Classifications

• • 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
  • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
• • 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
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • 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
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• • 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
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
  • G01N33/555—Red blood cell

Definitions

• the present invention relates to a lipid-vesicle-surface assay reagent, and to enzyme immunoassay methods using such a reagent.
• a variety of methods for determining the presence or concentration- of biochemical analytes is known.
• the analyte to be assayed typically is one which plays an important role in biochemical processes.
• Low molecular weight substances, such as peptide and steroid hormones, vitamins and the like, and high molecular weight substances such as carbohydrates and proteins are commonly assayed analytes.
• analyte assay techniques are based on a reaction between the analyte and an anti-analyte capable of binding the analyte with high affinity and specificity.
• Typical analyte anti-analyte binding pairs include antigen-antibody, immunoglobulin-protein A, carbohydrate-lectin, biotin-avidin, hormone-hormone receptor protein and complementary oligo- and polynucleotide strands.
• ligand and ⁇ antiligand will be used herein to designate the opposite binding members in such a binding pair.
• enzyme immunoassays provide a number of advantages in sensitivity, low cost and simplicity.
• an enzyme-ligand reagent is reacted in the presence of a ligand or ligand-like analyte with a solid support having anti-ligand binding sites carried on its surface, wherein the analyte and ligand-enzyme reagent compete for binding to the solid support.
• a ligand analyte is capable of binding both to anti-analyte binding sites on a solid support, and to an anti-ligand in a anti-ligand-enzyme reagent, to couple the reagent to the support in a sandwich fashion.
• the amount of analyte present is determined by separating the liquid and solid (support) phases, and measuring the enzyme activity associated with one or both phases.
• the enzyme activity associated with the support is inversely proportional to the concentration of analyte present.
• a direct relationship between the amount of analyte and the enzyme activity associated with the solid support is observed where the analyte functions to join the reagent to the support by sandwiching.
• the reagent used in an enzyme immunoassay test includes an enzyme molecule covalently coupled to a ligand or anti-ligand molecule to form a bi olecular pair.
• This type of reagent has limited sensitivity, inasmuch as each reagent binding event is ⁇ reported" by one enzyme molecule only.
• This limitation has prevented general application of the enzyme immunoassay technique to cell typing based on the detection of selected cell surface antigens, except in cases where antigen surface concentrations are quite large.
• Another limitation is that the bimolecular reagent must be formed from a relatively pure ligand preparation.
• An enzyme immunoassay reagent composed of lipid vesicles coated with ligand molecules and encapsulating enzymes within the interior vesicle spaces has been proposed in the prior art.
• a reagent of this type may be relatively expensive to manufacture due to the recognized problems of encapsulating enzymes within liposo es efficiently. Further, many enzymes appear to undergo loss of activity during encapsulation (reference 1).
• the encapsulating vesicles must be lysed before enzyme activity associated with the vesicles can be • measured.
• Complement has been used for lysing lipid vesicles, but this method often lacks reproducibility due to complement inactivation on storage.
• Detergent lysis has been used, but this approach may be unsuitable for applications —e.g., cell typing— where the cell "support" to which the vesicles are bound is itself susceptible to detergent lysis.
• a more specific object of the present invention is to provide such a reagent composed of particles which have been prepared to have a highly mobile, or fluid surface array of enzyme and ligand molecules.
• Yet another object of the invention is to provide such a reagent composed of lipid vesicles which, in the .05 to 10 micron diameter range, each has an average of at least about 15 ligand molecules and up to several thousand enzyme molecules attached to its surface.
• Another important object of the invention is to provide, for use in an enzyme immunoassay method, a reagent capable of giving a high signal-to-noise ratio in the method.
• a related object of the invention is to provide an enzyme immunoassay kit containing such a reagent, and a separable support having surface binding site molecules which may be directly bound to the support or may be carried on lipid vesicles which are themselves attached to the support surface.
• Still another object of the invention is to provide such a reagent which can be used in a wide variety of different enzyme immunoassay methods.
• the reagent of the invention is composed of lipid vesicle particles having a highly mobile, or fluid surface array of ligand and enzyme molecules.
• the particles are preferably lipid vesicles in the 0.05 to 10.0 micron diameter size range, and include an average of at least about 15 ligand molecules and up to several thousand enzyme molecules bound to each vesicle surface.
• the ligand molecules may include one or more substantially pure ligand species, or may include impure mixtures thereof.
• the method of the invention includes reacting the reagent with a separable support to produce -5-
• the invention also contemplates an assay kit including a separable support and the reagent.
• the assay reagent of the present invention is composed of closed lipid vesicles, each having attached to its outer surface a laterally mobile array of enzyme and ligand molecules.
• the lipid vesicles take the form of lipid bilayer structures encapsulating an aqueous interior region, such structures also being referred to as liposomes.
• the properties and methods of preparation of lipid vesicles have been detailed in the literature. The reader is referred particularly to above-numbered references 1 and 2, and references cited therein, for a comprehensive discussion of the topic. hat will be described herein are preferred methods of preparing liposomes used in forming the reagent of the invention, and liposome properties which contribute to the advantages of the invention.
• Lipid vesicles are prepared from lipid mixtures which typically include phospholipids and sterols.
• a list of phospholipids used typically in liposome preparations is given on page 471 of reference 1.
• One consideration which determines the choice of lipids used is the degree of fluid mobility and lipid packing density which is desired in the vesicles formed. As reported in a number of literature reports, these characteristics can be varied according to the lengths and degree of saturation of the aliphatic chains in the lipids, and the ratio of sterol to aliphatic chain
• lipids used The significance of surface fluid mobility in the the vesicle reagent of the invention will be seen below. Packing density characteristics are important to the success of reactions used to attach ligand and enzyme molecules covalently to the vesicle surfaces.
• the vesicle lipid composition is also selected to produce a requisite number of specific lipid head groups through which the surface-bound reagent components can be coupled to the vesicles.
• the head groups, or necessary modifications thereof, may be formed in the prepared liposomes, or in the individual lipids before incorporation into the liposomes. Examples of lipids used in preferred coupling reactions will be discussed below.
• the number of and type of polar lipid groups may also be selected to produce a desired charge distribution on the lipid vesicles at a selected pH and ionic strength.
• the charge distribution may affect the relative reactivities of enzyme and ligand molecules in their coupling to lipid vesicles, as will be seen in Example III, and is an important feature in minimizing non-specific binding of the reagent vesicles to charged solid supports, as will be discussed.
• a typical lipid composition used in preparing lipid vesicles for the reagent of the invention preferably includes between about 10 and 50% cholesterol or other sterol, between about 2 and 50% of glycolipid or phospholipid to which the enzyme and ligand molecules of the reagent can be individually coupled, with the remainder lipid composed of a neutral phospholipid, such
• OMPI as phosphatidylcholine, or a phospholipid mixture.
• Charged lipids such as phosphatidylserine, phosphatidic acid, glycolipids and charged cholesterol derivatives such as cholesterol hemisuccinate or cholesterol sulfate may be included to produce a desired surface charge in the lipid vesicles.
• the lipid vesicles may be formed by one of a variety of methods discussed particularly in reference 1.
• Multilamellar vesicles that is, vesicles composed of a ' series of closely packed bilayer lamellae— can be prepared by drying a mixture of lipids in a thin film and hydrating the lipids with an aqueous buffer.
• the size and number of lamellae in the formed lipid vesicles can be controlled, within limits, by varying the hydration time and amount of agitation used in hydrating the lipids. Where desirable, vigorous agitation, brief sonication or extrusion through polycarbonate membranes can be employed to obtain smaller and more uniformly sized multilamellar vesicles.
• Small unilamellar vesicles having diameters of about 0.05 micron or less can be formed by sonicating a suspension of large multilamellar vesicles, either by probe or bath sonication.
• Another technique for producing small unilamellar vesicles involves the removal of detergent from a detergent-phospholipid mixture by dialysis. Typical detergents include cholate and deoxycholate.
• An alternative method for the preparation of small unilamellar vesicles that avoids both sonication and detergents employs an ethanol injection step in which lipids dissolved in ethanol are rapidly injected into a buffer solution.
• a similar technique in which phospholipids dissolved in ether-containing solvents has been used to produce large unilamellar vesicles with a generally heterogenous size distribution.
• unilamellar vesicles referred to as reverse phase evaporation
• a desired composition of lipids is dissolved in a suitable organic solvent such as diethyl ether, isopropyl ether, or a solvent mixture such as isopropyl ether and chloroform (1:1).
• An aqueous solution is added directly to between about 3 and 6 volumes of the lipid-solvent mixture and the preparation is sonicated for a brief period to form a homogeneous emulsion.
• the organic solvent, or solvent mixture is removed under reduced pressure, resulting in the formation of a viscous, gel-like intermediate phase which spontaneously forms a liposome dispersion when residual solvent is removed by evaporation under reduced pressure.
• the size of the resulting vesicles may be varied according to the amount of cholesterol included in the lipid mixture.
• references 1 or 2 for further details concerning the reverse phase evaporation technique.
• the lipid vesicles prepared may be obtained in a defined size range by various techniques. Methods for reducing size heterogeneity in small unilamellar vesicles by gel filtration and ultra centrifugation have been described. A method of reducing " the size and the size heterogeneity of lipid vesicles by extrusion through polycarbonate filters having selected pore sizes is described in reference 2. The latter method is advantageous because of its simplicity and because essentially all of the vesicles are recovered, the larger ones being converted to desired-sized smaller vesicles by passage through the filter.
• lipid vesicles having a desired size range, morphology, deformability, fluid mobility and surface charge and reactivity characteristics may be prepared by proper selection of lipid components and preparative techniques.
• Enzyme and Ligand Coupling to Lipid Vesicles This section is concerned with techniques used to couple ligand and enzyme molecules covalently to surface lipids in lipid vesicles.
• the ligand molecules in the reagent function to bind the reagent to anti-ligand binding sites on a separable support.
• the term "ligand” refers broadly to either species in a binding pair composed of a target molecule having one or more specific epitopic features, and a target-binding molecule which recognizes such features to bind the target molecule specifically and with a high affinity.
• Anti-ligand refers to the other of the two species in the binding pair.
• the binding pairs which are contemplated by the present invention are antigen-antibody, immunoglobulin-protein A, carbohydrate-lectin, biotin-avidin, hormone-hormone receptor protein and complementary nucleotide strands. More generally, the ligand may include any fragment or portion of a ligand molecule which is capable of participating with the opposite member of the pair in specific, high affinity binding.
• the binding ligand may include the antigen binding F(ab')2 or Fab' fragments.
• the target ligand may include only the Fc.immunoglobulin fragments. According to an important feature of the present invention, relatively impure ligand mixtures containing as little as 0.5 to 20 mole percent of specific ligand molecules may be employed in the vesicle reagent.
• the enzyme in the reagent includes an enzyme which can function to produce a measurable enzyme activity in the presence of suitable -substrate(s) and necessary cofactor(s) with the enzyme covalently to the outer surface of a lipid vesicle.
• the enzyme ⁇ can be obtained in pure or near-pure form and is relatively stable on storage in solution, or is resistant to freezing or lyophilization. For most applications, enzymes whose activity can be expressed by an easily detectable color change will be preferred.
• oxidoreductases typified by luciferase, glucose oxidase, galactose oxidase and catalase
• hydrolases typified by various types of phosphatases
• glycoside hydrolases such as beta-galactosidase
• peptidases peptidases
• beta-galactosidase derived from a bacterial source.
• the enzyme is available in purified form with high specific activity; (2) the enzyme contains free thiol groups that can be joined to reactive lipids without affecting the enzyme activity; and (3) both fluorogenic and chromogenic substrates are available.
• the relatively low molecular weight of the enzyme with respect to lipid vesicles allows for the attachment of a relatively large number of enzyme molecules on each vesicle, as will be seen below.
• Two or more enzyme species may be attached to the lipid vesicles in accordance with the present invention.
• the plural enzymes may function independently, or cooperatively, as where the product generated by one enzyme is used as the substrate by another.
• the first method involves Schiff-base formation between an aldehyde group on the lipid or molecule to be coupled, and a primary amino group on the other of the two reactants.
• the aldehyde group is preferably formed by periodate oxidation.
• the coupling reaction after removal of the oxidant, is carried out in the presence of a reducing agent.
• the nonlipid molecule being coupled may be oxidized, more commonly it is the lipid group which is the aldehyde precursor since periodate treatment inactivates many proteins.
• Typical aldehyde-lipid precursors include lactosylceramide, trihexosylceramide, galactocerebroside, phosphatidylglycerol, phosphatidylinositol and gangliosides.
• the vesicles are oxidized by periodate for a period sufficient to produce oxidation of a majority of the oxidizable lipid groups, and thereafter the vesicles are separated from the periodate by column gel filtration.
• Aldehyde groups on the vesicle surfaces are conjugated with a primary amine, such as a lysine group in a protein, to form a Schiff*s
• oxidized lipid vesicles at a concentration of between about 5 and 10 micromoles of total lipid are mixed in 1 ml with 10 to 30 milligrams of protein at an alkaline pH. The reaction is carried out for about 2 hours at room temperature. For conjugation reduced with sodium cyanoborohydride, the reaction typically is carried out over longer reaction times. The reader is referred to reference 3 for additional details.
• lipid vesicles prepared by reverse phase evaporation and extruded through a 0.2 micron pore-size polycarbonate membrane, up to about 200 micrograms of immunoglobulin G (IgG) per micro ole of lipid vesicle lipid can be attached to the vesicle surfaces by the above method. Based on a calculated number of about 1.2 x 10 12 vesicles per micromole of vesicle lipid, this conjugation ratio corresponds to about 600 IgG molecules per lipid vesicle. Studies conducted in the support of the present application indicate that correspondingly smaller molecules can be coupled to lipid vesicles in correspondingly larger numbers.
• IgG immunoglobulin G
• Fab* antibody fragments per lipid vesicle in the 0.2 micron diameter range
• the method has wide applicability, due to the general availability of primary amine groups in proteins and other biomolecules which can be reacted with oxidation-produced aldehydes in selected lipids.
• a second general coupling technique is applicable to thiol-containing molecules, involving formation of a disulfide or thioether bond between a vesicle lipid and the molecule attached.
• the technique is particularly useful for coupling F(ab')2 and Fab* antibody fragments to lipid vesicles.
• phosphatidylethanolamine is modified to provide a pyridyldithio derivative which can react with an exposed thiol group in a protein or other biomolecule.
• the reader is referred to reference 4 for a detailed discussion of reaction conditions used in the method.
• the thioether coupling method which is described in detail in reference 5, is carried out by incorporating in the lipid vesicles a small proportion of a sulhydryl-reactive phospholipid derivative, such as N-(4 (p-maleimidophenyl) butyryl) phosphatidylethanolamine (MPB-PE) .
• MPB-PE N-(4 (p-maleimidophenyl) butyryl) phosphatidylethanolamine
• the lipid vesicles are reacted with a thiol-containing protein to form an essentially irreversible thioether coupling between the protein thiol group and the MPB-PE maleimide group.
• the requisite protein thiol group may be endogenous to the protein or may be introduced on the protein by amino-reactive thiol groups according to known methods. Coupling ratios of up to about 350 mg of sulfhydryl containing protein per micromole of lipid vesicle
• enzyme or ligand molecules can be separately and individually attached to lipid vesicles by first coupling the molecules covalently to free lipids dispersed in a detergent solution.
• the lipid-enzyme or lipid-ligand couples are then incorporated into lipid vesicles, either during vesicle formation or by diffusion into preformed vesicles according to known techniques.
• the ligand itself may contain an
• OMPI endogenous hydrophobic region for example, a hydrophobic stretch of a ino acids— by which the ligand can be incorporated into the surface of a lipid vesicle.
• human transplantation antigens can be attached to egg lecithin vesicles by anchoring hydrophobic peptide regions in the antigens to the vesicles (reference 6).
• immumoglobulin or immunoglobulin fragment ligand molecules can be attached to lipid vesicles through pair-specific binding to anti-immunoglobulin antibodies, or fragments thereof, or to protein A covalently attached to the vesicles.
• the enzyme and ligand molecules may be coupled to (or incorporated into) lipid vesicles either sequentially, in separate coupling reactions, or in simultaneous reactions. Sequential coupling is indicated where different reactions are used to couple enzyme and ligand molecules to the lipid vesicles, or where the relative conjugation reactivities of the two species is difficult to control. The latter problem may arise, for example, where the reactivity of either species varies significantly during the reaction period.
• ligand or a ligand-containing mixture is first reacted by an above-described coupling reaction, with vesicles to produce a desired vesicle surface concentration of the analyte-specific ligand.
• the vesicles are separated from unreacted ligand molecules, then reacted with enzyme molecules.
• One advantage of the present invention is that the vesicles may be easily separated from the unreacted solution components of the coupling reaction(s) by centrifugation, facilitating intermediate purification steps that may be required during reagent preparation.
• binding affinity generally increases as the surface concentration of ligand molecules increases from an average of about 10-15 molecules per vesicle up to about 50-100 ligand molecules per vesicle (of average diameter of about 0.2 microns).
• the number of ligand molecules bound to the lipid vesicles is increased, particularly where the ligand fraction coupled to the vesicle is relatively impure, the number of sites available for enzyme attachment to the vesicles is reduced.
• the vesicles can easily accomodate 100 or more ligand molecules and several times that number of enzyme molecules.
• Binding studies done in support of the present application indicate that at least two, and probably three or more reagent ligand molecules must bind specifically to the separable support binding sites in order to produce stable attachment of the vesicle to the support.
• the surface concentration of ligand molecules required to promote such stable multi-site vesicle binding to a macromolecular support can be quite low, on the order of about 15 molecules per vesicle. This feature is believed to be due to in part to the highly mobile, or fluid nature of the lipid-bound surface molecules on the reagent vesicle surfaces. Diffusion constants of the order of 10-H to 10-9 cm2/sec for
• Binding efficiency may be further enhanced where the binding sites on the solid support are themselves carried on lipid vesicles.
• the combined mobility of the ligand molecules on the reagent lipid vesicles and the binding site molecules on the separable support lipid vesicles would facilitate multi-site binding at low ligand and anti-ligand (binding site) surface concentrations.
• Another important advantage of the reagent invention is the relatively high surface packing of covalently attached molecules which is achievable in the reagent vesicles.
• a 0.2 micron vesicle having an average of about 50 Fab' fragments carried on its outer surface can carry nearly 100 times that number of an enzyme having about a 50,000 molecular weight.
• each vesicle can still accomodate up to several hundred or more enzyme molecules, producing a vesicle whose enzyme to ligand molar ratio is still substantially greater than one.
• Other advantages of the instant reagent in an enzyme immunoassay will be considered below.
• the method of the invention comprises reacting a liposome surface reagent with a separable support carrying analyte-related binding-site molecules.
• the reagent binds to the support in proportion to the amount of analyte present.
• separable support refers to any support structure capable of being readily separated — for example, by differential centrifugation, precipitation or electrophoretic separation — from analyte and vesicle reagent components not bound to the support. Another feature of the support is that binding-site molecules can be attached to its surface.
• the separable support may include a water-insoluble solid support, such as one formed of glass, cellulose, agarose, polystyrene and the like. Macromolecular tissue homogenate structures, intact cells, and cell membrane structures are other contemplated supports. Also as detailed above, the support may include surface-bound lipid vesicles to which the support binding site molecules are attached.
• the analyte-related binding site molecules on the support are selected to bind specifically to the reagent ligand molecules, or to the analyte molecules, or to both, depending on the type of enzyme immunoassay method, as considered below.
• the binding site molecules may be adsorbed to the solid support, or may be covalently attached thereto by means of a suitable coupling reaction which may involve the use of conventional linking agents such as glutaraldehyde.
• solid supports are known to have surface irregularities, such as cavities or crevices, which may be inaccessible to reagent liposome particles of the type contemplated herein.
• the fewer binding sites on the support available for liposome binding can result in a proportionate reduction in assay sensitivity.
• the solid support may have surface properties which tend to promote non-specific attachment of the liposome reagent, leading to a
• glass surfaces for instance glass tubes or controlled-pore glass beads
• glycerol activated with carbonyldiimidazole
• amino-glass is converted into pyridyl dithio glass by reaction with N-succinimidyl 3-(2-pyridyldithio) propionate.
• the pyridyl dithio glass is then reduced with dithiothreitol or 2-mercaptoethanol to yield a glass surface with thio functions.
• the lipid vesicles prepared to include N-(3-(2-pyridyldithio)propionyl)phosphatidylethanolamine (PDP-PE) synthesized according the method described in reference 4, are reacted with the thio glass at a pH between about 7.0 and 8.5.
• the disulfide bond which forms between the glass and the vesicles can be cleaved fay mild reduction, for example with dithiothreitol at low pH.
• Irreversible attachment of vesicles to a glass support may be achieved by reacting the thio glass with lipid vesicles prepared to contain MPE-PE, as described in reference 5, to form a thioether linkage.
• lipid vesicles may be attached to a solid support noncovalently through specific, high affinity ligand/anti-ligand binding.
• avidin molecules are attached covalently to a solid support using conventional methods.
• Lipid vesicles prepared to contain biotinylated surface lipids then bind with high affinity to the support.
• Binding site molecules may be attached to, incorporated into, or formed with the lipid vesicles, according to above-described techniques. Attachment of binding site molecules to lipid vesicles carried on a solid support may increase the accessibility of the liposome reagent to the binding sites on the solid support.
• the vesicles to which the binding site molecules are attached may themselves be prepared to have a selected surface charge character, with respect to the reagent liposome particles, for enhancing specific binding, and reducing non-specific binding, between the liposome reagent and the solid support. Because the binding site molecules are themselves supported in highly mobile vesicle-surface arrays, reagent binding to the solid support may be facilitated.
• the reagent vesicles carry analyte or analyte-like ligands which compete with analyte in solution for binding to anti-analyte binding sites on the solid support.
• the analyte, and the ligand attached to the liposome reagent may be a target-type antigen which compete for binding to an anti-analyte attached to the solid support, or the analyte may be a binding protein which competes with binding proteins on the reagent for binding to target-type binding sites on the solid support.
• the amount of liposome reagent binding to the solid support varies inversely with the amount of analyte present.
• the assay reaction is carried out in a suitable reaction medium which may include a biological specimen fluid, such as serum, containing the analyte.
• a biological specimen fluid such as serum
• the pH of the reaction medium is one which is compatible with ligand/anti-ligand binding reactions, and preferably between about 5 and 9. More specifically, the pH and/or ionic strength of the reaction medium may be adjusted to achieve a desired charge interaction between the liposome reagent and the solid support. Generally it can be said that the greater the charge repulsion between the reagent and the support, the less the reagent will bind both" specifically and nonspecifically, to the support.
• the reaction medium may also be adjusted to have a specific gravity which approximates the bouyant density of the reagent particles. Typically, this can be achieved in a medium having a specific gravity between about 1.0 and 1.2.
• the adjustment in specific gravity by reducing the tendency of the vesicles to float or sink in the medium, promotes the requisite contact between the vesicles and the solid support.
• Sensitivity in the assay requires reacting a defined amount of solid support with a known, selected amount of the liposome reagent. Ith too little liposome reagent added to the reaction mixture, the binding sites on the solid support can accomodate a substantial quantity of bound analyte without any observed analyte-dependent displacement of the liposome reagent from the support, with too much liposome reagent added, excess unbound liposome reagent competes with the analyte for binding to any displaced binding sites on the support.
• the assay background also tends to be high with too much liposome reagent, due to nonspecific binding to the solid support and excess reagent in the liquid phase. In most assays, the optimal amount of liposome reagent is determined by titrating a given amount of solid support with liposome reagent to an end point which just indicates saturation or near saturation of the support binding sites.
• the method can be carried out as a single reaction in which the solid support, a defined amount of liposome reagent, and the analyte are coincubated for a period sufficient to produce binding equilibrium among the reaction components.
• Typical reaction times range from about 5 minutes to several hours, at temperatures ranging preferably from about room temperature up to 37°C or somewhat higher.
• the assay may be performed as a two-step reaction in which the analyte is reacted first with the solid support alone, after which the separated solid support is reacted with the liposome reagent.
• the two-step test may be advantageous where the volume of original solution to be assayed is quite large, or where that solution contains substrates or inhibitors of the liposome reagent enzyme.
• the two-step reaction also has the advantage that the second reaction in which the liposome particles bind to the solid support can be carried out in a selected reaction medium having a desired pH, ionic strength and specific gravity.
• the solid support Upon completion of the assay reaction, the solid support is separated from the liquid phase of the reaction medium, including the unbound suspended liposomes, and the support or the liquid phase, or both. are assayed for enzyme activity. To reduce the level of non-specifically bound liposome reagent, the separated support is preferably washed one or more times with a washing solution whose pH and ionic strength act to increase charge repulsion between the solid support and the liposome reagent.
• the solid support carries an analyte or analyte-like binding molecule which competes with the analyte for binding to the ligand on the liposome reagent.
• the analyte, and the binding-site molecules on the support may be either a target-type antigen which compete for binding to a target-binding ligand on the liposome, or the analyte may be a target-binding molecule which competes with the solid support for binding to an antigen-like ligand on the liposome reagent.
• Various considerations relating to the pH, ionic strength and specific gravity of the reaction solution which have been discussed above are applicable to the instant method. Likewise the procedure used for optimizing the amount of liposome reagent added to a given amount of solid support is similar to that already discussed.
• a third general assay type is a sandwich technique in which the liposome reagent is bound to a support through a multivalent analyte.
• the assay is preferrably performed as a two-step method in which analyte is first reacted with the support, after which the separated support is reacted with the liposome reagent.
• the analyte may be either an antibody or an antigen, with the solid support and liposome reagent each carrying an opposite.binding pair of the analyte.
• One advantage of this method is that the amount of liposome reagent bound to the solid support is directly- proportional to the amount of analyte present. The method can thus be used to detect very small quantities of analyte.
• the liposome reagent is bound to the solid support through analyte sandwiching, the amount of liposome added to a given quantity of solid support can be less than the saturating or near saturating amounts required in the previously described _ tests. In turn, the lower concentration of liposome reagent leads to an improved signal-to-noise ratio in the test.
• an immunoassay kit which includes the liposome reagent of the invention. Also included in the kit is a separable support of the type described above, having surface-attached, analyte-related binding site molecules, which may be directly bound to the support or attached to the support surface through lipid vesicles, as described above.
• the binding site molecules may be analyte or analyte-like molecules which compete with the analyte for binding to anti-analyte ligand molecules in the surface reagent.
• the binding site molecules may be an anti-analyte species, where the analyte and analyte-like reagent ligand compete for binding to the support, or where the reagent is bound to the support in sandwich fashion through the ligand.
• the assay methods just described are intended for detecting the presence or concentration of a free analyte in a sample solution.
• the reagent is used in an enzyme immunoassay for determining the presence or concentration of cell-specific surface antigen analytes.
• the separable support includes a biological cell, and more specifically, a cell membrane
• OMP whose outer surface carries the antigen.
• Typical analytes include blood-type specific antigens carried on the surface of blood cells, species and strain specific surface antigens carried on the surface of various animal tissues, and surface antigens characteristic of particular cellular transformation states in various tissues or in tissue culture.
• the analyte may include anti-cell surface antigen antibodies attached to the cell by incubation with the free antibody.
• the- cell sample to be assayed is added to defined amounts of liposome reagent having analyte-recognition molecules.
• liposome reagent having analyte-recognition molecules.
• the cells and bound liposome reagent particles may be separated from the unbound liposomes which remain suspended in solution by differential centrifugation. The separated cells are then washed to remove non-specifically bound liposomes and the enzyme activity associated with the washed cells is determined.
• antigen-bearing supports such as viral particles, spores, tissue structure, or other suspendible particulate matter which can be separated readily from unbound reagent and soluble components —for example by differential centrifugation or precipitation— are also contemplated herein.
• the liposome reagent of the invention contributes to the improved signal-to-noise ratio achievable in various types of enzyme immunoassay tests.
• a high signal level is achieved by virtue of the large number of enzyme molecules which "report" each binding event in the assay, S seen, up to several thousand enzyme molecules C an be attached to a vesicle which binds to a support through a small number of binding sites on the support.
• the fact that the enzyme whose activity is being measured is bound to a liposome surface may further enhance the signal level.
• Several enzymes are known to have increased activity in an immobilized state, a phenomenon thought to be related to favorable surface reaction kinetics. Immobilized enzymes are often less susceptible to inactivation as well.
• the noise level in the assay methods described is reduced by limiting non-specific binding of the liposome reagent to a separable support. This may be accomplished, according to the invention, by reacting the liposome reagent with a solid support under pH and ionic strength conditions which favor specific binding between the two, followed by exposing the separated support to a washing medium which removes non-specifically bound liposomes through a charge repulsion effect.
• the improved signal-to-noise ratio is observed where the ligand molecules in the reagent represent only a small fraction of the total non-enzyme molecules carried on the reagent vesicles, and where the liposome reagent carries two or more distinct types of ligand molecules or more than one type of enzyme.
• Example I Lipid Vesicle Preparation The following procedure was used to produce a suspension of lipid vesicles containing the sulfhydryl-reactive phospholipid derivative MPB-PE.
• MPB-PE The synthesis of MPB-PE was performed substantially as described in reference 5. Briefly, transesterified egg PE was reacted with freshly distilled triethylamine and succinimidyl 4-(p-maleimidophenyl) butyrate in anhydrous methanol under an argon atmosphere at room temperature for two hours. The MPB-PE formed was purified by silicic acid chromotography.
• the lipid vesicle preparation was examined by electron microscopy. Most of the vesicles were in the 0.2 micron diameter size range and had one or a few bilayer lamellae. Based on the microscopic examination of the vesicles, and the known lipid concentration thereof, a vesicle concentration of about 1.2 x 10 ⁇ 2 vesicles per micromole of lipid was calculated.
• This example examines optimal conditions for coupling antibody Fab' fragments to lipid vesicles formed in accordance with Example I.
• Rabbit anti-human i munoglobulin G (IgG) antibodies were isolated and purified according to conventional methods.
• F(ab')2 dimers were prepared by pepsin digestion of the purified antibodies.
• the dimer fragments were reduced with dithiothreitol at a pH of about 4.8 to produce Fab* monomer fragments.
• the pH of the reduction reaction is important in-that when the reaction is performed significantly above pH 4.8 " , (i.e.. pH 5.0) overreduction may occur which leads to inactivation of the antibody fragments, while underreduction, which may occur which at a lower pH (i.e., pH 4.5), is characterized by relatively poor coupling efficiency to the vesicles.
• Freshly prepared vesicles at a concentration of about 1 micromole of phospholipid per ml were reacted with freshly prepared Fab' fragments at a concentration selected between 0.5-4.0 mg per ml.
• the reaction was carried out in a pH 6.5 buffer under a stream of argon for up to 12 hours at room temperature.
• the vesicles were separated from unconjugated antibody fragments by differential centrifugation.
• the amount of protein conjugated to the vesicles varied according to the initial concentration of antibody fragments in the reaction. At an initial protein concentration of 4 mg/ml, approximately 500 micrograms of Fab' per micromole lipid were coupled to the vesicles in eight hours. For vesicles in the 0.2 micron diameter size,.
• Fab'-liposome reagent Rh+ human red blood cells were sensitized with human anti-D IgG and incubated with the anti-IgG carrying vesicles. Vesicle concentrations between 10 and 125 nanomoles of phospholipid per ml were mixed with an equal volume of a 2 percent suspension of the sensitized erythrocytes. The erythrocytes were incubated with the reagent for two hours at room temperature, after which they were separated and washed by low speed centrifugation in a clinical centrifuge. Radioactivity associated with the cell-bound vesicles increased quantitatively with increasing amounts of the liposome added, up to a saturation point corresponding to about 5,000 liposome vesicles per red blood cell. Binding of liposomes to human erythrocytes not coated with human IgG was less than about 5 percent of that of antibody-specific liposome binding.
• Example III Coupling Fab' Fragments and Beta-Galactosidase to Lipid Vesicles This example demonstrates the effect of lipid vesicle surface charge on the relative amounts of coupling of Fab* fragments and beta-galactosidase to lipid vesicles.
• Lipid vesicles were prepared according to the procedure described above, except that the vesicles were prepared to contain either 10 percent or 20 percent phosphatidylglycerol and proportionately less phosphatidylcholine.
• Enzyme and freshly reduced ligand preparations were reacted with one of the two lipid vesicle preparations under reaction conditions similar to those described in Example II. Specifically, 1 mg per ml Fab' and 0.25 mg per ml beta-galactosidase were reacted with 1 micromole vesicle lipid in one ml reaction buffer containing 20 mM citric acid, 35 mM disodium phosphate, 108 mM NaCl, and 1 mM EDTA, adjusted to pH 6.5 with 1 N NaOH. The reaction was stirred under a stream of argon for 14 hours at room temperature. The vesicles were twice pelleted by centrifugation at 20,000 G for 20 minutes to remove unreacted proteins.
• the pelleted and resuspended reagent vesicles were assayed for beta-galactosidase activity using the chromogenic substrate ortho-nitrophenol-3-D- galactopyranoside (nitrophenyl galactoside) .
• the relative specific activity values shown in Table I represent, respectively, 54 percent and 64 percent of 5 the total beta-galactosidase activity added to the coupling reaction mixture that was found to be associated with the vesicles.
• the data indicate greater enzyme coupling reactivity toward the vesicle preparation containing the higher concentration of 10 phosphatidylglycerol (PG), apparently resulting from the greater charge attraction between the negatively charged phosphatidylglycerol surface groups and the positively charged enzyme.
• PG phosphatidylglycerol
• the signal-to-noise ratio for each of -35 the two vesicle preparations was calculated by dividing the enzyme activity obtained for the IgG coated tubes by
• Reagent vesicles having different ratios of surface attached enzyme and ligand molecules were prepared, and their immunospecific binding to erythrocytes examined.
• Lipid vesicles containing MPB-PE thiol-reactive surface groups were prepared in accordance with the method described in Example I. Rabbit anti-human IgG antibodies were obtained in a form purified according to standard methods. • Reduced Fab* fragments, and beta-galactosidase were provided in accordance with Example III. Vesicle preparations, containing about 1 micromole of vesicle lipid, were reacted with selected amounts of reduced Fab' and beta-galactosidase, as indicated in Table II.
• the reactant concentrations of Fab* were 0.75, 1.0 or 1.25 mg per ml (column 1), and those of beta-galactosidase, were 0.1, 0.3 or 0.5 mg per ml (column 2) for each Fab* concentration.
• the coupling reactions were carried out at pH 6.8 under a stream of
• beta-galactosidase that the increased reagent protein concentration is attributable to increased amounts of coupled beta-galactosidase can be seen from the data in column 4 in Table II, showing specific activities of beta-galactosidase (expressed as arbitrary beta-galactosidase activity units per micromole of vesicle lipid). It is interesting to note that the amount of beta-galactosidase coupled to the vesicle lipids was relatively independent of the initial reactant concentration of Fab' in the Fab' concentration range shown (column 4).
• OMPI depends both on the initial reactant concentration of ligand, and on the relative reactant concentrations of ligand and enzyme.
• Example V Enzyme immunoassay to detect human subgroup antibodies on sensitized erythrocytes.
• This example illustrates use of the liposome reagent of the invention in an enzyme immunoassay to detect the presence of anti-subgroup antibodies on human erythrocytes, and in particular, anti-D, anti-Jka, and anti-Fya IgG antibodies carried on sensitized erythrocytes.
• lipid vesicles containing MPB-PE were prepared in accordance with Example I.
• Immunopurified anti-human IgG antibodies were prepared and treated as described above to produce Fab' fragments, each of which contains at least 1 thiol group. These fragments (0.75 mg per ml), and beta-galactosidase, (0.4 mg per ml) were reacted with the lipid vesicles (1.0 micromole of phospholipid per ml) for 18 hours at room temperature, pH 6.8.
• the reagent was separated from unreacted protein by centrifugation and resuspended in a suitable reaction buffer.
• Erythrocyte samples typed according to either D, Fya or Jka subgroup type were used. To sensitize each cell type, freshly washed cells were subdivided and each sample incubated with one of a series of two-fold serial dilutions of the appropriate anti-D, anti-Fya, or anti-Jka typing sera at 37°C for 30 minutes. The cells were washed three times with a saline solution. D, Fya, and Jka positive control cells were washed three times with the above saline solution and resuspended in phosphate buffered saline.
• the sensitized cells were treated.with liposome surface reagent, (5 nanomoles liposomes per 5 x 10 7 cells) for 30 minutes at room temperature, with rocking every few minutes. The cells were subsequently washed 5 times with saline/BSA, resuspended in 10% sucrose containing the enzyme substrate nitrophenyl galactoside, and incubated for 10 minutes at room temperature. Cells were pelleted and the enzyme activity determined by measuring the spectrophotometric absorption of the supernatant at 405 nm.
• the immunoassay was between about 8 and 32 times more sensitive, in terms of the minimum number of erythrocyte-bound antibodies which were detectable, than a standard anti-globin agglutination test which is used commonly for the determination of erythrocyte subgroup antigens.
• a lipid vesicle surface reagent was prepared essentially according to the method described in Example IV. Specifically, 0.75 mg per ml of immunopurified anti-human IgG Fab' fragments and 0.4 mg per ml of beta-galactosidase were reacted with lipid vesicles containing MPB-PE, under the reaction conditions described in Example IV.
• a control sample containing a known amount of Rubella antibody was prepared in 4 different sample concentrations, namely, an undiluted sample and 1:5, 1:25 and 1:125 serial dilutions thereof.
• the antibody was then reacted with solid support discs coated with Rubella antigens (Cordis Laboratories) for about 45 minutes at room temperature.
• the support discs were washed with saline/BSA, then placed in 0.5 ml of a high salt solution containing 250 mM NaCl, 100 mM phosphate, and a 25 icroliter aliquot of the liposome surface reagent (0.1 micromoles per ml).
• the liposome reagent was incubated with the support for about 2 hours at room temperature.
• the support discs were then washed two times with saline/BSA, and the enzyme activity associated with the discs determined according to the method noted above.
• Table III shows the relative enzyme activity, expressed in units per ml, associated with each of the different-concentration samples indicated in the table.
• the data show increasing levels of enzyme activity associated with increasing amounts of Rubella antibody added to a solid support.
• the negative control serum contained no Rubella antibody.

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