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WO1992007959A1 - Procede de dosage immunologique lytique homogene sur membrane utilisant des formulations de liposomes sensibles au contact - Google Patents

Procede de dosage immunologique lytique homogene sur membrane utilisant des formulations de liposomes sensibles au contact Download PDF

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Publication number
WO1992007959A1
WO1992007959A1 PCT/US1991/008293 US9108293W WO9207959A1 WO 1992007959 A1 WO1992007959 A1 WO 1992007959A1 US 9108293 W US9108293 W US 9108293W WO 9207959 A1 WO9207959 A1 WO 9207959A1
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Prior art keywords
liposome
analyte
mole percent
molecules
contact sensitive
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PCT/US1991/008293
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English (en)
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Bruce P. Babbitt
Larry R. Hillis
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Biowhittaker Inc.
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Publication of WO1992007959A1 publication Critical patent/WO1992007959A1/fr

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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
    • G01N33/58—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

  • Liposomes are microscopic unilamellar or multilamellar phospholipid bilayer vesicles which enclose an aqueous compartment. They are uniquely suited as carriers for a variety of compounds, both biological and nonbiological. Such compounds can be encapsulated in the interior aqueous compartment of the liposome, or, they may be inserted in or attached to the lipid bilayers, which bound and limit the internal aqueous space. Due to their relatively simple composition, and their flexibility for chemical, physical, and immunological manipulations, liposomes are a favorite material for membrane lytic assays, especially immunoassays.
  • RIA radioimmunoassay
  • FFA fluorescence immunoassay
  • ELISA enzyme - based technologies
  • McConnel et al. in U.S. Patent No. 3,887,698, described the use of liposomes containing stable free radicals in an EPR monitored immunoassay. However, the sensitivity of this assay, compared to other detection systems was poor.
  • Mandle et al. in U.S. Patent No. 4,372,745, described the use of- liposome encapsulated fluorescent dyes useful in an immunoassay. However, this method required detergent lysis of analyte captured liposomes to release the entrapped fluorescent signal, a cumbersome step which, if avoidable, should be avoided.
  • Huang & Ho constructed phosphatidylethanolamine (PE) - based liposomes which typically under physiological conditions, are unable to form intact liposomes (lipid bilayer vesicles) but instead, form what is referred to as "hexagonal phases" in aqueous buffers. These so- called hexagonal phases are totally incapable of entrapping small or large molecules (i.e., "reporter molecules”) , as normal bilayer vesicles do.
  • An example of a hexagonal phase forming lipid is an unsaturated PE, such as egg PE or dioleoyl -PE.
  • Lipids in addition to PE which can form the hexagonal phase include monogalac osyl diglyceride, cardiolipin, and phosphatidic acid (see, U.S. Patent No. 4,708,933).
  • Cardiolipin and phosphatidic acid form the hexagonal phase in the presence of a divalent cation, such as Ca ++ »
  • a divalent cation such as Ca ++ »
  • No other neutral lipids have been reported to form the hexagonal phase under physiological conditions of temperature and salt concentration. It appears that the general requirement for the structure of a hexagonal phase forming lipid is that it possess a relatively poorly hydrated polar head group coupled to a bulky hydrophobic moiety; the overall shape of the molecule is therefore that of a cone. It has been postulated that molecules having a complementary, so called “inverted cone” shape, may form stable, bilayer liposomes when appropriately mixed with hexagonal phase forming lipids. Such stabilizer molecules should contain a bulky hydrophilic head group coupled to a small, hydrophobic moiety (see Cullis and DeKruijff, Biochem. Biophvs. Acta. 559 (1979), page 339.)
  • Huang et al. were able to stabilize the PE phospholipids in a bilayer form by the addition of 12 mole percent of a hapten-phospholipid conjugate, whose molecular shape approximated that of an inverted cone.
  • the hapten coated liposomes were disrupted, and released dye into the media.
  • the proposed mechanism was one in which an antibody induce d phase separation of the haptenated phospholipid molecules (served the dual role of stabilizer and targeting molecule) away from the bulk PE lipids at the area of contact between the liposomes and the solid surface, induced a rapid and leaky bilayer-to-hexagonal lipid phase transition.
  • Huang in U.S. Patent No. 4,957,735, described an advancement of this technology, in which a stabilized and targeted PE-based immunoliposome was prepared by the incorporation of an acylated, anti-herpes monoclonal antibody into the vesicle bilayer. Upon binding to virus infected cells, or to partially purified virus particles devoid of cells, they were able to show multivalent, antigen specific destabilization and lysis of the liposomes, in a totally homogeneous immunoassay format.
  • the Huang liposomes are stabilized and targeted by the same molecule (a very restrictive parameter, in terms of broad applicability to a range of analytes and manufacturability of the reagents) and. 2. there is the requirement for a multivalent binding interaction between the target analyte and the liposome, in order to induce vesicle lysis.
  • the present invention is based upon the discovery that "contact sensitive" liposomes, composed of a hexagonal phase forming lipid as their major component, may be employed in a one or multistep, competitive or noncompetitive, homogeneous membrane lytic immunoassay format, with the following key performance characteristics: 1. liposome membrane lysis, and subsequent reporter molecule readout, is induced/inhibited either directly or indirectly by specific linker molecule - analyte interaction(s) , at the contact sensitive liposome surface,
  • the liposomal immunoassay system of the present invention is an excellent alternative for homogeneous phase detection of low molecular weight analytes (such as therapeutic drugs, drugs of abuse,etc.), which does not rely upon or mimic any systems such as, Syntex (EMIT R technology) , Abbott (TDx R technology) , and Microgenics (CEDIATM technology) , to name but a few. More importantly, this invention may represent the only commercially viable method to perform homogeneous immunoassays on a wide variety of large molecular weight analytes, solution phase and particulate (tumor markers, hormones. lymphokines, antibodies, viruses, bacteria, mycoplasma, etc.) .
  • DOPA DIOLEOYL PHOSPHATIDIC ACID
  • BIOTIN-CAP-DPPE BIOTIN CAPROYL DIPALMITOYL
  • DOPS DIOLEOYL PHOSPHATIDYLSERINE MPB - PE : P - MALEIMIDOPHENYL - BUTYRYL - PE PBS : PHOSPHATE BUFFERED SALINE 3H - CE : HEXADECYL [ 3 H]
  • CHOLESTANYL ETHER DMPS DIMYRISTOYL PHOSPHATIDYLSERINE N-GPE : N-GLUTARYL PHOSPHATIDYLETHANOLAMINE
  • CHEMS CHOLESTERYL HEMISUCCINATE DOSG: DIOLEOYL SUCCINYL GLYCEROL DMPG : DIMYRISTOYL PHOSPHATIDYLGLYCEROL DOPG : DIOLEOYL PHOSPHATIDYLGLYCEROL OA: OLEIC ACID
  • inverted hexagonal phase forming lipids is use d to refer to the so-called “cone” shaped lipids that in aqueous buffers under physiological conditions of temperature,salt, pH, etc., self aggregate into nonbilayer/nonlamellar, reverse/inverted micelles, consisting of hexagonally/closed packed extended lipid cylinders with a hydrophilic core.
  • targeting molecule(s) is used to refer to any compound, molecule, or composition on the surface of the liposome, that either directly or indirectly binds the analyte. These molecules can be either analyte specific or nonspecific. Examples of commercially available materials include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, and antibodies.
  • the term “stabilizing molecule(s) " is used to refer to any amphipathic compound, molecule, or composition, that upon addition to an inverted hexagonal phase forming lipid, allows the mixture to form a lipid bilayer phase (liposome) , enclosing an aqueous compartment.
  • the stabilizer molecule is/can be independent of both the analyte(s), and the targeting molecule(s) ; it does not necessarily bind the analyte, either directly, or indirectly. Examples of commercially available materials include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, and antibodies.
  • the term “amphipathic”, is used to refer to any compound, molecule, or composition, that contains both a hydrophobic and a hydrophilic region.
  • contact sensitive is used to refer to a liposome formulation which renders the phospholipid vesicles capable of a membrane lytic event, which is dependent upon analyte induced effects at the surface of the liposome(s) , and, independent of exogenously added lytic molecules or ions.
  • reporter molecule(s) is used to refer to any compound, molecule, or composition, which, upon release from the liposome, is itself directly capable, or able to generate another compound, molecule, or composition, capable of detection by eye or machine.
  • examples of commercially available materials include colorogenic or fluorogenic dyes, enzymes and their substrates, chemiluminescent species, and bioactive compounds, such as nucleic acids, hormones, lymphokines, etc., together with their appropriate cellular and noncellular targets.
  • analyte(s) is used to refer to any compound, molecule, or composition to be qualitatively or quantitatively detected in the test sample media.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member, or, for which a specific binding member can be prepared. Examples include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, antibodies, viruses, and bacteria.
  • the term “homogeneous immunoassay” is used to refer to an antibody - antigen binding mediated diagnostic assay, in which a single step or a sequence of steps, are performed without any intervening washing or separation steps required.
  • linker molecule(s) is used to refer to an analyte specific or analyte nonspecific molecule(s) , present in solution (or in suspension, or on a solid support) together with the liposome reagent and the analyte, that binds directly or indirectly, to either the targeting molecule on the liposome surface, or the analyte, or both, or other linker molecules, allowing the analyte to induce a "contact sensitive" liposome lysis.
  • Examples include carbohydrates, proteins, nucleic acids, lipids, drugs, hormones, metabolites, antigens/haptens, antibodies, receptors and ligands.
  • TM targeting molecule(s) on the liposome surface
  • LM linker molecule(s)
  • AN analyte(s)
  • Figures 1A and IB illustrate the effect of analyte addition on the size of LUV composed of 90 mole percent DOPE, 10 mole percent MPB-PE as stabilizer, and 1.0 mole percent Biotin-PE as targeting molecule.
  • Five ⁇ l (50 nmol) of LUV were added to 10 ⁇ l of PBS, in the presence (IB) or absence (1A) of 2.5 ⁇ g of polyclonal goat anti-biotin antibody. After 1 hour at 37°C, samples were analyzed by electron microscopy, and sized using photographically enlarged micrographs. The magnification shown is 20,OO0X and the bar insert in Fig. 1A represents a length of 500 nm.
  • Figure 2 depicts LUV containing 50 mM calcein and composed of 90 mole percent DOPE, 10 mole percent MPB-PE as stabilizer, and either 0.1 (ref. line 20) or 1.0 (ref. line 22) mole percent Biotin-PE as the targeting molecule.
  • the LUV were incubated at 37°C for 1 hour with various amounts of goat anti-biotin antibody (analyte) .. Percent quenching was measured as described in the examples.
  • Figure 3 illustrates the effect of temperature on analyte induced lysis of LUV containing 50 mM calcein, composed of 85 mole percent of either DOPE (ref. line 30) or DOPC (ref. line 32) , stabilized with 15 mole percent DOPS, and containing 1 mole percent Biotin-PE as the targeting molecule.
  • Ten ⁇ g of goat anti-biotin antibody (analyte) was added to the liposome suspension at a variety of temperatures.
  • DOPE controls (ref. line 34) and DOPC controls (ref. line 36) were run in the absence of analyte. Percent quenching was measured after a 5 minute incubation as described in the examples.
  • Figure 4 illustrates the effect of analyte concentration on lysis of LUV containing 50 mM calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1 mole percent Biotin-PE as targeting molecule. Incubation was at 70°C for 5 minutes. Percent quenching was measured as described in the examples.
  • Figure 5 illustrates the effect of free theophylline on the antibody mediated lysis of LUV containing 50 mM calcein, composed of 85 mole percent DOPE, 15 mole percent DOPS as stabilizer, and 1 mole percent theophylline - DOPE as the targeting molecule/analyte.
  • a mouse monoclonal anti-theophylline antibody was pre-incubated for 5 minutes at room temperature with a range of free theophylline in PBS (0-500 nmoles) , followed by addition of liposomes for 5 minutes at 70°C. Percent quenching was measured as described in the examples.
  • Figure 6 illustrates the detection of streptavidin in human serum samples, utilizing LUV containing 70 mM entrapped calcein, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent Biotin-Cap-DPPE as targeting molecule.
  • LUV 146 nmol
  • SUV composed of 20 mole percent DOPA and 80 mole percent DOPE
  • Figure 7 illustrates the correlation of the Lipogen contact sensitive, liposome based competitive immunoassay for serum theophylline, as compared to the commercially available Dupont Petinia R assay.
  • Figure 8 illustrates the detection of HCG in human serum samples, utilizing LUV containing glucose oxidase, and composed of 75 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.1 mole percent Biotin-Cap-DPPE as targeting molecule on the liposome surface.
  • LUV (10.5 nmol) were added to sera that had been spiked with HCG, and preincubated briefly with 1 ⁇ mol of "empty bags", composed of 25 mole percent DOPA and 75 mole percent DOPE.
  • Biotinylated mouse monoclonal anti-HCG (75 ng) together with streptavidin (9 ng) was added to the test samples as "linker molecules”.
  • Figure 9 illustrates the detection of antibodies in human serum samples, utilizing LUV containing 70 mM calcein, and composed of 74.5 mole percent DOPE, 25 mole percent CHEMS as stabilizer, and 0.5 mole percent theophylline-DOPE conjugate as targeting molecule on the liposome surface.
  • LUV 200 nmol
  • mice monoclonal anti-theophylline antibody (1-5 ⁇ g)
  • percent quenching was measured as previously described.
  • Figure 10 depicts the virus induced lysis of LUV containing 50 mM calcein, composed of 80 mole percent DOPE, 20 mole percent DOPA as stabilizer, and 0.06 mole percent of anti-herpes mouse monoclonal antibody (conjugated to NGPE) as targeting molecule.
  • Liposomes 500 ⁇ M were incubated at room temperature with HSV (2 mg viral protein/ml) in the presence (ref. line 100) or absence (ref. line 110) of excess antibody, Sendai virus (ref. line 120) , or uninfected Vero cell supernatant (ref. line 130) . At various time intervals samples were measured for percent dye released as described in the examples.
  • Control liposomes, containing DOPE/DOPA, but without targeting antibodies, were also tested for lysis by HSV (ref. line 140) .
  • Figure 1 (B) shows that the contact sensitive liposomes are extensively aggregated by the appropriate antibody, to form a heterogeneous mixture of multi-liposome particles, with an average diameter of between 1,000 - 2,500 nm.
  • Leakage of entrapped calcein dye occurred subsequent to the agglutination step(s) , and the final fluorescent signal was proportional to the degree of liposome aggregation (data not shown) .
  • FIG. 1 (A) shows an even dispersion of contact sensitive liposomes, incubated in the absence of antibody, with an average size (diameter) of 250 nm.
  • Figure 2 shows the results of an experiment described in Example 3, that shows that the degree of lysis of extruded liposomes containing DOPE as their bulk lipid is dependent upon both the concentration of analyte and the targeting molecule surface density, when temperature, liposome concentration, and time, are held constant. Specificity of the lysis reaction was demonstrated by the complete inhibition of dye leakage when an excess of free/soluble biotin was added to the mixture (data not shown) .
  • the Figure 2 results lead to two important conclusions:
  • the DOPE liposome system can detect a 150,000 dalton, nonmultivalent analyte in a "contact sensitive" homogeneous format, and,
  • Figure 3 shows that results of an experiment described in Example 4, that shows that a rapid and strongly temperature dependent analyte induced lysis occurred when liposomes composed of DOPE were utilized, while no such dye release was seen when conventional DOPC based liposomes, of otherwise identical composition and size were employed.
  • DOPC based liposomes were aggregated by analyte, no lysis was seen for up to 30 minutes of incubation, further supporting the uniqueness of the PE lipid formulation in the present "contact sensitive" liposomal immunoassay.
  • Figure 4 shows the results of an experiment described in Example 5, which shows that across a wide range of analyte concentration, there exists a bell shaped liposome lysis curve, similar to that seen for classical antigen - antibody agglutination reactions.
  • the significance of such a dose - response is the flexibility it allows in terms of immunodiagnostic assay design.
  • the sharpness/slope of the curve could be controllably fine - tuned by manipulation of the identity and mole percent of the liposome stabilizer molecule, and the mole percent of the targeting molecule (data not shown) .
  • Manipulations of divalent cation concentrations, salt concentrations (osmotic pressures), and pH levels, in the assay incubation buffer were found to decrease both the required time, and incubation temperature, of the assay (data not shown) .
  • Figure 5 shows the results of an experiment described in Example 6 in which an analyte of actual commercial and therapeutic interest, theophylline, is coupled to DOPE as a "targeting molecule" on the surface of the contact sensitive liposome.
  • DOPE an analyte of actual commercial and therapeutic interest
  • Increasing amounts of free theophylline pre-mixed with the liposome reagent were able to inhibit mouse monoclonal anti-theophylline antibody induced liposome agglutination and lysis in a dose dependent manner.
  • Such a homogeneous two step competition assay for small drugs is similar to commercially available technologies such as the Syva EMIT R system and the ABBOTT TDx R system.
  • FIG. 6 shows that the results of an experiment described in Example 7, that shows the use of SUV (and, in subsequent experiments, EV; data not shown) composed of DOPE and DOPA (containing no reporter molecule(s) , and no surface targeting molecule(s); referred to as "empty bags”) , allows the detection of a high molecular weight, model "analyte", streptavidin, in human serum, in the ng/ml range of sensitivity, utilizing contact sensitive LUV (60) .
  • contact sensitive LUV 60
  • the absence of SUV (62) there is no dose response proportional to analyte concentration, due to a large degree of nonspecific LUV lysis by the serum samples.
  • the major significance of these data is the ability to broaden the use of the contact sensitive liposome reagent to detection of analytes in human serum, employing the empty bags to adsorb out any interfering substances.
  • the ability to quantitatively detect streptavidin in 5 minutes, in serum, or in a variety of conventional buffers, by a single step, homogeneous immunoassay format allows the contact sensitive liposome formulation to be utilized in a large number of conventional ELISA protocols, currently utilizing biotin/streptavidin bridges, in- the diagnostics field.
  • An interesting possibility is the use of the contact sensitive liposome to quantitate the amount of DNA synthesized by the enzymes used in the PCR gene amplification technology.
  • Another interesting possibility is the use of the liposomes of the present invention in conjunction with a variety of biosensor probe technologies.
  • Figure 7 shows the results of an experiment described in Example 8, that shows that the present contact sensitive liposome formulation compares quite favorably in performance to the commercially well established Dupont Petinia R theophylline immunoassay kit.
  • the present inventors were able to achieve almost a 90% correlation with the Dupont assay, while evaluating 21 human patient sera obtained from a local hospital.
  • the present homogeneous, liposome based membrane lytic immunoassay was linear over the entire therapeutically relevant range of theophylline (0-40 ⁇ g/ml) tested.
  • results of an experiment in which theophylline levels were quantitated in serum, which is the relevant biological fluid for the detection of this particular drug (and most other therapeutic drugs) .
  • PBS aqueous buffer
  • Figure 8 shows the results of an experiment described in Example 9, that shows that the contact sensitive DOPE-based liposome is able to detect, by a totally homogeneous (one or two step) immunoassay format, a high molecular weight, solution phase hormone analyte, human chorionic gonadotropin (HCG: 33,000 daltons) , in human serum.
  • HCG human chorionic gonadotropin
  • a “generic” liposome formulation was used, employing a biotinylated phospholipid as the phospholipid vesicle surface handle ("targeting" molecule) , together with the appropriate analyte specific (biotinylated monoclonal antibodies) , and analyte nonspecific (streptavidin), "linker molecules”, added in solution phase.
  • targeting biotinylated phospholipid
  • analyte nonspecific streptavidin
  • the present inventors have since been able to detect LH, FSH and TSH, utilizing the same generic liposome formulation, simply by changing to the appropriate linker molecules in each case (data not shown) .
  • this contact sensitive liposome formulation is able to release latent enzyme activity proportional to analyte concentration, at 37° C in 5-10 minutes, (or at room temperature in 20 min. - data not shown) , without any washing or separation steps.
  • "antigen capture" is accomplished in the solution/suspension phase, with no need to manufacture any solid phase antibody coated microwells, for example.
  • liposome reagents should be compatible with a wide variety of ELISA based immunoassays; ranging from microwell to dedicated equipment formats; at the same time performing in much shorter time frames, homogeneous immunodiagnostic assays for a wide variety of small/single epitope, and large/multiepitope analytes, requiring little or no training of laboratory personnel, and highly simplified clinical equipment.
  • Figure 9 shows the results of an experiment described in Example 10, that shows the detection of specific antibodies in human serum, once again utilizing the contact sensitive liposome reagent(s), in a totally homogeneous immunoassay format. Although only ⁇ g quantities of serum antibody were homogeneously detected in this case, it is expected that one will be able to increase the detectability to the ng and pg levels by replacement of enzymes for fluorescent dyes as reporter molecules (see HCG results. Figure 8) . These results extend the use of the liposomes of the- present invention to the quantitative measurement of analytes as large as 150,000 daltons, as compared to HCG (and related hormones) at approximately 33,000 daltons.
  • these data indicate the potential to utilize the contact sensitive liposomes as a screen for serum antibodies.
  • Figure 10 shows the results of an experiment described in Example 11, that shows an extension of the contact sensitive liposome immunoassay to the detection of multivalent, particulate analytes.
  • these results were the first obtained utilizing as reagents true "immunoliposomes", in that the analyte specific linker molecules/antibodies were covalently attached to the surface of the liposome.
  • a cellular extract of HSV was added to the immunoliposomes at room temperature, a rapid lysis of the vesicles, leading to an increase in fluorescent signal, was observed. The lysis could be partially blocked by the addition of a 100-fold excess of solution phase antibody, and appeared to be specific in that Sendai virus at the same protein concentration gave little dye release.
  • Table I shows a list of stabilizers that have been utilized in the various immunoassays described in this text. This list is not meant to be exhaustive, but instead, is included to point out the broad range of stabilizers available to construct/formulate the "contact sensitive" liposome of this invention.
  • percent stabilizer shown in this table simply represents the most commonly used mole percent of each compound utilized in the present inventors attempt to most controllably and conveniently manufacture and store the liposomes, as well as to optimize, up to this point in time, analyte detection, in terms of assay time, temperature, sensitivity, sample fluid media compatibility, etc.
  • All synthetic phospholipids were purchased as a chloroform solution from Avanti Polar Lipids, Inc. Goat anti - biotin antibody, and calcein were purchased from Sigma Chemical Co.
  • the mouse monoclonal anti - theophylline antibody was purchased from Medix. Human chorionic gonadotropin and streptavidin were purchased from Scripps Laboratories.
  • the 3 H - hexadecyl cholestanyl ether was a gift from Dr. Leaf Huang at the University of Tennessee.
  • Theophylline - DOPE was synthesized and purified as described below.
  • Lipid preparations were composed of either DOPC or DOPE as the bulk phospholipid (80-90 mole percent) , 10-20 mole percent of an amphipathic stabilizer compound, and 0.1-1.0 mole percent of a surface targeting molecule(s) .
  • 3H-CE was included in the lipid mixture as a tracer.
  • Lipid films were generated by evaporation of solvent from a chloroform/phospholipid solution utilizing a N 2 gas stream, followed by desiccation of the lipids at reduced pressure for a minimum--of four hours. Lipids (10 mM) were hydrated at room temperature for at least 30 minutes in PBS containing 50 mM calcein dye.
  • Calcein 2 / ,7'-[[bis(carboxymethyl)amino]methyl] - fluorescein, is a self - quenching dye that, at concentrations greater than 5 ⁇ M, undergoes progressive collissional fluorescence quenching. It has been widely used for analysis of liposome lysis/leakage , especially in the design of homogeneous phase systems.
  • the calcein solution was prepared at 50 mM in distilled water and the pH adjusted to 7.4 with 10 N NaOH. The osmolarity of the solution was adjusted with 10X PBS to that of human serum, approximately 330 milliosmoles.
  • the lipid suspensions were formed by rigorous vortexing and sized by repeated extrusion (10 times) through Nucleopore polycarbonate filters (0.2 ⁇ m pore size) at 200 psi utilizing a Lipex Extruder.
  • Percent quench [(F(t) - F(i))/F(t) ] X 100 , where F(i) is the initial fluorescence, F(t) is the total fluorescence after the addition of detergent (which releases all of the entrapped calcein) .
  • Initial calcein fluorescence was measured by adding a 5 ⁇ l lipid sample to 3 ml of PBS in a quartz cuvette, and reading light emission intensity on a Perkin Elmer LS - 5B Luminescence Spectrophotometer at 490 nm excitation/ 520 nm emission. Total calcein fluorescence was monitored after the addition of 10 ⁇ l of 10% Triton X - 100 to completely lyse the vesicles and release all entrapped dye.
  • the size of the liposomes was determined by laser light scattering using the Coulter N4MD submicron particle analyzer.
  • the theophylline - DOPE conjugate was prepared by the condensation of 5,6-diamino-l,3 - dimethyluracil with succinic anhydride to form 8-(2-carboxyethyl)- theophylline.
  • This theophylline derivative in turn, generated the desired lipid conjugate upon condensation with DOPE.
  • the drug conjugate was mixed with the appropriate phospholipids and liposomes were formed by the extrusion method as previously described.
  • the anti-HSV monoclonal antibody onto the surface of the liposome, it was necessary to conjugate the antibody to N-glutaryl-PE in the presence of carbodiimide and N-hydroxy sulfosuccinimide (S-NHS) . Following the coupling reaction, the NGPE-IgG mixture was dialyzed against PBS, and then co-sonicated with the appropriate phospholipid mixture, in order to insert the antibody-lipid complex into the liposome bilayer.
  • S-NHS N-hydroxy sulfosuccinimide

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Abstract

Formulation de liposome sensible au contact comprenant environ 0,1 à environ 99,9 mole pourcent d'un ou de plusieurs lipides formant des phases hexagonales inversées, ledit liposome stabilisé dans la phase à deux couches comportant environ 0,1 à environ 99,9 pourcent en moles d'une ou de plusieurs molécules stabilisatrices amphipatiques indépendantes de l'analyte ou de mélanges de ces dernières, ledit liposome comprenant en outre une ou plusieurs molécules de ciblage sur sa surface.
PCT/US1991/008293 1990-11-05 1991-11-05 Procede de dosage immunologique lytique homogene sur membrane utilisant des formulations de liposomes sensibles au contact WO1992007959A1 (fr)

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WO2013093891A1 (fr) 2011-12-22 2013-06-27 Nuvo Research Gmbh Compositions liposomales comprenant des chlorites ou chlorates
US9297035B2 (en) 2008-05-27 2016-03-29 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
WO2018087720A1 (fr) 2016-11-14 2018-05-17 Novartis Ag Compositions, méthodes et utilisations thérapeutiques associées à une protéine fusogène minion
US10662465B2 (en) 2011-09-30 2020-05-26 Agilent Technologies, Inc. Hybridization compositions and methods using formamide
US11118226B2 (en) 2011-10-21 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
US11795499B2 (en) 2009-02-26 2023-10-24 Agilent Technologies, Inc. Compositions and methods for performing hybridizations with separate denaturation of the sample and probe

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US9297035B2 (en) 2008-05-27 2016-03-29 Dako Denmark A/S Compositions and methods for detection of chromosomal aberrations with novel hybridization buffers
US11118214B2 (en) 2008-05-27 2021-09-14 Agilent Technologies, Inc. Hybridization compositions and methods
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US12209276B2 (en) 2008-05-27 2025-01-28 Agilent Technologies, Inc. Hybridization compositions and methods
US11795499B2 (en) 2009-02-26 2023-10-24 Agilent Technologies, Inc. Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
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