-14 | E. I. Du Pont De Nemours And Company | Specific binding assays utilizing analyte-cytolysin conjugates |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4636479A (en) * | 1983-04-20 | 1987-01-13 | Cooper-Lipotech, Inc. | Enhanced agglutination method and kit |
US4605630A (en) * | 1983-07-27 | 1986-08-12 | Cooper Lipotech Inc. | Large-liposome agglutination reagent and method |
-
1987
- 1987-01-23 WO PCT/US1987/000222 patent/WO1987004795A1/en not_active Application Discontinuation
- 1987-01-23 EP EP19870901814 patent/EP0258388A4/en not_active Withdrawn
- 1987-01-23 JP JP62501616A patent/JPH01500848A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4483929A (en) * | 1982-05-03 | 1984-11-20 | Liposome Technology Incorporated | Liposomes with glycolipid-linked antibodies |
US4517303A (en) * | 1982-10-20 | 1985-05-14 | E. I. Du Pont De Nemours And Company | Specific binding assays utilizing analyte-cytolysin conjugates |
Non-Patent Citations (4)
Title |
---|
CHEMICAL ABSTRACTS, vol. 103, no. 1, 8th July 1985, page 2469, abstract no. 2467b, Columbus, Ohio, US; R.M. EPAND: "High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines", & CHEM. PHYS. LIPIDS 1985, 36(4), 387-93 * |
JOURNAL OF IMMUNOLOGICAL METHODS, vol. 75, no. 1, 1984, pages 351-361, Elsevier, Amsterdam, NL; Y. ISHIMORI et al.: "Liposome immune lysis assay (LILA)" * |
See also references of WO8704795A1 * |
THE JOURNAL OF IMMUNOLOGY, vol. 134, no. 6, June 1985, pages 4035-4040, The Official Journal of the American Association of Immunologists, San Diego, California; R.J.Y. HO et al.: "Interactions of antigen-sensitized liposomes with immobilized antibody" * |
Also Published As
Publication number | Publication date |
---|---|
JPH01500848A (en) | 1989-03-23 |
WO1987004795A1 (en) | 1987-08-13 |
EP0258388A4 (en) | 1990-01-26 |
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