WO1987002778A1 - Systeme d'immunoanalyse a base de liposomes en phase solide - Google Patents

Systeme d'immunoanalyse a base de liposomes en phase solide Download PDF

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Publication number
WO1987002778A1
WO1987002778A1 PCT/US1986/002233 US8602233W WO8702778A1 WO 1987002778 A1 WO1987002778 A1 WO 1987002778A1 US 8602233 W US8602233 W US 8602233W WO 8702778 A1 WO8702778 A1 WO 8702778A1
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Prior art keywords
molecules
analyte
substrate
reagent
binding
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PCT/US1986/002233
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English (en)
Inventor
Luke Guo
Alan S. Michaels
Francis J. Martin
Ned M. Weinshenker
Pedro Huertas
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Cooper-Lipotech
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Publication of WO1987002778A1 publication Critical patent/WO1987002778A1/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Definitions

  • the present invention relates to a solid-phase liposome immunoassay system, and to an improved reagent for use in the system.
  • One system employs a solid support having bound surface molecules, and a reporter/ligand bimolecular conjugate which binds to the solid support in proportion to the concentration of analyte present in the assay system.
  • the conjugate may compete with the analyte for binding to the support, in a competitive- inhibition type assay, or may bind to the support through a bifunctional analyte. in a sandwich-type assay.
  • the solid (support) and liquid phases are separated, and the reporter activity associated with one or both phases, typically the solid phase, is measured to determine the presence and/or amount of analyte.
  • the bimolecular conjugate immunoassay system has limited sensitivity, inasmuch as each analyte-binding event is "reported" by one reporter molecule only, e.g., a single enzyme molecule. Another limitation is that the conjugate must be formed from a relatively pure ligand preparation. Otherwise, a significant amount of the reporter/ligand conjugate formed will not show analyte-specific binding to the support, and relatively high levels of non-specific binding and/or interference with specific binding may occur.
  • the limitations of the bimolecular conjugate system just described are overcome, in part, in a solid-phase system employing a liposome assay reagent in place of the reporter/ligand conjugate.
  • the liposomes are prepared to include a surface array of analyte-related binding molecules (analogous to the ligand moiety of the reporter/ligand conjugate), to produce binding to a solid support in proportion to the amount of analyte present, either analyte-mediated sandwich binding or direct binding to the support in competition with the analyte.
  • the reporter molecules typically enzyme molecules, are entrapped in the liposomes at relatively high concentration, either in liposome-encapsulated form, or bound to the surface of the liposomes.
  • a solid-phase immunoassay system employing a liposome reagent having encapsulated reporter enzyme has been disclosed in U.K. patent application GB 215360.
  • Co-owned patent application for "Lipid-Vesicle Surface Assay Reagent and Method", Serial No. 452.798. filed 23 December 1982. describes a liposome immunoassay containing surface-bound reporter enzymes.
  • Experiments conducted in support of the latter application indicate that each liposome can bind to the support through a relatively small number of surface binding events, e.g., individual antigen/antibody binding events. Accordingly, this system has the potential for very high sensitivity due to the.high ratio of reporter molecules to analyte-related binding events.
  • a more specific object of the invention is to provide in such a system, a novel surface reagent which gives specific-to-nonspecific liposome binding ratios which are several times higher than those achievable using prior art surface reagents.
  • Another object of the invention is to provide methods for making such a surface reagent, and for using the same in a liposome immunoassay.
  • the invention includes, in one aspect, a surface reagent for use in a solid-phase immunoassay based on immunospe ⁇ ific liposome attachment to the reagent.
  • the reagent comprises a cellulose acetate substrate whose surface has been treated to replace surface acetate groups with hydroxyl groups, and an array of surface molecules attached to the substrate through the hydroxyl groups.
  • the reagent is base-hydrolyzed under conditions which result in a hydrophilic surface shell of hydroxyl groups that protects the material from dissolution by organic solvents capable of dissolving cellulose acetate, and the surface molecules are attached to the substrate through bifunctional linking groups which are attached to the support in the presence of an organic solvent.
  • the immunoassay system of the invention includes the surface reagent and liposomes containing surface-bound, analyte-related binding molecules and entrapped reporter molecules.
  • the liposomes are adapted to bind to the surface reagent by direct binding to the reagent surface molecules, in a competitive-inhibition assay, or by binding to a bifunctional analyte in a sandwich-type assay.
  • Reporter groups in the liposome reagent are preferably surface-bound and/or encapsulated enzymes
  • Figure 1 shows a reaction scheme for producing a solid surface reagent having surface-bound ligand molecules coupled to the reagent substrate by means of a cyanuric chloride-activating agent
  • Figure 2 shows a reaction scheme for producing a solid surface reagent having surface molecules attached to the reagent substrate through a carbonyl diimidazole coupling agent.
  • the reagent of the invention is prepared from a cellulose acetate solid-support substrate. According to one finding of the invention, this type of substrate gives significantly lower levels of non-specific liposome binding than a number of other substrate materials which are available. To illustrate, the binding of two different types of liposomes to each of four different types of bead substrates was examined.
  • One liposome type (#1 in Table 1 below) was prepared to contain surface-bound anti-hCG antibodies and ⁇ -galactosidase.
  • a second liposome type (#2) was prepared to include encapsulated alkaline phosphatase and surface-bound anti-hCG antibody, as described in Example VI.
  • the liposomes were incubated with three beads of each bead type in 50 mM phosphate buffer, pH 7.4, for 30 minutes at room temperature, then washed four times with incubation buffer. Each bead was assayed separately for associated enzyme activity, according to procedures discussed below, and the mean value obtained for three beads for each bead and liposome type, expressed in OD unit/bead, was determined. The results, seen in Table 1 below, show a severalfold improvement in non-specific binding levels obtainable with the cellulose acetate substrate.
  • the cellulose acetate substrate used in preparing the surface reagent may be beads, strips, rods, or other substrate forms which are readily separated from the liquid phase of an assay mixture.
  • the assay vessel itself such as a tube, may be formed of cellulose acetate.
  • the substrate is treated with a strong base to replace surface acetate groups with hydroxyl groups.
  • the surface molecules may be coupled to the surface-hydroxyl groups through chemical coupling reagents which can be attached to the substrate hydroxyl groups in an aqueous reaction medium.
  • the sur ace-treated substrate can be reacted with an alkalating agent, such as iodoacetic acid, to attach carboxyl groups to the substrate surface in an aqueous reaction.
  • Amine-containing surface molecules could then be coupled to the acid groups through amide linkages by reaction in the presence of a water-soluble carbodiimide. according to known methods. In such applications, where only aqueous solvents are used in coupling the surface molecules to the substrate, it is unnecessary to treat the substrate to resist solvent attack.
  • the molecules to the surface by first activating the substrate surface with a bifunctional linking agent, then reacting the molecules with the activated, washed substrate.
  • the activation reaction used to couple the bifunctional agent to the substrate is typically carried out in an organic solvent or solvent mixture, to minimize hydrolysis of the activated reactive group in the bifunctional agent, and, in many cases, to increase compound solubility.
  • cellulose acetate has not been a suitable substrate for surface activation reactions which need to be carried out in organic solvents, because the polymer material is soluble in most solvents, such as acetone, dioxane. dimethyl sulfoxide, which are used in surface activation reactions.
  • solvents such as acetone, dioxane. dimethyl sulfoxide, which are used in surface activation reactions.
  • This limitation has been overcome, according to the present invention, by treating the cellulose acetate substrate under conditions which produce extensive base hydrolysis, to form a hydrophilic shell of hydroxyl groups on the substrate surface.
  • the hydrophilic shell confers substantial resistance against dissolution in organic solvents such as dioxane and acetone, during the course of such activation reactions.
  • the hydrolysis conditions used in forming the solvent-resistant shell may be determined empirically, for example, by carrying out the base hydrolysis until the substrate material acquires the requisite solvent re ⁇ istance.
  • the surface-treated substrate may be dried or stored in solution.
  • the substrate-activation reaction just described is carried out by suspending the surface-treated substrate from above in a solution of the bifunctional agent in a suitable organic solvent. After a reaction period typically between a few seconds up to 1-2 hours, depending on the concentration and reactivity of the agent, the substrate is washed several times to remove bifunctional agent and is dried. The dried substrate may be resuspended in a suitable solvent, such as benzene, to remove trace amounts of non-covalently attached bifunctional agent.
  • Example II below describes the activation of modified cellulose acetate beads with cyanuric chloride and Example V, activation of the beads with carbonyl diimidazole.
  • the washed, activated substrate is reacted in aqueous medium with a selected concentration of molecules which will form the surface molecules in the surface reagent.
  • the surface molecules may be either (1) analyte or analyte-like molecules capable of competing with the analyte for binding to anti-analyte molecules carried on liposomes, or (2) anti-analyte molecules capable of binding a bifunctional analyte to the support, preferably through only one of two or more analyte epitopic sites.
  • analyte and anti-analyte refer to the opposite members of a binding pair composed of a target molecule having one or more specific epitopic features, and a target-binding molecule which recognizes at least one such feature to bind the target molecule specifically and with high affinity.
  • the analyte/anti-analyte binding pairs which are suitable for use in the invention are antigen-antibody, immunoglobulin-protein A, carbohydrate-lectin, biotin-avidin, hormone-hormone receptor protein, and complementary nucleotide strand pairs, where the analyte may be either member of the pair, and the anti-analyte. the opposite member.
  • the anti-analyte may include any portion of an anti-analyte molecule which is capable of participating with the analyte in specific, high-affinity binding, for example, in an antibody-antigen pair, an anti-analyte antibody may include either analyte-binding F(ab') or Fab- f agments.
  • concentration of surface molecules in the coupling reaction is selected to produce a desired surface concentration of coupled molecules on the support, under the reaction conditions employed.
  • the coupling reaction is usually carried out at a pH of about 7.5 at 2-8°C for one hour, at a protein concentration between about 0.5 and 5 mg/ml.
  • the substrate is washed and may be incubated with an amine, such as ethanolamine, to tie up any unreactive activating groups.
  • the substrate can be stored in standard physiological buffer, and is typically incubated overnight with 1% serum albumin before, use.
  • Figure 1 illustrates the reactions used in coupling a protein (P-NH ) to a modified cellulose acetate surface with cyanuric chloride.
  • the reaction which follows the scheme described in reference 1. involving initial covalent attachment of cyanuric chloride to the support substrate (S-OH) through an ether linkage, and subsequent reaction of the dichlorotriazine with a protein amine group to couple the protein to the triazine ring.
  • the initial activation reaction is carried out in acetone, and the protein-coupling reaction in an aqueous buffer.
  • a similar series of reactions, illustrated in Figure 2, are used in coupling a protein to a . base-modified cellulose acetate surface by means of a carbonyl diimidazole linking agent.
  • the diimidazole reacts with a surface hydroxyl group to form an imadazolyl carbamate which can then react with the amine group of a protein to attach the protein to the substrate.
  • the general reaction scheme is described in reference 2.
  • the coupling of hCG and anti-hCG antibody to a modified cellulose acetate substrate activated with cyanuric chloride is described in Examples II and III below, and the coupling of hCG to the modified substrate activated with carbonyl diimidazole, in Example IV.
  • the activated substrate may also be reacted with an "intermediate" coupling compound which has one functional group, such as an amine group, adapted to react with the surface activating agent and another group such as an amine, carboxyl, or sulfhydryl group, to which the surface molecules can be conveniently coupled, according to well-known coupling methods.
  • the intermediate coupling compounds may provide a spacer arm, e.g., a 3-20 atom carbon or carbon-nitrogen chain with the desired reactive end group. Having the surface molecules attached to the support through a spacer arm may reduce steric binding constraints near the substrate surface and thereby provide enhanced im unospecific binding at the substrate surface.
  • the liposomes in the immunoassay system of the invention are composed of lipid vesicles having surface-bound, analyte-related binding molecules and reporter molecules which are entrapped in the vesicles, either in encapsulated or surface-bound form.
  • Lipid vesicles are prepared from lipid mixtures which preferably include phospholipids at a mole ratio between about 40 and 90%, and sterols, at a mole ratio between about 10 and 50 mole percent.
  • the lipid mixture may include one or more negatively charged lipids, such as phosphatidyl glycerol, typically at a mole percent between about 5 and 20%. to impart a negative surface charge.
  • the vesicles also preferably include one or more lipid components having reactive or activated polar head groups which can be used in coupling analyte-related binding molecules to the vesicle surfaces.
  • lipids include those having reactive polar groups such as amine, carboxyl, hydroxyl. or sulfhydryl groups, or activated groups capable of reacting directly with hydroxyl, carboxyl, amine. or sulfhydryl groups on the molecules to be attached to vesicles.
  • lipid vesicle components at a mole ratio preferably between about 0.1 and 10%.
  • the lipid components used in preparing the liposomes described in Examples VI and VII include 40% cholesterol. 48% phosphatidylcholine (PC). 6% phosphatidylglycerol (PG). and 6% (MPB-PE). a thiol-reactive phophatidyl ethanolamine (PE) described in references 3 and 4.
  • the lipid vesicles may be formed by any of a variety of methods, such as those detailed in reference 5.
  • One preferred method referred to as a reverse-phase evaporation method, involves initial formation of an emulsion of aqueous particles in an organic solvent containing the vesicle lipids. Removal of the organic solvent by evaporation leads to a reverse-phase emulsion that can be converted to reverse-evaporation vesicles (REVs) by agitation in an aqueous medium.
  • REVs reverse-evaporation vesicles
  • the REV method produces predominantly uni- and oligolamellar vesicles having relatively large sizes, i.e.. greater than about 1 micron.
  • Vesicles of this type may be readily converted to a population of substantially homogeneous sizes by extrusion through a unipore polycarbonate membrane having selected pore sizes, e.g., between 0.1 and 1 micron.
  • a unipore polycarbonate membrane having selected pore sizes e.g., between 0.1 and 1 micron.
  • Examples VI and VII below describes the preparation of REVs and subsequent vesicle sizing by extrusion through 0.4 micron unipore membranes
  • vesicles can be formed readily by hydrating a film of lipid vesicle components in an aqueous medium, and these vesicles can also be made homogeneous in size by extrusion through a unipore membrane filter.
  • This liposome formation technique is described in reference 6.
  • the vesicles may be prepared under conditions which lead to encapsulation of reporter molecules, for use in preparing liposomes having surface-bound binding molecules and encapsulated reporter.
  • reporter molecules such as fluophores. chromophores. spin-labeled molecules, and enzymes may be used. Of these, enzymes generally provide greater assay sensitivity due to high enzyme turnover numbers in converting a substrate to a detectable product.
  • the enzyme preferably is one which: (1) is capable of producing an easily measured colorometric or fluorometric effect in the presence of a suitable substrate; (2) is available in pure or nearly pure form; (3) can be encapsulated in lipid vesicles without significant loss of activity; and (4) is stable in encapsulated form on storage in solution, or is resistant to freezing and lyophilization in encapsulated form.
  • the two lipid vesicle preparation methods mentioned above are both suitable for preparing liposomes having encapsulated enzyme reporter molecules, as are a variety of other liposome preparation methods.
  • the initial water and oil emulsion is formed using an aqueous enzyme solution, leading to lipid vesicles which may encapsulate up to about 50% of the enzyme solution used in forming the emulsion.
  • a high encapsulation efficiency is a significant advantage of the REV method, but enzyme inactivation by organic solvents may limit use of this method for some enzymes.
  • Co-owned patent application for "Encapsulated Enzyme Liposome Reagent" Serial No. 699,860. filed 8 February 1985. describes a novel method for stabilizing glucose-6-phosphate dehydrogenase against inactivation by organic solvents in the REV procedure and against inactivation on storage in liposome-encapsulated form.
  • the reporter may be entrapped either in encapsulated form or surface-bound form or both.
  • Methods for forming liposomes having surface-bound reporter molecules, such as enzyme molecules, in addition to surface-bound binding molecules, such as antibodies, are described in the above-noted patent application for "Lipid-Surface Vesicle Reagent". Protein-to-liposome coupling methods described in that application, and methods described below for attaching analyte-related binding molecules to liposome surfaces are applicable.
  • One enzyme which has been used advantageously in forming a "co-conjugate" liposome having surface-bound enzyme and binding molecules is bacterial-derived ⁇ -galactosidase. Among the advantages of this enzyme are: (1) the enzyme is available in purified form with high specific activity:
  • the enzyme contains free thiol groups which can be used in coupling the enzyme to thiol-reactive lipids in liposomes. without affecting the enzyme activity;
  • Example VII describes the preparation of co-conjugate lipisomes having surface-bound ⁇ -galactosidase.
  • the liposomes are further prepared to include surface-bound molecules adapted to produce lipid binding to the solid-support reagent in proportion to the amount of analyte present in an immunoassay reaction mixture.
  • the analyte-related binding molecules are typically anti-analyte molecules which form one member of an analyte/anti-analyte binding pair, as defined above.
  • the analyte compete with analyte or analyte-like molecules carried on the surface reagent for binding to anti-analyte carried on the liposomes.
  • the liposome-bound anti-analyte is typically an anti-antigen antibody or antibody fragment.
  • the anti-analyte is one which typically has binding specificity for one binding moiety (epitopic site) of the analyte, with the anti-analyte carried on the reagent preferably having binding specificity for a second, distinct binding moiety.
  • the reagent molecules may include anti-A site antibodies, and the liposome binding molecules, anti-B site antibodies.
  • a third efficient method which has been described in co-owned patent application for "Carboxylated Lipid Coupling Reagent”. Serial No. 692.679, filed 18 January 1985, involves coupling of proteins to a carboxyl group carried at the end of a 3-18 atom spacer arm attached to the vesicle through a hydrophobic anchor.
  • the first two methods mentioned above are capable of producing protein-to-vesicle coupling ratios of up to 200-300 ⁇ g protein per ⁇ mol lipid: the third method, ratios of up to about 600 ⁇ g protein/ ⁇ mole lipid.
  • Example VI describes the coupling of anti-hCG antibody to lipid vesicles containing encapsulated alkaline phosphatase. In Example VII.
  • the antibody and enzyme are coupled to the enzyme in a single coupling reaction containing thiol-reactive liposomes and antibody and enzyme, at a selected concentration of each.
  • This section describes the use of the above solid-phase immunoassay system in two general types of solid-phase immunoassays.
  • the first type referred to above as a competitive inhibition assay
  • the analyte competes with analyte and analyte-like surface molecules on the solid-phase reagent for binding to anti-analyte molecules on the liposomes.
  • the extent of the liposome binding to the solid support is therefore inversely proportional to the amount of analyte present in the reaction mixture.
  • the initial binding reaction is carried out in a reaction medium whose pH and ionic strength are compatible with analyte/anti-analyte binding, typically in a buffered medium, pH 5-8.
  • the concentration of liposome vesicles in the assay medium is preferably adjusted such that the extent of the liposome binding to the support is inversely proportional to the amount of analyte present in the mixture within the concentration range of analyte to be tested.
  • Typical liposome vesicle concentrations are in the range between about 10 -12 and 10-7 mole lipid per ml total assay.
  • the binding reaction is typically carried out at room temperature for a period of between about 5 and 60 minutes.
  • the assay is calibrated using serially diluted analyte solutions.
  • analyte solutions such as diluted serum control samples containing known concentrations of analyte, to establish a standard curve of reagent-associated enzyme activity versus analyte concentration over a selected analyte range.
  • the test sample itself may also be assayed at each of a series of different concentrations to give at least some, sample points which are within the standard curve range.
  • the solid support is washed, e.g., with the binding-reaction buffer, to remove non-specifically bound liposomes.
  • the solid support bearing the lipsomes is placed in an enzyme-assay medium containing enzyme substrate and. where the encapsulated enzyme liposome reagent is being used, a detergent such as Tween-20 effective in solubilizing the liposomes and releasing the encapsulated enzyme for reaction.
  • a detergent such as Tween-20 effective in solubilizing the liposomes and releasing the encapsulated enzyme for reaction.
  • the level of enzyme in the reaction mixture is measured conventionally, either spectrophotometrically or in a qualitative test by visible detection of color change.
  • Example VIII illustrates a competitive-inhibition assay for determination of hCG using a liposome reagent containing surface-bound anti-hCG antibodies and ⁇ -galactosidase.
  • the measured enzyme activity expressed as OD/bead. ranged from about 1.6 in the absence of analyte to 0.112 at the highest concentration of analyte added.
  • Example IX describes a similar type of competitive-inhibition assay using a liposome reagent having surface-bound anti-hCG antibodies and encapsulated alkaline phosphatase.
  • the OD/bead levels ranged from 1.2 in the absence of added analyte to a low of 0.033 at high analyte concentration.
  • a second general type of immunoassay involves sandwich-type binding of liposomes to the solid-phase reagent through analyte bridging.
  • the analyte is a bivalent antigen having two or more epitopic binding sites which combine simultaneously to anti-analyte molecules carried on the support and to liposome-bound anti-analyte binding molecules.
  • This assay type gives greater liposome binding to the solid support in the presence of greater amounts of analyte.
  • the assay involves an initial reaction step which is carried out under conditions like those used in the competitive-inhibition assay described above.
  • the concentration of liposomes in the reaction mixture is preferably selected to give proportionally more binding to the solid support with increasing amounts of added analyte. over the desired analyte concentration range.
  • the assay is usually carried out by preparing a standard curve from known analyte controls, in a linear range of analyte concentrations, and assaying for the analyte at one of a number of serial dilutions.
  • Example X describes a sandwich-type assay for determination of hCG using liposomes containing either surface-bound polyclonal anti-hCG or monoclonal anti-hCG antibodies.
  • the test can accurately measure hCG to a sensitivity level of about 15 mlU/ml. and shows a concentration dependence in the range from about 15 to 250 mlU/ml.
  • An OD range of more than 2 OD units/reagent bead over the analyte range tested. and a maximum signal-to-noise ratio of about 30 were attained.
  • modified cellulose acetate substrate of the invention is readily prepared for attachment of surface molecules through surface hydroxyl groups, and the hydrophilic surface shell formed in accordance with the invention allows surface molecule coupling by means of and initial surface activation reaction carrie out in an organic solvent.
  • non-specific liposome binding to the modified-substrate reagent is severalfold less than to other types of solid phase reagent which have been used heretofore.
  • the signal-to-noise ratio and hence assay sensitivity attainable in a solid-phase liposome immunoassay. is enhanced severalfold.
  • the wider range of detectable reporter signal achievable with the reduced non-specific (background) binding levels markedly enhances the accuracy and reliability of solid-phase liposome immunoassay tests.
  • the immunoassay system thus overcomes a serious limitation in prior art liposome immunoassay systems. without compromising the inherent advantages of such a system, particularly high signal levels, due to the large number of reporter molecules which report each analyte/anti-analyte binding event on the reagent surface.
  • the following examples illustrate various aspects of the invention, but are in no way intended to limit the scope thereof.
  • Example I Preparing Modified Cellulose Acetate Beads Cellulose acetate beads (1/8 in. diameter) were obtained from Precision Plastic Ball Co. (Chicago. IL). The beads were suspended in 3 N NaOH for 36 hours, and then washed extensively with water until the pH of the bead suspension dropped to between about 6 and 7. The beads were dried in a Buchner funnel under vacuum.
  • the IgG was radiolabeled with 125I according to a chloramine T iodination method.
  • the specific activity of the radiolabeled protein was about 6 ⁇ Ci/mg.
  • the I radioactivity associated with each batch was determined. The results are shown in Table 2 below, along with the calculated protein coupling ratios for activated (A) to non-activated (NA) material. The total radioactivity associated with the activated beads corresponds to about 1 ⁇ g protein/bead (1/8").
  • Example III Coupling hCG to Activated Substrate Human chorionic gonadotropin (hCG) was obtained from Calbiochem-Behring (San Diego. CA) .
  • Liposomes having surface-bound anti-hCG in either encapsulated alkaline phosphatase (preparation #1) or surface-bound ⁇ -galactosidase (preparation #2) were prepared as described in Examples VI and VII. respectively. Levels, of specific binding to the solid surface support were measured by incubating preparation #1 (approximately 50 nmole phospholipid) or #2 (approximately 10 nmole phospholipid) in 50 mM. hosphate buffer, pH 7.4. with 3 beads for each liposome type. Non-specific binding levels were determined similarly, using the two liposome preparations which were first preincubated with excess hCG for 30 minutes to block anti-hCG antibody binding sites on the liposomes.
  • Beads having bound preparation #1 liposomes were assayed by addition to each bead of 0.5 ml assay solution containing 0.5% Tween-20 (to solubilize the bound liposomes) and 1.9 mM of the substrate p-nitrophenyl phosphate in 1.0 M diethanolaraine. 0.5 rriM MgCl . pH 9.8. The reaction was followed at 405 nm.
  • Beads having bound preparation 4.2 liposomes were assayed by addition to each bead of 0.5 ml of assay solution containing 2.7 mM of the substrate orthonitrophenyl-3- D-galactopyranosides (Calbiochem-Behring. San Diego, CA) (ONGP) in phosphate buffer, pH 7.8. The reaction was followed at 405 nm.
  • Mouse monoclonal anti-hCG antibody was obtained from Bioclone Australia Pty (Marrickville. Australia).
  • Example III A 1 mg/ml solution of the antibody (5 ml) in 50 mM phosphate buffer, pH 7.4. was added to freshly activated cellulose beads (2-5 g) from Example II. The coupling reaction was carried out at 2-8°C for 1 hour with moderate shaking, after which the anti-hCG solution was removed. Any reactive groups remaining on the surface of the beads were blocked for incubation with ethanolamine. as in Example III. The antibody-coupled beads were washed and stored overnight with 1% BSA. as in Example III. For control purposes, a bead reagent having surface-bound mouse immunoglobulin was similarly prepared.
  • Example V Coupling hCG to Beads Activated with Carbonyl Diimidazole
  • Previously hydrolyzed cellulose beads (2-5 g) from Example I were reacted with 5 ml dry acetone containing 0.12 g carbonyl diimidazole. After a reaction period of 15 minutes, the mixture was filtered and the beads were washed 3 times with cold acetone and then dried in a Buchner funnel under suction. The dried beads were resuspended in benzene to remove trace amounts of non-specifically attached diimidazole, and again dried.
  • the f eshly activated beads were reacted with 5 ml of hCG (1 mg/ml) in 50 mM phosphate buffer, pH 7.4, under substantially the same reaction conditions as those described in Example III. except that the reaction was carried out overnight. After removing the hCG solution, the beads were incubated with ethanolamine. to tie up free amine groups, and the beads washed and stored according to the method described in Example III.
  • Liposomes containing encapsulated alkaline phosphatase and surface-bound anti-hCG antibodies were prepared as in Example VI.
  • the binding of the liposomes to beads having surface-bound hCG coupled to the substrate surface either through cyanuric chloride (Example II) or carbonyl diimidazole was compared.
  • a suspension of the liposomes in 20 mM phosphate buffer, 150 mM NaCl. pH 7.4. was serially diluted with the same buffer and added to each of 4 assay tubes, for each surface substrate, in the total amount of liposomes shown at the left in Table 5 below.
  • Liposome (nmole pL) (cyanuric chloride) (diimidazole)
  • the sulfhydryl-reactive phospholipid derivative MPB-PE was prepared as substantially as described in reference 3. Alkaline phosphatase was obtained from Boehringer Mannheim Biochemicals (Indianapolis. IN).
  • Lipid vesicles were prepared by a reverse evaporation phase method as described in reference 6. Briefly, a lipid mixture containing phosphatidylcholine (8 ⁇ mol), cholesterol (6.7 ⁇ mol) . phosphatidyl- glycerol (1 ⁇ mol), and MPB-PE (1 ⁇ mol) was dissolved in 1.0 ml diethylethe . An enzyme solution (8.5 mg/ml) in 40 mM phosphate buffer, 0.5 mM MgCl 2 , 0.1 mM ZnCl , pH 6.0 was added (350 ⁇ l) and two phases emulsified by sonication for 1 min. Ether was removed under reduced pressure at room temperature and resulting dispersion was extruded through 0.4 micron unipore polycarbonate membranes (Bio-Rad Laboratories, Richmond, CA) .
  • Example VII Preparing Liposomes Containing Surface-bound Anti-HCG Antibody and ⁇ -Galactosidase ⁇ -galactosidase was obtained from Boehringer Mannheim Biochemicals (Indianapolis. IN). Lipid vesicles were prepared by a reverse phase evaporation method as described in Example VI. The enzyme solution used in preparing the liposomes in Example VI was replaced by a buffer containing 150 mM NaCl. and ImM EDTA. pH 6.0. The buffer (300 ⁇ l) was added to a lipid mixture dissolved in 1 ml diethylether and the two phases emulsified by sonication for one minute at 25°C in a bath sonicator.
  • Ether was removed under reduced pressure at room temperature and the resulting dispersion was extruded successively through 0.4 micron unipore polycarbonate membranes (Bio-Rad Laboratories. Richmond. CA) .
  • Freshly reduced antibody (0.1 mg) and ⁇ -galactosidase (0.375 mg) was reacted with 1 ⁇ raol vesicle lipid in 1 ml reaction buffer described in Example VI.
  • the vesicles were separated from the unreacted proteins by ultracentrifugation at 50,000 rpm for 2 hr using a metrizamide gradient.
  • Example VIII Competitive Inhibition Assay A surface reagent having surface-bound hCG molecules was prepared as in Example III. A suspension of liposomes having surface-bound ⁇ -galactosidase and anti-hCG was prepared as in Example VII.
  • test sample containing hCG at one of the concentrations indicated at the left in Table 6 below, was added to one of six assay tubes containing 3 nmoles liposomes to a final liquid volume of 0.25 ml in 20 mM phosphate buffer. 150 mM NaCl. pH 7.4. Each assay mixture was pre-incubated at 25°C for 30 minutes. Three surface reagent beads were then added to each assay tube and incubated at 25°C for 1 hr. The reagent beads were washed 2 times with the assay buffer.
  • the washed reagent from each sample was placed in 0.5 ml of assay solution containing phosphate buffer, pH 7.8, and 2.7 mM ONGP (Example III).
  • the conversion of the substrate was followed at 405 nm, and enzyme activity expressed in terms of OD per bead.
  • the results obtained are shown in the second column in Table 6 below. Each data point is the mean value for three separately measured beads.
  • Example IX Competitive Inhibition Assay for HCG
  • a competitive inhibition assay for determination of hCG was carried out substantially as described in Example VIII. but using liposomes from Example VI having surface-bound anti-hCG and encapsulated alkaline phosphatase.
  • the assays were carried out in a 20 mM phosphate buffer. 150 mM NaCl. pH 7.4. the analyte being initially introduced to one of the concentrations shown at the left in Table 1 below in an initial analyte volume of 0.05 ml.
  • To each of the ten samples was added 0.3 nmole of liposome reagent in 0.2 ml. Each mixture was preincubated at 25°C for 30 min. 3 surface-reagent beads were added. The reaction mixtures were incubated at 25°C for 1 hr.
  • Example III After removing the reaction fluid by aspiration, the beads were washed four times with reaction buffer and surface-bound enzyme was assayed as in Example III. Each data value shown in Table 7 is the mean value for three separately determined beads. The results show a substantially linear relationship between the log of the analyte concentration and the enzyme activity measured. in the analyte range between about 16 and 3.200 IU/ml.
  • Mouse monoclonal antibodies specific against either the ⁇ or ⁇ subunits of hCG were obtained from Bioclone Australia Pty (Marrickville, Australia). Rabbit polyclonal anti-hCG antibody was obtained from New England Immunology (Cambridge. MA).
  • a solid support reagent composed of cellulose acetate beads having surface-bound antibody against the subunit was prepared as in Example III. reacting cyanuric chloride-activated substrate with a solution of 1 mg/ml antibody.
  • Liposomes having encapsulated alkaline phosphatase enzyme and either surface-bound monoclonal antibodies against the ⁇ -subunit of hCG or polyclonal anti-hCG antibodies were prepared substantially as in Example VI.
  • Two samples containing each of the increasing concentrations of hCG. in a sample volume of 0.05 ml were prepared.
  • To each sample was added 0.2 ml of the monoclonal- or polyclonal-antibody liposomes to a final liposome concentration of about 0.3 nmole PL/ml. and 3 beads of the surface reagent.
  • Each sample was incubated at 25°C for 1 hr in a final assay mixture containing 50 mM phosphate buffer. pH 7.4.
  • the sample beads were washed 4 times with the assay buffer and each bead suspended in 0.5 ml of the enzyme assay medium employed in Example III.

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Abstract

Le système d'analyse pour la détermination d'un analyte comprend des liposomes ayant, liées en surface, des molécules de liaison relatives à l'analyte et des molécules reporters piégées, ainsi qu'un réactif de surface d'acétate de cellulose ayant des molécules de surface adaptées pour lier les liposomes au réactif proportionellement à la quantité d'analyte présente. Le réactif est prépare selon l'invention en traitant un substrat d'acétate de cellulose avec une base pour former une enveloppe hydrophile de groupe hydroxyle sur la surface du substrat, en faisant réagir le substrat modifié avec un réactif bifonctionnel dans un solvant organique, et en couplant, dans une solution aqueuse, les molécules de surface au substrat activé.
PCT/US1986/002233 1985-10-22 1986-10-22 Systeme d'immunoanalyse a base de liposomes en phase solide WO1987002778A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006293A2 (fr) * 1987-02-17 1988-08-25 Lewis Donald G Immunoanalyse liposomique pour detecter des antigenes et des immunoglobulines specifiques des antigenes
US4855240A (en) * 1987-05-13 1989-08-08 Becton Dickinson And Company Solid phase assay employing capillary flow
WO2007101661A1 (fr) 2006-03-09 2007-09-13 F.Hoffmann-La Roche Ag Dosage d'anticorps antimédicament
WO2008031532A1 (fr) 2006-09-12 2008-03-20 F. Hoffmann-La Roche Ag Essai d'anticorps anti-médicament
WO2009077127A1 (fr) 2007-12-15 2009-06-25 F. Hoffmann-La Roche Ag Dosage de distinction
WO2010072384A1 (fr) 2008-12-22 2010-07-01 F. Hoffmann-La Roche Ag Anticorps anti-idiotype dirigé contre un anticorps dirigé contre le peptide amyloïde β
WO2012022774A1 (fr) 2010-08-19 2012-02-23 Roche Diagnostics Gmbh Dosage pour la mesure d'anticorps se liant à un anticorps monoclonal thérapeutique
WO2013092611A2 (fr) 2011-12-19 2013-06-27 F. Hoffmann - La Roche Ag Procédé pour la détection d'un partenaire de liaison libre d'un liant multispéficique
WO2013113663A1 (fr) 2012-02-01 2013-08-08 F. Hoffmann-La Roche Ag Procédé de détection d'un partenaire de liaison d'un liant multispécifique
WO2014009474A1 (fr) 2012-07-13 2014-01-16 F. Hoffmann-La Roche Ag Procédé de détection d'un liant multispécifique
US8809005B2 (en) 2006-11-21 2014-08-19 Hoffmann-La Roche Inc. Conjugate and its use as a standard in an immunoassay
US11340234B2 (en) 2014-11-05 2022-05-24 Hoffmann-La Roche Inc. Method for the determination of anti-drug antibodies against an effector function suppressed human or humanized drug antibody

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3824150A (en) * 1967-07-14 1974-07-16 Nat Res Dev Enzyme bound to polymeric sheet with a triazine bridging group
US4012571A (en) * 1971-02-11 1977-03-15 Peter Duncan Goodearl Dean Enzyme separation
US4033822A (en) * 1975-11-07 1977-07-05 Gregor Harry P Enzyme-coupled ultrafiltration membranes
US4090022A (en) * 1976-04-22 1978-05-16 Purdue Research Foundation Porous cellulose beads
US4195128A (en) * 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4357311A (en) * 1980-10-03 1982-11-02 Warner-Lambert Company Substrate for immunoassay and means of preparing same
US4483929A (en) * 1982-05-03 1984-11-20 Liposome Technology Incorporated Liposomes with glycolipid-linked antibodies
US4514505A (en) * 1982-05-21 1985-04-30 The Trustees Of Columbia University In The City Of New York Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2502786B1 (fr) * 1981-03-24 1985-06-21 Stallergenes Laboratoire Procede de fixation d'antigenes et d'anticorps sur support de polysaccharides, et utilisation du produit ainsi obtenu pour les dosages immunologiques
US4666866A (en) * 1983-04-19 1987-05-19 Krauth Gary H Immunoassay for antigens employing supported binder

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3824150A (en) * 1967-07-14 1974-07-16 Nat Res Dev Enzyme bound to polymeric sheet with a triazine bridging group
US4012571A (en) * 1971-02-11 1977-03-15 Peter Duncan Goodearl Dean Enzyme separation
US4033822A (en) * 1975-11-07 1977-07-05 Gregor Harry P Enzyme-coupled ultrafiltration membranes
US4090022A (en) * 1976-04-22 1978-05-16 Purdue Research Foundation Porous cellulose beads
US4195128A (en) * 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4357311A (en) * 1980-10-03 1982-11-02 Warner-Lambert Company Substrate for immunoassay and means of preparing same
US4483929A (en) * 1982-05-03 1984-11-20 Liposome Technology Incorporated Liposomes with glycolipid-linked antibodies
US4514505A (en) * 1982-05-21 1985-04-30 The Trustees Of Columbia University In The City Of New York Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0243471A4 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006293A3 (fr) * 1987-02-17 1988-09-22 Donald G Lewis Immunoanalyse liposomique pour detecter des antigenes et des immunoglobulines specifiques des antigenes
WO1988006293A2 (fr) * 1987-02-17 1988-08-25 Lewis Donald G Immunoanalyse liposomique pour detecter des antigenes et des immunoglobulines specifiques des antigenes
US4855240A (en) * 1987-05-13 1989-08-08 Becton Dickinson And Company Solid phase assay employing capillary flow
KR101047207B1 (ko) * 2006-03-09 2011-07-06 에프. 호프만-라 로슈 아게 항-약물 항체 분석
WO2007101661A1 (fr) 2006-03-09 2007-09-13 F.Hoffmann-La Roche Ag Dosage d'anticorps antimédicament
WO2008031532A1 (fr) 2006-09-12 2008-03-20 F. Hoffmann-La Roche Ag Essai d'anticorps anti-médicament
US8809005B2 (en) 2006-11-21 2014-08-19 Hoffmann-La Roche Inc. Conjugate and its use as a standard in an immunoassay
EP2573568A1 (fr) 2007-12-15 2013-03-27 F. Hoffmann-La Roche AG Analyse de distinction
US8227195B2 (en) 2007-12-15 2012-07-24 Hoffman-La Roche Inc. Distinguishing assay
WO2009077127A1 (fr) 2007-12-15 2009-06-25 F. Hoffmann-La Roche Ag Dosage de distinction
US8530176B2 (en) 2007-12-15 2013-09-10 Hoffmann-La Roche Inc. Distinguishing assay
US8614297B2 (en) 2008-12-22 2013-12-24 Hoffmann-La Roche Inc. Anti-idiotype antibody against an antibody against the amyloid β peptide
WO2010072384A1 (fr) 2008-12-22 2010-07-01 F. Hoffmann-La Roche Ag Anticorps anti-idiotype dirigé contre un anticorps dirigé contre le peptide amyloïde β
KR101777899B1 (ko) 2008-12-22 2017-09-13 에프. 호프만-라 로슈 아게 아밀로이드 β 펩티드에 대한 항체에 대한 항-이디오타입 항체
US9081016B2 (en) 2010-08-19 2015-07-14 Roche Diagnostics Operations, Inc. Assay for measurement of antibodies binding to a therapeutic monoclonal antibody
WO2012022774A1 (fr) 2010-08-19 2012-02-23 Roche Diagnostics Gmbh Dosage pour la mesure d'anticorps se liant à un anticorps monoclonal thérapeutique
WO2013092611A2 (fr) 2011-12-19 2013-06-27 F. Hoffmann - La Roche Ag Procédé pour la détection d'un partenaire de liaison libre d'un liant multispéficique
WO2013113663A1 (fr) 2012-02-01 2013-08-08 F. Hoffmann-La Roche Ag Procédé de détection d'un partenaire de liaison d'un liant multispécifique
US9945869B2 (en) 2012-02-01 2018-04-17 Hoffmann-La Roche Inc. Method for the detection of a binding partner of a multispecific binder
US10761097B2 (en) 2012-02-01 2020-09-01 Hoffmann-La Roche Inc. Method for the detection of a binding partner of a multispecific binder
WO2014009474A1 (fr) 2012-07-13 2014-01-16 F. Hoffmann-La Roche Ag Procédé de détection d'un liant multispécifique
US11340234B2 (en) 2014-11-05 2022-05-24 Hoffmann-La Roche Inc. Method for the determination of anti-drug antibodies against an effector function suppressed human or humanized drug antibody

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EP0243471A4 (fr) 1989-07-11

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