CA1239582A - Lipid-vesicle-surface assay reagent and method - Google Patents

Lipid-vesicle-surface assay reagent and method

Info

Publication number
CA1239582A
CA1239582A CA000455271A CA455271A CA1239582A CA 1239582 A CA1239582 A CA 1239582A CA 000455271 A CA000455271 A CA 000455271A CA 455271 A CA455271 A CA 455271A CA 1239582 A CA1239582 A CA 1239582A
Authority
CA
Canada
Prior art keywords
molecules
reagent
lipid
vehicles
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA000455271A
Other languages
French (fr)
Inventor
Pedro E. Huertas
Francis J. Martin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
COOPER-LIPOTECH Inc
Original Assignee
COOPER-LIPOTECH Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by COOPER-LIPOTECH Inc filed Critical COOPER-LIPOTECH Inc
Priority to CA000455271A priority Critical patent/CA1239582A/en
Application granted granted Critical
Publication of CA1239582A publication Critical patent/CA1239582A/en
Expired legal-status Critical Current

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

LIPID-VESICLE-SURFACE ASSAY REAGENT AND METHOD

ABSTRACT

An improved enzyme immunoassay reagent composed of lipid vesicles coated with a mobile surface array of ligand and enzyme molecules is described. The reagent is adapted for use in an enzyme immunoassay in which the reagent partitions between a separable support and a liquid phase, in proportion to the amount of analyte present. The amount of reagent in the liquid or support phase is determined by measuring the enzyme activity associated with the reagent in that phase. Also described is an immunoassay employing such a reagent.

Description

f Jo 1~39~8~

LIPID-VESICLE-SU~.FACE ASSAY Regilt AND METHOD
. _ Background and Summary The following publications are referred to by corresponding number in this application:
1. Seiko, F. or., and Papahajopoulos, D., Ann. Rev.
Boyce. Bit I, 9:467 508 (1980).
2. Seiko, F ., Jr. and Papahajopoulos~ D., Pro. Nat.
clue. USA, 75:4194-4198 (1978).
lo 3. Heath, T. D., Matcher, B. A. and ~apahadjopoulos, 3., Biochimica et Biophysics Act, 640:66-~1 ~1981).
4. Martin, F. J., Hubbell, W. L., and Papahadjopoulos, D., iochem sty, 20:4229-4238 ~1~81).
5. Martin, F. J. and Papahadjopoulos, D., J. Blot.
Chum., 257:286-2B8 (1982).
6. Furman, B., Klareskog, L., and Peterson, P. A , J tot Chum., 255:7820-7826 (1980).
7. Smith, B. A. and McConnell, H. M., Pro. Nat. Aged.
Sat. USA, 75, 2759-2763 (1978).
8. Lowry, O. H., Rosebrough, N. J., Fern, I. I,., and Randall, R. J., J. Biol._Chem., 193:265-275 tl951).
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 analyzes is known. The analyze to be assayed typically is one which plays an important role in biochemical processes. Dow molecular weight substances, such as peptize and steroid hormones, vitamins and the like, and high molecular weight substances such as carbohydrates and proteins are commonly assayed analyzes.
Several important analyze assay techniques are based on a reaction between the analyze and an 35 anti-analyte capable of binding the analyze with high affinity and specificity. Typical analyze anti-analyte ., .

~3958~

binding pairs include an~igen-antibody, immunoglobulin-protein A, carbohydrate-lectin, biotin-avidin, hormone-hormone receptor protein and complementary oligo- and polynucleotide strands. The terms ~ligand~ and ~antiligand~ will be used herein to designate the opposite binding members in such a binding pair Among the various types of assays which employ specific binding reactions, enzyme immunoassay provide a number of advantages in sensitivity, low cost and simplicity. In one type of enzyme immunoassay, an enzyme-ligand reagent is reacted in the presence of a ligand or ligand-like analyze with a solid support having anti-ligand binding sites carried on its surface, lo wherein the analyze and ligand-enzyme reagent compete for binding to the solid support. In another type of enzyme immunoassay, a ligand analyze 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. In both assay types, the amount of analyze present is determined by separating the liquid and solid (support) phases, and measuring the enzyme activity associated with one or both phases. Where the reagent and analyze compete for binding sites on the solid support, the enzyme activity associated with the support is inversely proportional to the concentration of analyze present. On the other hand, a direct relationship between the amount of analyze and the enzyme activity associated with the solid support is observed where the analyze functions to join the reagent to the support by sandwiching.
Commonly, the reagent used in an enzyme immunoassay test includes an enzyme molecule covalently coupled to a ligand or anti-ligand molecule to form a bimolecular 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. Otherwise, a significant portion of the reagent (the portion composed of enzyme coupled to non-ligand molecules) will not bind, or will bind nonspecifically, to the solid support. As a result, a high background attributable to unbound reagent (in the liquid phase) and nonspecifically bound reagent (in the solid phase) will be observed.
An enzyme immunoassay reagent composed of lipid vehicles coated with ligand molecules and encapsulating enzymes within the interior vehicle 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 liposomes efficiently. Further, many enzymes appear to undergo loss of activity during encapsulation (reference 1). The encapsulating vehicles must be lucid before enzyme activity associated with the vehicles can be measured. Complement has been used for lying lipid vehicles, but this method often lacks reproducibility due to complement inactivation on storage. Detergent louses has been used, but this approach may be unsuitable for applications --e.g., cell typing-- where the cell Cypriot to which the vehicles are bound is itself susceptible to detergent louses.
3~5~3~
According to an aspect of the invention there is provided a method for detecting the presence of analyze molecules carried on biological cells, comprising: providing a suspension of lipid vehicles having surface bound anti-analyze molecules, at a surface concentration of at least about 15 molecules per vehicle, and surface-bound reporter molecules, incubating the suspension with the sample cells, to bind the vehicles to the cell surfaces by specific analyte/anti-analyte binding interactions, separating the cells from unbound vehicles, and detecting the presence of reporter associated with the cells.
According to a further aspect of the invention there is provided a system for determining the presence of a selected cellular antigen carried on the surface of a cell comprising a soluble antibody specific against the antigen, and a suspension of lipid vehicles having surface bound antibody or antibody-fragment molecules which are specific against the soluble antibody, at a surface concentration of at least about 15 molecules per vehicle, and surface-bound reporter molecules.
The reagent of the invention is composed of lipid vehicle particles having a highly mobile, or fluid surface array of ligand and enzyme molecules. The particles are preferably lipid vehicles 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 vehicle 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 -partitioning of the reagent between the support and liquid reaction phases according to the concentration of analyze present.
The invention also contemplates an assay kit including a separable support and the reagent.
These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention.
Detailed Description of the Invention Preparation of Lipid Vehicles The assay reagent of the present invention is composed of closed lipid vehicles, each having attached to its outer surface a laterally mobile array of enzyme and ligand molecules. Typically, the lipid vehicles take the form of lipid Baylor structures encapsulating an aqueous interior region, such structures also being referred to as liposomes. The properties and methods of preparation of lipid vehicles 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.
What 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 vehicles are prepared from lipid mixtures which typically include phospholipids and strolls. 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 vehicles formed. As reported in a number of literature reports, these characteristics can ye varied according to the lengths and degree of saturation of the aliphatic chains in the lipids, and the ratio of stroll to aliphatic chain lipids used The significance of surface fluid mobility in the the vehicle reaccent of the invention will be seen below. Packing density characteristics are important to the success of reactions used to attach Lund and enzyme molecules covalently to the vehicle surfaces.
For example, it has been found that the inclusion of at least about 10% mole per cent of cholesterol is important for the success of certain protein-coupling reactions which will be described below. The fluidity and packing characteristics will also affect the size and number of bowlers in the vehicles produced.
The vehicle 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 vehicles. 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 vehicles at a selected pi and ionic strength. The charge distribution may affect the relative reactivities of enzyme and ligand molecules in their coupling to lipid vehicles, as will be seen in Example III, and is an important feature in minimizing non-specific binding of the reagent vehicles to charged solid supports, as will be discussed.
A typical lipid composition used in preparing lipid vehicles for the reagent of the invention preferably includes between about 10 and 50% cholesterol or other stroll, 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 as phosphatidylcholine, or a phospholipid mixture.
Charged lipids, such as phosphatidylserine/ phosphatidic acid, ylycolipids and charged cholesterol derivatives such as cholesterol hemisuccinate or cholesterol sulfate may be included to produce a desired surface chary in the lipid vehicles.
he lipid vehicles may be formed by one of a variety of methods discussed particularly in reference 1. Multilamellar vehicles --that is, vehicles composed of a series of closely packed Baylor lamely-- 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 lamely in the formed lipid vehicles 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 vehicles.
Small unilamellar vehicles having diameters of about 0.05 micron or less can be formed by sonicating a suspension of large multilamellar vehicles either by probe or bath sonication. Another technique for producing small unilamellar vehicles involves the removal of detergent from a deteryent-phospholipid mixture by dialysis. Typical detergents include shalt and deoxychola~e. An alternative method for the preparation of small unilamellar vehicles that avoids both sonication and detergents employs an ethanol injection step in which lipids dissolved in ethanol are rapidly injected into a buffer solution. similar technique in which phospholipids dissolved in ether-containing solvents has been used to produce large unilamell~r v~sicles with a generally heterogeneous size distribution on one preferred method of preparing large ~395~3~

unilamellar vehicles, referred to as reverse phase evaporation, a desired composition of lipids is dissolved in a suitable organic solvent such as deathly ether, isopropyl ether, or a solvent mixture such as isopropyl ether and chloroform I n 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 I 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 vehicles may be varied according to the amount of cholesterol included in the lipid mixture. The reader is referred to references 1 or 2 for further details concerning the reverse phase evaporation technique.
The lipid vehicles prepared may be obtained in a defined size range by various techniques. Methods for reducing size heterogeneity in smell unilamellar vehicles by gel filtration and ultra centrifugation have been described. A method of reducing the size and the size heterogeneity of lipid vehicles 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 vehicles are recovered, the larger ones being converted to desired-sized smaller vehicles by passage through the filter.
It can be appreciated from the foregoing that lipid vehicles 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.

I

Enzyme and Ligand Coupling to Lipid Vehicles This section is concerned with techniques used to couple ligand and enzyme molecules covalently to surface lipids in lipid veslcles. The ligand molecules in the reagent function to bind the reagent to anti-ligand binding sites on a separable support. ho used herein, 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. Among 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 nucleated 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. For example, in an antibody-antigen paint the binding ligand may include the antigen binding Phoebe or Fob' fragments. As another example, in the protein A-immunoglobulin pair, 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 vehicle 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 vehicle. Preferably the enzyme I

can be obtained in pure or near-pure form and is relatively staple on Starkey 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.
Representative classes of enzymes contemplated herein include oxidoreductases, typified by luciferase, glucose oxidize, galactose oxidize and kettles; hydrolyses, typified by various types of phosphatases; glycoside hydrolyses, such as beta-galactosidase; peptidases; and leases.
One enzyme which has been used advantageously in the reagent of the invention is beta-galactosidase derived prom a bacterial source. Among the advantages of this enzyme are: (1) the enzyme is available in purified Norm with high specific activity; (2) the enzyme contains free they'll 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 vehicles allows for the attachment of a relatively large number of enzyme molecules on each vehicle, as will be seen below.
Two or more enzyme species may be attached to the lipid vehicles 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.
Several methods are available for coupling biomolecules covalently to the polar head groups of lipids. As a general consideration, it is important to select a coupling reaction which does not significantly reduce the enzymatic activity or ligand binding activity of the molecules being coupled. At the same time it is important to select a method which produces a relatively 3~3t5~;~

high coupling efficiency. In this regard, where the enzyme and ligand components are coupled to the vehicles in simultaneous reactions, the relative reactivities of the two species toward the lipid sites must be taken into account. finally, care must be exercised to avoid reactions which would produce significant cross linking of the vehicle lipid components to each other, or of the individually - coupled ligand or enzyme molecules Jo one another, since any cross linking of the reagent components (except for the individual lipid surface component conjugations would reduce the fluid mobility of the surface lipids and attached molecules. Without intending to limit the scope of the invention, two preferred methods of coupling biomolecules, particularly proteins, to lipid vehicles will be described herein The first method involves Schiff-base formation between an alluded group on the lipid or molecule to be coupled, and a primary amino group on the other of the two reactants. The alluded group is preferably formed by peridot oxidation. The coupling reaction, after removal of the oxidant, is carried out in the presence of a reducing agent. although the non lipid molecule being coupled may be oxidized, more commonly it is the lipid group which is the alluded precursor since peridot treatment inactivates many proteins. Typical aldehyde-lipid precursors include lactosylceramide, trihexosylceramide, galactocerebroside, phosphatidylglycerol, phosphatidylinositol and gangliosides.
In practice, the vehicles are oxidized by peridot 'or a period sufficient to produce oxidation of a majority of the oxidizable lipid groups, and thereafter the vehicles are separated from the peridot by column gel filtration. Alluded groups on the vehicle surfaces are conjugated with a primary amine, such as a Lawson group in a protein, to form a Showoffs {
I 1%

! -12-base which is subsequently reduced with sodium bordered or sodium cyanoborohydride to form a more stable bond. Typically, for conjugation reduced with sodium bordered, oxidized lipid vehicles at a concentration of between about 5 and 10 micro moles of total lipid are mixed in 1 ml with 10 to 30 milligrams of protein at an alkaline phi 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.
Using lipid vehicles prepared by reverse phase evaporation and extruded through a 0.2 micron pore-size polycarbonate membrane, up to about 200 micrograms of I immunoglobulin G (Gig) per micro mole of lipid vehicle lipid can be attached to the vehicle surfaces by the above method. Based on a calculated number of about 1.2 x 1012 vehicles per micro mole of vehicle lipid, this conjugation ratio corresponds to about 600 Gig molecules per lipid vehicle. Studies conducted in the support of the present application indicate that correspondingly smaller molecules can be coupled to lipid vehicles in correspondingly larger numbers. Thus up to about 1800 Fob' antibody fragments per lipid vehicle (in the 0.2 micron diameter range) can be attached. 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.
second general coupling technique is applicable to thiol-containing molecules, involving formation of a disulfide or thither bond between a vehicle lipid and the molecule attached. the technique is particularly useful for coupling Phoebe and Fob' antibody fragments to lipid vehicles.

In the disallowed interchange reaction, phosphatidylethanolamine is modified to provide a pyridyldithio derivative which can react with an exposed they'll 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.
us reported there, a coupling ratio of up to 600 micrograms of Fob' antibody ruminates per micro mole of phospholipid can be achieved Based on calculations lo similar to those presented above, this number corresponds to about 6000 Fob' antibody molecules per JO . 2 micron diameter vehicle.
The thither coupling method, which it described in detail in reference 5, is carried out by 115 incorporating in the lipid vehicles a small proportion of a sulhydryl-reactive phospholipid derivative, such as No (p-maleimidophenyl) bitterly) phosphatidylethanolamine MOPE The lipid vehicles ware reacted with a thiol-containing protein to form an essentially irreversible thither coupling between the protein isle group and the MPB-PE maleimide group. It is noted that the requisite protein they'll group may be endogenous to the protein or may be introduced on the protein by amino-reactive they'll groups according to known methods. coupling ratios of up to about 350 my of sulfhydryl containing protein per micro mole of lipid vehicle phospholipid have been obtained.
It is also contemplated herein that enzyme or ligand molecules can be separately and individually attached to lipid vehicles by first coupling the molecules covaiently to free lipids dispersed in a detergent solution. The lipid-enzyme or lipid-ligand couples are then incorporated into lipid vehicles, either during vehicle formation or by diffusion into preformed vehicles according to known techniques.
Alternatively the ligand itself may contain an endogenous hydrophobic region --for example, a hydrophobic stretch of amino acids-- by which the Lund can be incorporated into the surface of a lipid vehicle. As an example, it has been shown that human transplantation antigens can be attached to egg lecithin vehicles by anchoring hydrophobic peptize regions in the antigens to the vehicles (reference 6).
t is further contemplated that immumoglobulin or immunoglobulin fragment Lund molecules can be attached to lipid Yesicles through pair-specific binding to anti-immunoglobulin antibodies, or fragments thereof, or to protein A covalently attached to the vehicles.
! The enzyme and ligand molecules may be coupled to (or incorporated into) lipid vehicles 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 vehicles, 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.
In one typical protocol, ligand or a ligand-containing mixture, is first reacted by an I above-described coupling reaction, with vehicles to produce a desired vehicle surface concentration of the analyte-speciEic ligand. after the initial coupling reaction has been completed, the vehicles are separated from unrequited Lund molecules, then reacted with enzyme molecules. One advantage of the present invention is that the Tuscaloosa may be easily separated from the unrequited solution components of the coupling reaction(s) by centrifugation, facilitating intermediate purification steps that may be required during reagent preparation.
The ratio of analyze specific ligand to enzyme :~3~582 molecules coupled to the vehicles is generally selected to maximize the signal-to-noise ratio in an enzyme immunoassay employing the reagent. Studies on the kinetics and specificity of vehicle reagent binding to various types of separable supports suggest that two countervailing factors are important in maximizing reagent performance. on one hand, a minimum surface concentration of analyte-specific ligands is required to effect stable reagent binding to a support. Binding affinity generally increases as the surface concentration of ligand molecules increases from an average of about 10-15 molecules per vehicle up to about 50-100 ligand molecules per vehicle (of average diameter of about 0.2 microns). On the other hand, as the number of ligand molecules bound to the lipid vehicles is increased, particularly where the ligand traction coupled Jo the vehicle is relatively impure, the number of sites available for enzyme attachment to the vehicles is reduced. Ideally, as in the case where a relatively pure ligand preparation is coupled to the lipid vehicles, the vehicles can easily accommodate 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 vehicle to the support. The surface concentration of ligand molecules required to promote such stable multi site vehicle binding to a macro molecular support can be quite low, on the order of about I molecules per vehicle. 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 vehicle surfaces. Diffusion constants of the order of owe o 10-9 cm~Jsec for I

phospholipid diffusion within lipid bowlers have been measured reference I
Binding efficiency may be further enhanced where the binding sites on the solid support are S themsel Vow carried on lipid vehicles. Here the combined mobility of the ligand molecules on the reagent lipid vehicles and the binding site molecules on the separable support lipid vehicles would facilitate multi-site binding a low ligand and anti-liyand (binding site) surface concentrations.
nether important advantage of the reagent invention is the relatively high surface packing of covalently attached molecules which is achievable in the reagent vehicles. As noted above, it is possible to attach up to several thousand protein molecules on a vehicle surface, based on a protein molecular weight of around 50,000 and a vehicle diameter size of about 0.2 microns. Thus a 0.2 micron vehicle having an average ox about 50 Fall fragments carried on its outer surface can carry nearly 100 times that number of an enzyme having about a 50,000 molecular weight.
Alternatively, where the analyte-specific ligand being coupled to the vehicles constitutes as little as one percent of the total non-enzyme molecules attached to the surface, each vehicle can still accomoda~e up to several hundred or more enzyme molecules, producing a vehicle whose enzyme to ligand molar ratio is still substantially greater than one.
Other advantages of the instant reagent in an enzyme 0 immunoassay will be considered below.
Assay Methods The method of the invention comprises reacting a liposome surface reagent with a separable support carrying ~nalyte-related binding-site molecules. The reagent binds to the support in proportion to the amount of analyze present.

I

.

s used herein, separable support refers to any support structure capable of being readily separated --for example, by differential centrifugation, precipitation or electrophoretic separation -- from S analyze and vehicle 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, agrees, polystyrene and the like. Macro molecular tissue homogenate structures, intact cells, and cell membrane structures are other contemplated supports.
Also as detailed above, the support may include surface-bound lipid vehicles to which the support bindincJ site molecules are attached.
he analyte-related binding site molecules on the support are selected to bind specifically to the reagent Lund molecules, or to the analyze molecules, or to both, depending on the type of enzyme immunoassay I 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.
Methods of forming solid supports having a wide variety of attached molecules, being well known to those skilled in the art, will not be detailed herein.
Some types of 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. Further, the solid support may have I surface properties which tend to promote non-specific attachment of the liposome resent, leading to a :~3i9~

decreased signal-to-noise ratio in the assay.
The two problems just mentioned may be reduced or eliminated by employing a solid support in which the binding site molecules are carried on lipid vehicles which are themselves attached to the support. Two representative methods by which lipid vehicles can be attached to solid supports will now be described.
In first method, glass surfaces, for instance lass tubes or controlled-pore glass beads, are I derivitized with glycerol, activated with carbonyldiimidazole and converted into amino-ylass by reaction with excess Damon Al Kane. The amino-glass is converted into pyridyl depth glass by reaction with N-succinimidyl 3 (2-pyridyldithio~ preappoint. The pyridyl depth glass is then reduced with dithiothreitol or 2-mercaptoethanol to yield a glass surface with trio functions.
To achieve reversible attachment of liposomes, the lipid vehicles, prepared to include N-(3-(2-pyridyldithio)propionyl)phosphatidylethanooilmen POPE synthesized according the method described in reference 4 r are reacted with the trio glass at a pub between about 7.0 and 8.5. The disulfide bond which forms between the glass and the vehicles can be cleaved by mild reduction, for example with dithiothreitol at low pi.
Irreversible attachment of vehicles to a glass support Max be achieved by reacting the trio glass with lipid vehicles prepared to contain MPE-PE, as described in reference 5, to form a thither linkage.
It is also contemplated that lipid vehicles may be attached to a solid support nonequivalently through specific, high affinity ligand/anti-ligand binding. As one illustration, avid in molecules are attached covalently to a solid support using conventional methods. Lipid vehicles prepared to contain I

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 vehicles, according to above described techniques.
Attachment of binding site molecules to lipid vehicles carried on a solid support may increase the accessibility of the liposome reagent to the binding sites on the solid support. Another advantage inherent in this approach is that the vehicles 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.
Three general types of enzyme immunoassay tests employing a solid support in conjunction with the reagent vehicles will now be described. In a first type of test the reagent vehicles carry analyze or analyte-like ligands which compete with analyze in solution for binding to anti-analyte binding sites on the solid support. The analyze, 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 analyze may be a binding protein which competes with binding proteins on the reagent fox binding to target type binding sizes on the solid support. In both instances, the amount of liposome reagent binding to the solid support varies inversely with the amount of analyze present.

The assay reaction is carried out in a suitable reaction medium which may include a biological specimen fluid, such as serum, containing the analyze. The pi of the reaction medium is one which is compatible with ligand/anti-ligand binding reactions, and preferably between about S and 9. More specifically, the pi 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. It is often an advantage to carry out the binding reaction at a pi and ionic strength which minimizes charge repulsion between the support and the reagent, and to remove nonspecifically-bound reagent later by a washing step, usually with a low-ionic strength, high pi washing solution.
The reaction medium may also be adjusted to have a specific gravity which approximates the buoyant - 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 vehicles to float or sink in the medium, promotes the requisite contact between the vehicles and the solid support.
Alternatively, it may be advantageous in some assay methods to employ a liposome surface reagent which is either more or less dense than the reaction medium, to facilitate separation of the liposome reagent from the support, or to achieve some other advantage related to reagent partitioning.
Sensitivity in the assay requires reacting a defined amount of solid support with a known selected amount of the liposome reagent. With too little liposome reagent added to the reaction mixture, the binding sites on the solid support can accommodate a substantial quantity of bound analyze 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 Anita for binding to any displaced binding S sites on the support. The assay background Allah 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 analyze 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 37C or somewhat higher.
Alternatively, the assay may be performed as a two-step reaction in which the analyze 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 phi ionic strength and specific gravity.
Upon completion of the assay reaction, the solid support is separated from the liquid phase of the reaction medium/ including the unbourld suspended liposomes, and the support or the liquid phase, or both, are assayed for enzyme activity. To reduce the level of nonspecifically bound liposome reagent, the separated support is preferably washed one or more times with a washing solution whose pi and ionic strength act to increase charge repulsion between the solid support and the liposome reagent.
In a second type of enzyme immunoassay contemplated herein, the solid support carries an analyze or analyte--like binding molecule which competes lo with the analyze for binding to the ligand on the liposome reagent. The analyze, and the binding-site molecules on the support may be either a target-type antigen which compete for binding to a ~arget~binding ligand on the liposome, or the analyze 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 phi ionic strength and specific gravity of the reaction solution which have been discussed above are applicable to the instant method. Lookers 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 analyze. The assay is preferably performed as a two-step method in which analyze is first reacted with the support, after which the separated support is reacted with the liposome reagent. The analyze may be either an antibody or an antigen, with the solid support and liposome reagent each carrying an opposite binding pair of the analyze.
One advantage of this method is that the amount of liposome reagent bound to the solid support is directly proportional to the amount of analyze present. The method can thus be used to detect very small quantities I

of analyze Additionally/ since the liposome reagent is bound to the solid support through analyze 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 siynal-to-noise ratio in the test The considerations relating to pi, ionic strength and specific gravity in the reaction medium are similar to those discussed above and will not be described further here.
One aspect of the invention which can be appreciated from the above is the provision of 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 vehicles, as described above. The binding site molecules may be analyze or analyte-like molecules which compete with the analyze for binding to anti-analyte ligand molecules in the surface reagent. Alternatively, the binding site molecules may be an anti-analyte species, where 'the analyze 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 analyze in a sample solution. According to another aspect of the invention, the reagent is used in an enzyme immunoassay for determining the presence or concentration of cell-specific surface antigen analyzes. were the separable support includes a biological cell, and more specifically, a cell membrane .
i,, ' 3S~32 whose outer surface carries the antigen. Typical analyzes 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. Alternatively, the analyze may include anti-cell surface antigen antibodies attached to the cell by incubation with the free antibody.
In the cell-surface antigen assay, the cell sample to be assayed is adder to defined amounts of liposome reagent having analyte-recognition molecules.
After a suitable reaction time, under reaction conditions which may be selected in accordance with the considerations mentioned above, the ceils 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. Other 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 From the foregoing it can be appreciated how 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 AS seen, up to several thousand enzyme molecules can be attached to a vehicle 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 pi and ionic strength conditions which favor specific binding between the two, followed by exposing the separated lo 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 vehicles, and where the liposome reagent carries two or more distinct types of ligand molecules or more than one type of enzyme.
The following examples describe particular S embodiments of making and using the invention.
Example I
Lipid Vehicle Preparation The following procedure was used to produce a suspension of lipid vehicles containing the sulfhydryl-reactive phospholipid derivative MPB-PE.
The synthesis of MPB-PE was performed substantially as described in reference S. Briefly, transesterified egg Pi was reacted with freshly distilled triethylamine and succinimidyl 4-(p-maleimidophenyl) bitterroot in an hydrous methanol under an argon atmosphere at room temperature for two hours The POPE formed was purified by 3~35~

silicic acid chromatography.
Large, unilamellar vehicles were prepared by a reverse phase evaporation method described generally in references 1 and 2. Cholesterol (10 micro mole), phosphatidylcholine (9.5 micro mole), MPB~PE (.5 micro mole) and a trace amount of initiated dipalmitoylphosphatidylcholine were dissolved in 1 ml of deathly ether. A buffer, at pi I containing 20 my citric acid, 35 my disodium phosphate, 108 my sodium chloride and 1 my ETA was added (300 micro liter) r and the two phases emulsified by sonication for one minute at 25C in a bath sonicator. Ether was removed under reduced pressure at room temperature and the resulting dispersion was extruded successively through 0.4 micron and 0.2 micron Unhip polycarbonate membranes (Byrd Laboratories, Richmond, CA).
The lipid vehicle preparation was examined by electron microscopy. Most of the vehicles were in the 0.2 micron diameter size range and had one or a few Baylor lamely. Based on the microscopic examination of the vehicles, and the known lipid concentration thereof, a vehicle concentration of about 1.2 x 1012 vehicles per micro mole of lipid was calculated.
Example II
_ uplin~ of Fob' Fragments to Vehicles This example examines optimal conditions for coupling antibody Fob' fragments to lipid vehicles formed in accordance with Example It Rabbit anti-human immunoglobulin G (Gig) antibodies were isolated and purified according to conventional methods. Libya divers were prepared by pepsin digestion of the purified antibodies. The diver fragments were reduced with dithiothreitol at a pi of about 4.8 to produce Fob' monomer fragments. The pi of the reduction reaction is important in that when tune reaction is performed significantly above pi 4.8~ (i.e.

' i i r 3 9 5 I

pi OWE) over reduction may occur which leads to inactivation of the antibody fragments; while under reduction, which may occur which at a lower pi (i.e., pi o'er is characterized by relatively poor coupling efficiency to the vehicles.
Freshly prepared vehicles at a concentration of about l micro mole of phospholipid per ml were reacted with freshly prepared Fob' fragments at a concentration selected between 0.5-4.0 my per ml. The reaction was lo carried out in a pi 6.5 buffer under a stream of argon for up to 12 hours at zoom temperature. The vehicles were separated from unconjugated antibody fragments by differential centrifugation. The amount of protein conjugated to the vehicles 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 Fob' per micro mole lipid were coupled to the vehicles in eight hours. For vehicles in the 0.2 micron diameter size, this corresponds to about 5,000 molecules of Fall monomer fragments per vehicle. This number is somewhat higher than that reported in the literature and may be due it part to the reducing conditions used to reduce Phoebe divers to Fob' monomers.
To test the specificity of binding of the Fab'-liposome reagent, ho human red blood cells were sensitized with human anti-D Gig and incubated with the anti-IgG carrying vehicles. Vehicle 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 vehicles increased quantitatively with increasing amounts of the liposom~e added, up to a saturation point corresponding to about 5,000 liposome vehicles per red blood cell.
Binding of liposomes to human erythrocytes not coated with human Gig was less than about 5 percent of that of antibody-specific liposome binding.
Example III
Coupling ens and Beta-Galactosidase to Lipid Vehicles This example demonstrates the effect of lipid vehicle surface charge on the relative amounts of coupling of Fob' fragments and beta-galactosidase to lipid vehicles.
ibid vehicles were prepared according to the procedure described above, except that the vehicles were prepared to contain either 10 percent or 20 percent phosphatidylglycerol and proportionately less phosphatidylcholine.
Purified rabbit anti-human Gig antibodies were pepsin-digested and reduced with dithiothreitol for twenty minutes at pi 4.8, according to the method above. Beta-galactosidase (847 IU/mg), was obtained from Boehringer-Mannheim.
Enzyme and freshly reduced loosened preparations were reacted with one of the two lipid vehicle preparations under reaction conditions similar to those described in Example II. Specifically, 1 my per ml Fob' and 0.25 my per ml beta-galactosidase were reacted with 1 micro mole vehicle lipid in one ml reaction buffer containing 20 my citric acid, 35 my disodium phosphate, 108 my Nail, and l my DATA adjusted to pi 6.5 with 1 N
Noah. The reaction was stirred under a stream of argon for 14 hours at room temperature. The vehicles were twice pelleted by centrifugation at 20,000 G for 20 minutes to remove unrequited proteins.
The pelleted and resuspended reagent vehicles were assayed for beta-galactosidase activity using the ~395~3~
I

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 the total beta-galactosidase activity added to the coupling reaction mixture that was found to be associated with the vehicles. The data indicate greater enzyme coupling reactivity toward the vehicle preparation containing the higher concentration of phosphatidylglycerol (PUG), apparently resulting from the greater charge attraction between the negatively charged phosphatidylglycerol surface groups and the positively charged enzyme.
TABLE I
15 PUG B-Gal Couplingsignal-to-noise Efficiency ratio 10% 393 54 I
20% 431 64 4.8 The specific binding activity of the two reagent preparations toward human Gig adsorbed on a polypropylene solid support was measured to determine roughly the relative amounts of Fob' carried on each of the two vehicle reagents. Polypropylene tubes were coated with human Gig according to known procedures. An Alcott containing 1 nanomole of each vehicle preparation was added to a coated tube, and the suspension was incubated for a half hour at room temperature in a phosphate-buffered saline buffer, pi 7.4. As a control, the two vehicle preparations were also incubated with tubes coated only with bovine serum albumin (USA). After incubation, the tubes were washed one time with a low-salt buy f or and the enzyme activity color development) associated with each of tune tubes was determined. The signal-to-noise ratio for each of the two vehicle preparations was calculated by dividing the enzyme activity obtained for the Gig coated tubes by ~.~3958%

that for the tubes coated only with BRA. As seen in Table I, a greater signal-to-noise ratio, presumably attributable to a greater surface concentration of Fob molecules, was associated with the vehicle preparation containing less phosphatidylglycerol.
The data in Table I suggest that an increased negative charge on the surface of lipid vehicles favors beta-galactosidase coupling, resulting in enzyme coupling efficiencies of up to about 60 percent. The lo data also indicate that nanomolar amounts of the liposome reagent are sufficient to produce a strong signal-to-noise ratio in a binding assay, and that the ratio of enzyme and ligand molecules in tune vehicles can be adjusted to produce an optimal signal-to-noise ratio.
EXAMPLE IV
Preparation of a Reagent availing Different Ligand to Enzyme ratios Reagent vehicles having different ratios of surface attached enzyme and ligand molecules were prepared, and their immunospecific binding to erythrocytes examined.
Lipid vehicles containing MPB-PE thiol-reactive surface groups were prepared in accordance with the method described in example I.
Rabbit anti-human Gig antibodies were obtained in a form purified according to standard methods.
Reduced Fob' fragments, and beta-galactosidase were provided in accordance with Example III. Vehicle preparations, containing about l micro mole of vehicle lipid, were reacted with selected amounts of reduced Fob' and beta-galactosidase, as indicated in Table II.
The reactant concentrations of Fob' were 0.75, lo or 1.25 my per ml (column lo, and those of beta-galactosidase, were 0.1, 0.3 or 0.5 my per ml (column 2) for each Fob' concentration. The coupling reactions were carried out at pi 6.8 under a stream of Jo I

argon gas for 12 hours at room temperature, similar to what has been described above. The vehicles were twice washed to remove unrequited Fall and beta-galactosidase, and the protein concentration associated with each vehicle preparation was determine according to the method of Lowry (reference 8). Column 3 in Table II
shows the measured protein concentrations, expressed in micrograms of protein per micro mole vehicle phospholipid. The data show that, for each reactant concentration of jab' r increasing amounts of beta-galactosidase resulted in increasing amounts of protein covalently coupled to the vehicles. 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 micro mole of vehicle lipid). It is interesting to note that the amount of beta-galactosidase coupled to the vehicle lipids was relatively independent of the initial reactant concentration of Fob' in the Ebb' concentration range shown (column 4).
The binding of the 9 different reagent preparations to IgG-coated erythrocytes was determined in accordance with the method described in Example III.
Sensitized (IgG-coated) red cells, after incubation with a vehicle reagent were separated by centrifugation at low speed and washed in a low-salt solution, made isotonic with sucrose, to remove nonspecifically bound reagent. Enzyme activity associated with the red blood cells was used to determine the percent of vehicle reagent which bound to the cells. The values, which are shown in column 5 in Table lit confirm that liposomes having treater immunospecific binding capacity can be prepared by coupling greater amounts of ligand to tile vehicle surfaces, and that the amount of Lund bound depends both on the initial reactant concentration of ligand, and on the relative reactant concentrations of ligand and enzyme.
TABLE II

Reagent Fob' B-Gal Reagent Reagent Binding Protein B-Gal to Cells 0.75 0.1 330 51 36 0.3 3~0 1~4 20 0.5 545 2g6 25 1.0 0.1 355 29 I
r 0.3 439 120 30 0.5 500 265 31 151.25 0.1 370 50 a o. 3 450 140 52 0.5 565 246 27 Example V
Enzyme immunoassay to detect human Siberia 20 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 Gig antibodies carried on sensitized erythrocytes.
To prepare the liposome reagent, lipid vehicles containing MPB-PE were prepared in accordance with Example I. Immunopurified anti-human Gig antibodies were prepared and treated as described above to produce Fob' fragments, each of which contains at least 1 they'll group. These fragments (0.75 my per ml), and beta-galactosidase, (0.4 my per ml) were reacted with tube lipid vehicles ~1.0 micro mole of phospholipid per ml) for 18 hours at room temperature, pi 6.8. The reagent was separated from unworked protein by .3~58~

centrifuqation and resuspended in a suitable reaction buffer.
Erythrocyte samples typed according to either D, Foe 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 sofa at 37~C or 30 minutes. The cells were washed three times Whitehall a saline solution. D, Foe, 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, (S nanomoles liposomes per 5 x 107 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 g31actoside, 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 no.
A linear relationship between the reagent enzyme activity observed ~supern3tant absorption) and number of surface specific Gig molecules (antisera dilution) was observed over a wide dilution range for all three cell types. 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-slobin agglutination test which is used commonly for the determination of erythrocyte subgroup antigens.
Example VI
Enzyme immune for determination of Rubella antibodies A lipid vehicle surface reagent was prepared 3~582 essentially according to the method described in Example IV. Specifically, 0.75 my per ml of immunopurified anti-human IyG Fob' fragments and 0.4 go per ml of beta-galactosidase were reacted with lipid vehicles containing MPB-PE, under the reaction ondi~ions 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 (Cords laboratories) for about I
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 my Nail, 100 my phosphate, and a 25 micro liter Alec of the liposome surface reagent (0 1 micro moles per my The liposome reagent was incubated with the support Pro about 2 hours at room temperature. The support discs were then washed two times with saline/BSA, and Ike 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 I 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.
Table III
. _ .
Antibo dilutionB-&al. active (Odyssey) 1:0 0-49 1:5 0.38 1:~5 ~.26 1:12~ 0.16 negative control 0.15 ~;~39582 While the invention has been described with particular reference to specific examples, it will be understood that these examples are in no way intended to limit the scope of the invention. Various changes and modifications may be made without departing from the spirit of the invention.

Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for detecting the presence of analyte molecules carried on biological cells, comprising:
providing a suspension of lipid vesicles having surface bound anti-analyte molecules, at a surface concentration of at least about 15 molecules per vesicle, and surface-bound reporter molecules, incubating the suspension with the sample cells, to bind the vesicles to the cell surfaces by specific analyte/anti-analyte binding interactions, separating the cells from unbound vesicles, and detecting the presence of reporter associated with the cells.
2. The method of claim 1, for typing cells according to the presence of specific cell-surface antigens, wherein the surface bound analyte molecules are anti-antigen antibody or antibody fragment molecules which are attached immunospecifically to the surface antigens.
3. The method of claim 2, for typing human erythrocyte cells according to their surface antigens, wherein the analyte molecules are IgG antibody molecules specific against a selected surface antigen, and the anti-analyte molecules are anti-IgG antibody or antibody fragment molecules.
4. The method of claim 1, wherein the reporter is .beta.-galactosidase.
5. A system for determining the presence of a selected cellular antigen carried on the surface of a cell comprising a soluble antibody specific against the antigen, and a suspension of lipid vesicles having surface bound antibody or antibody-fragment molecules which are specific against said soluble antibody, at a surface concentration of at least about 15 molecules per vesicle, and surface bound reporter molecules.
6. The system of claim 5, wherein the soluble antibody is an IgG antibody, and the antibody molecules on the lipid vesicles are anti-IgG antibody, or antibody fragment molecules.
7. The system of claim 5, wherein the reporter is .beta.-galactosidase.
8. A system for determination of human subgroup IgG antibodies specific against a selected erythrocyte subgroup antigen, comprising erythrocytes having the selected surface bound subgroup antigen, and a suspension of lipid vesicles having surface bound anti-human IgG
antibody molecules, present at a surface concentration of at least about 15 molecules per vesicle, and surface-bound reporter molecules.
9. The system of claim 8, for determination of anti-D, anti-Jka, or anti-Fya IgG molecules, wherein the erythrocytes have the corresponding D. Jka, or Fya surface antigents, respectively.
CA000455271A 1984-05-28 1984-05-28 Lipid-vesicle-surface assay reagent and method Expired CA1239582A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA000455271A CA1239582A (en) 1984-05-28 1984-05-28 Lipid-vesicle-surface assay reagent and method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CA000455271A CA1239582A (en) 1984-05-28 1984-05-28 Lipid-vesicle-surface assay reagent and method

Publications (1)

Publication Number Publication Date
CA1239582A true CA1239582A (en) 1988-07-26

Family

ID=4127953

Family Applications (1)

Application Number Title Priority Date Filing Date
CA000455271A Expired CA1239582A (en) 1984-05-28 1984-05-28 Lipid-vesicle-surface assay reagent and method

Country Status (1)

Country Link
CA (1) CA1239582A (en)

Similar Documents

Publication Publication Date Title
US4636479A (en) Enhanced agglutination method and kit
US4783400A (en) Homogeneous enzyme immunoassay system and method
US4605630A (en) Large-liposome agglutination reagent and method
US4704355A (en) Assay utilizing ATP encapsulated within liposome particles
US4598051A (en) Liposome conjugates and diagnostic methods therewith
EP0900382B1 (en) Immunoassays to detect antiphospholipid antibodies
US4752572A (en) Lipid vesicles containing labeled species and their analytical uses
WO1984002579A1 (en) Lipid-vesicle-surface assay reagent and method
US4745074A (en) Blood-fluid composition for cell lysis system
US4839276A (en) Interference - resistant liposome specific binding assay
US5210040A (en) Process for coupling antibodies or antibody fragments to liposomes
EP0243471A1 (en) Solid-phase liposome immunoassay system
US4731324A (en) Viral lysis assay
US4806466A (en) Cell agglutination reagent comprising conjugates of antibody covalently bound to liposomes
CA1239582A (en) Lipid-vesicle-surface assay reagent and method
EP0276165A1 (en) Targetted liposomes and their use in immunoassay
US4971916A (en) Liposome based homogeneous immunoassay for diagnostic tests
CA1309344C (en) Liposome based homogeneous immunoassay for diagnostic tests
JP4250704B2 (en) Dry immunoassay reagent
JP2003149246A (en) Method for detecting substance
JP2639573B2 (en) Liposome for measuring complement and method for measuring complement using the same
JPH01229970A (en) Complement measuring method
EP0184701A2 (en) A method for determining a ligand
Kung et al. Recent Developments in the use of Liposomes in in vitro Diagnostic Assays
JP2000235034A6 (en) Dry immunoassay reagent

Legal Events

Date Code Title Description
MKEX Expiry