[11]
`
`[-15]
`
`4,374,925
`
`3
`
`Feb. 22, 1983
`
`4,059,685 I1/19?? Johnson
`4,067,959
`1/1973 Bolz
`4.134.792
`1/I979 Boguslaslti et al. .
`4,193,983 3/ I930 Ullman et nl.
`4,263,663 5/I98] Skold ....... ..
`4,275,149
`6/1981 Lilman etal. ......
`
`
`
`435/?
`435/7
`435.’?
`23/230 B
`435/7x
`-135/7
`
`omen PUBLICATIONS
`
`Wingard et al.. Applied Biochemistry and Bioengineering.
`vol. 1. Academic Press._NY (1976), pp. 135-133.
`
`Primary EIxam:'ner~—'I'homas G. Wiseuaau
`Attorney, Agent. or Fi'rm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Method and compositions are provided for performing
`protein binding assays involving a homologous pair
`consistipg of ligand and receptor for the ligand. The
`method employs a label conjugated to a member of said
`homologous pair and a uniformly dispersed discontinu-
`ous phase of discrete particles in a continuous aqueous
`phase. where the discrete particles create n1icroenviron—
`ments which allow for discrimination between the label
`
`associated with the partic1e—in a discontinuous phase-
`—and the label in the continuous phase.
`
`Various conjugates and particles are provided which
`find use in the subject method.
`
`4Clm'ms,NoDrawiIIss
`
`SANOFI V. GENENTECH
`SANOFI v. GENE(cid:49)TECH(cid:3)
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2045
`EXHIBIT 2045
`
`United States Patent [191
`
`Litman et al.
`
`[54]
`
`[75]
`
`[73]
`
`[ ‘ l
`
`MACRDMOLECIJLAR ENVIRONMENT
`CONTROL IN SPECIFIC RECEPTOR
`ASSAYS
`
`Inventors: David J. Litman, Palo Alto; Zvi
`Harel, Stanford; Edwin F. Ullruan.
`Atherton, all of Calif.
`
`Assignee:
`Notice:
`
`Syva Company, Palo Alto, Calif.
`
`The portion of the term of this patent
`subsequent to Jun. 23, 1998, has been
`disclaimer].
`
`[21]
`
`{I21
`
`Appl. No.:
`Filed:
`
`232,???
`
`Feb. 9, 1981
`
`[621
`
`[511
`[52]
`
`[53]
`
`[56]
`
`Related US. Application Data
`Division of Ser. No. 964.099, Nov. 24, 1978, Pat. No.
`4,215,149.
`Int. Cl.’
`U.S. Cl.
`
`GDIN 33/5-I
`........................................... 435/7; 435/5;
`435/177; 435/310; 436/529; 436/300
`Field of Search .................. .. 23/230 B; 424/3, 12;
`435/5, 1'. 310, 177. 178
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3.191.932 2/ I974 Schuurs et al.
`4.us2,n1o in/I9-n Bakeret al.
`
`......................... 435/7
`424/12
`
`

`
`1
`
`4,374,925
`
`MACROMOLECULAR ENVIRONMENT
`CONTROL IN SPECIFIC RECEPTOR ASSAYS
`
`This is a divisional of application Ser. No. 964,099,
`filed on Nov. 24, 1973. which issued as U.S. Pat. No.
`4,275,149, on June 23, 1981.
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The measurement of trace amounts of a wide variety
`of organic compounds has become essential in medi-
`cine. ecology, quality control, and the lilte. One class of
`methods commonly referred to as ilnmunoassays is de-
`pendent upon the use of a compound or receptor which
`specifically binds to another compound having a partic-
`ular spatial and polar organization. The compound and
`its receptor form a homologous pair, referred to as
`ligand and receptor. where t.he receptor is normally
`antibody. One of the members of the homologous pair is
`bound to a label which is capable of providing a detecti-
`ble signal.
`The category of imrnunoassays may be further bro-
`ken down into what is referred to as heterogeneous and
`homogeneous. The heterogeneous techniques are de-
`pendent upon separating associations or complexes of
`the homologous pair from members of the pair which
`are not associated. Since the complexes will substan-
`tially differ in molecular weight from the dissociated
`members, techniques such as centrifugation can be used
`to separate the associated from the dissociated mem-
`bers. One can then measure the label either in the phase
`containing the dissociated members or the phase con-
`taining the associated members. For the most part the
`labels which have found me in the heterogeneous meth-
`ods are radiolabels, enzymes, and fluorescent molecules.
`An alternative to physical separation is to bind one of
`the members of the homologous pair to a solid support,
`which may or may not absorb the aqueous medium. The
`solid support can then provide for the separation since
`the complerred or associated ligand and receptor is
`bound to the solid support. This allows for relatively
`easy separation between the aqueous assay medium and
`the solid support.
`The homogeneous methods rely on the formation of
`complexes to modulate the signal obtained from the
`label. The dissociated conjugated label provides for a
`different level of signal from the associated conjugated
`label with its receptor. For example where the ligand is
`conjugated to a stable free radical, the association of the
`conjugate with its homologous receptor results in a
`substantial flattening of the esr pealts. With enzymes as
`labels to which ligands have been conjugated. the bind-
`ing of receptor to the ligands can result in steric inhibi-
`tion of the approach of substrate to the active site of the
`enzyme or allosteric modification of enzyme activity.
`The presence of ligand in the assay medium reduces the
`amount of available receptor for binding to the label
`conjugate and thus affects the amount of the label con-
`jugate which becomes associated with receptor. There-
`fore, by measurement of the signal from the label, one
`can relate the level of signal to the amount of ligand in
`the assay medium.
`An alternative to employing the receptor to directly
`affect the signal by its bulk is the opportunity to bring
`together two labels which interact. Where a ligand is
`polyepitopic or a polyepitopic ligand is formed from
`rrronoepitopic ligands, the opportunity exists to allow
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`receptors which are labeled differently to be
`for
`brought together when bound to the ligand or to have
`ligand with one label and receptor with a different label,
`which when the ligand and receptor are associated
`bring the labels into close spatial proximity. Where the
`different labels interact to affect the amount of signal
`observed, the associated ligand and receptor will pro-
`vide for a different signal level from the dissociated
`labeled receptor.
`This technique has been employed with chromo-
`phores which are related by one of the chromophores
`lluoresclng at a wavelength of an energy which is ac-
`cepted by the other chromophore, which acts as a
`quencher. Also. by employing two different enzym,
`where the product of one enzyme is the substrate of the
`other enzyme. one can observe an enhanced turnover in
`the complex, as compared to the dissociated label.
`The focus of effort in the homogeneous immunoassay
`area has been directed to either employ the properties of
`the complex to modulate the signal or to provide for the
`complex to bring together in close spatial proximity
`different labels which are related and provide for differ-
`ent degrees of interaction in relation to their distance
`from each other.
`In developing immunoassays, there are many consid-
`erations, not the least of which is sensitivity. For mea-
`suring extremely small amounts of a ligand, it is either
`necessary to have a label which is detected at very low
`levels with high accuracy or to provide for a plurality
`of events associated with an individual ligand. Another
`consideration is interference by the foreign materials
`present and the degree to which the interference can be
`minimized or removed.
`
`Another problem associated with imrnunoassaya is
`labeling, particularly where the ligand or receptor is
`impure. The background resulting from conjugation of
`the label to compounds other than those of the homolo-
`gous pair must be maintained at a minimum in order to
`obtain a satisfactorily sensitive assay. Other consider-
`ations include simplicity of protocol, ease of measure-
`ment, reproducibility, sensitivity to extraneous factors
`and the like.
`
`2. Description of the Prior Art
`Engasser and Horvath, Applied Biochem. Bioengi-
`neering, Vol. 1, 12'! (1976) Academic Press. report the
`kinetic and diffusion effects on the immobilization of
`enzymes U.S. Pat. No. 3,817.33”! describes a homogene-
`ous enzyme immunoassay. U.S. Pat. No. 3,996,345 de-
`scribes a homogeneous immunoassay employing two
`chrornophores related by being a fluorescer and a
`quencher. Copending application Ser. No. 893,650, filed
`Apr. 5, 1973, describes a technique employing a plural-
`ity of enzymes, where the substrate of one enzyme is the
`product of the other enzyme. Copending application
`Ser. No. 815,636. filed July 14, 19'l7, describes a homo-
`geneous immunoassay employing a non-enzymatic cata-
`lyst as a label. Co-pending application Ser. No. 906,514,
`filed May 16, 1978, describes a labeled liquid discontinu-
`ous phase for use in irnmunoassays. Application Ser.
`No. 667.996, filed Mar. 18, 1976, describes a homogene-
`ous immunoassay employing as a label an enzyme sub-
`strate. See also ‘U.S. Pat. No. 3,853,937, which discloses
`particles to which are conjugated radioactive and fluo-
`rescent labels and antibodies. See also U.S. Pat. No.
`4,001,400.
`
`

`
`3
`
`SUMMARY OF THE INVENTION
`
`4,374,925
`
`Methods and compositions are provided for the de-
`termination of an analyte which is a member of a spe-
`cific binding pair—liga.nd and homologous receptor—-
`where no separation or segregation is required for the
`determination. The method does not rely on a "bulk
`effect where one observes the sum of the signal from the
`labels of associated members, but rather relies on an
`enhancement or diminution of the signal as a result of
`association. The method employs a substantially uni«
`torn-ily dispersed discontinuous phase of discrete solid
`(includes solvent swelled) particles (beads) in an aque-
`ous assay medium. The particles are labeled with one of
`the members of the specific binding pair.
`The particles create a physical or chemical environ-
`ment distinctively different from the continuous aque-
`ous phase. A signal producing system is provided which
`produces a substantially different
`level of detectible
`signal depending upon whether the signal producing
`system operates in the solid or liquid aqueous phase. By
`causing the distribution between the solid and liquid
`phase of the signal producing system to be related to the
`amount of analyte in the assay medium, the observed
`signal will be a function of the amount of analyte in the
`assay medium.
`Conjugates to particles are provided for use in the
`method, as well as reagent compositions and kits. Also,
`specific compounds are provided as special substrates.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`A method is provided for determining low concen-
`trations of organic compounds in a wide variety of
`media. particularly having physiological activity, either
`being naturally present in physiological fluids, or ad-
`ministered to vertebrates. The method employs as an
`assay medium a continuous liquid aqueous phase and a
`discontinuous solid phase comprised of discrete small
`particles having relatively slow settling rates and being
`capable of providing an environment different from the
`environment of the continuous phm.
`The particles are large discrete solid beads providing
`an environment for a label which may be distinguished
`from the environment of the bull: solution, preferably
`po'rou.s, providing channels or surface indentations of
`substantial depth where the liquid environment in the
`channel or indentation is significantly affected by the
`substantially encompassing solid phase. A signal pro-
`ducing system is provided, where the signal producing
`system, in whole or in part, is partitioned between the
`two phases in relation to the amount of analyte present
`in the assay medium. Since the observed signal will be
`substantially different depending upon the degree to
`which the signal producing system is partitioned be-
`tween the liquid and the solid phase. the measured sig-
`nal will reflect the amount of anulyte in the assay me-
`dium.
`The analyte will be a member of a specific binding
`pair consisting of the ligand and its homologous recep-
`tor. The solid phase particles or heads will be bound,
`directly or indirectly, covalently or non-covalently to
`one of the members of the specific binding pair. There is
`an exception where a specific type of receptor to a
`specific ligand is the analyte.
`three specific binding
`components are required, viz receptor, antireceptor or
`ligand. which may be bound to the particle, and ligand
`or antireceptor respectively, employed for other label-
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`ing. Thus receptor as an analyte allows for a number of
`alternative conjugates. In addition,._one of the members
`ofthe signal producing system ‘will be bound or become
`bound to the reciprocal member of the specific binding
`pair. By appropriate choice of specific binding pair
`conjugates,
`the amount of signal producing member
`bound to the particle can be relatedjto the amount of
`analyte in the assay medium.
`In carrying out the method, one combines the analyte
`containing sample,
`the labeled particles,
`the labeled
`specific binding pair member. as well asany additional
`reagents and determines the signal from the assay me-
`dium. By comparing the observed signal with 3 signal
`obtained from an assay medium having aknown amount
`of analyte, one can qualitatively or quantitatively deter-
`mine the analyte of interest. One can use the properties
`of the discrete particles in a number of different ways.
`Arbitrarily will be divided into two categorim: (1) diffu-
`sion; and (2) physical effects:
`By appropriate choice of porous particles. one can
`affect the rate at which a molecule or molecular assem-
`bly moves through the volume of the liquid phase adja-
`cent to.the solid particle surface. The effect of the steric
`bulk and narrow channels of the particles is to reduce
`the rate of migration of a molecule or molecular assem-
`bly toward and away from the particle surfaces, as
`compared to the rate of migration in the bulk solution.
`by virtue of physical constraint, and the like. Thus. one
`can create a substantial concentration gradient between
`the bulk liquid aqueous phase and the liquid portion
`adjacent the solid phase surface. A signal producing
`system which is sensitive to the concentration of a spe-
`cies will give substantially different signal levels in the
`bulk liquid phase as compared to the solid phase.
`By having two members of the signal producing sys-
`tem which cooperate, that is. one member provides a
`compound which interacts with the second member.
`one can greatly enhance the localized concentration of
`the compound in the solid phase as compared to the
`bulk liquid phase. in these situations, the particle would
`not only be labeled with a member of the specific bind-
`ing puir, but also a member of the signal producing
`system.
`The second effect is a physical effect as I result of the
`chemical nature of the particle. The physical effect can
`be observed as pll, spectroscopic properties, and the
`like. In effect. the environment created by the particle
`surfaces, particularly in the channels or pores, for a
`molecule is substantially different from the environment
`in the bulk solution. Where the signal producing mem-
`ber is sensitive to its environment, there will be a sub-
`stantially ilifferent signal depending upon whether the
`signal producing member is in the solid phase or in the
`bulk solution. For example, the activity of an enzyme is
`pH dependent. By appropriate choice of buffer and an
`ion exchange resin, the pH at the surface of the solid
`phase can be distinctively different from the pH in the
`bulk solution. The enzymatic activity would therefore
`vary depending upon the partitioning of the enzyme
`between the two phases.
`The polarity between the particle and the bulk solu-
`tion can be greatly varied by employing a hydrophobic
`particle. The hydrophobic character could activate or
`deactivate an enzyme or chromogen e.g. lluorescer.
`The spectroscopic effect can be exemplified by em-
`ploying opaque.
`transparent or partially transparent
`(within a limited wavelength range) particles. One
`could therefore control the light that entered or exited
`
`

`
`4,374,925
`
`5
`la-
`from the particle. Alternatively, phosphorescent
`beled (includes embedded) particles could be employed
`or particles having labels capable of energy transfer to a
`chrornogen.
`In performing the subject method, there will be at
`least two reagents: the particle conjugate; and the spe-
`cific binding pair member conjugate. These conjugates
`will vary depending upon the nature of the analyte, the
`nature of the signal producing system. and the nature of
`the particle. In addition. by covalently bonding mole-
`cules. particularly enzymes to the particle, one can
`create a concentration gradient, where the bulk solution
`has a relatively low concentration of the particular
`compound or enzyme product. These molecules can be
`part of the signal producing system or merely provide
`an environment which affects the signal producing sys-
`tem.
`
`DEFINITIONS
`
`Analyte—the compound or composition to be mea-
`sured. which may be a ligand. which is rnono- or
`polyepitopic. antigenic or haptenic. a single or plurality
`of compounds which share at least one common epi-
`topic site or a receptor.
`Specific binding pair—two dilferent molecules,
`where one of the molecules has an area on the surface or
`in a cavity which specifically binds to a particular spa-
`tial and polar organization of the other molecule. The
`members of the specific binding pair are referred to as
`ligand and receptor fantiligand).
`Ligand—any organic compound for which a recep-
`tor naturally exists or can be prepared.
`Receptor (antiligand)—any compound or composi-
`tion capable of recognizing a particular spatial and polar
`organization of a molecule i.e. epitopic site. Illustrative
`receptors include naturally occurring receptors. e.g.
`thyroxine binding globulin. antibodies. enzymes. Fab
`fragments. lectins and the like.
`Ligand Analog—a modified ligand which can com-
`pete with the analogous ligand for a receptor. the modi-
`fication providing means to join a ligand analog to an-
`other molecule. The ligand analog will normally differ
`from the ligand by more than replacement of a hydro-
`gen with a bond which links the ligand analog to a hub
`or label.
`Polyfligand-ana.log)—a plurality of ligands or ligand
`analogs joined together covalently. normally to a hub
`nucleus. The hub nucleus is a polyfunctional material.
`normally polymeric. usually having a plurality of func-
`tional groups e.g. hydrory. amino, mercapto. ethylenic,
`etc. as sites for linking. The hub nucleus may be water
`soluble or insoluble. preferably water soluble, and will
`normally be at least about 35.000 molecular weight and
`may be'l0 million or more molecular weight, but usu-
`ally under 600.000. more usually under 300.000. Illustra-
`tive hub nucleii include polysaccharides, polypeptides.
`including proteins, nucleic acids. ion exchange resins
`and the like. Water insoluble hub nucleii can be the
`same as those indicated for the particle.
`Particle (solid phsse)—the particle is a discrete solid
`particle. which may be swelled or remain unswelled by-
`the liquid phase. and composed of a wide variety of
`both hydrophobic and hydrophilic materials. The parti-
`cles will be solid. hollow or porous. having a substan-
`tially smooth or irregular surface. having a primarily
`concave or convert surface. preferably being porous and
`having channels or indentations, which can be widely
`varied as to the size of molecule or assembly which is
`
`6
`excluded. defining an environment different from the
`medium in which the particles aredispersed. The parti-
`cles will be readily dispersiblejn an aqueous medium.
`and either polyfunctionalizcd-or capable of polyftInc-
`tionalization for linking to other-.mo1ecules. Depending
`on the signal producing system, the particles may be
`substantially transparentto light in a substantial wave-
`length range between 300 and 300 um. preferably
`through the range or be opaque over the entire ultravio-
`let and visible range.
`Signal producing system—the signal producing sys-
`tem may have one or more components. at least one
`component being conjugated to a specific binding pair
`member. The signal producing system produces a mea-
`surable signal which is detectible by external means,
`usually the measurement of electromagnefic radiation.
`and depending on the system employed. the level of
`signal will vary to the extent the signal producing sys-
`tem is iu the environment of the solid phase particles.
`For the most part, the signal producing system will
`involve enzymes and chrornophores. where chromo-
`phores include dyes which absorb light in the ultravio-
`let or visible region., phosphors,
`fluorescers. and
`chemiluminescers. While for the most part, the signal is
`conveniently the absorption or emission of electromag-
`netic radiation. usually in the ultraviolet or visible
`range, electrochemicalchanges, thermal changes, neph-
`elornetric changes. and the like may also find applica-
`tion.
`
`Label—the label may be any molecule conjugated to
`another molecule and is arbitrarily chosen as to which
`molecule is the label. In the subject invention. the labels
`will be the specific binding pair molecule that is conju-
`gated to the particle or a molecule which is part of the
`signal producing system that is conjugated to a member
`of the specific binding pair or to a particle.
`Particle conjugate—the particle to which is bound.
`directly or indirectly a member of the specific binding
`pair, and. as appropriate one or more members of the
`signal" producing system. A substantial proportion of the
`labels conjugated to the particle will be influenced by
`the particle surface, usually within the channels and
`pores of the particle when these are present. so that
`where the signal producing member is bound to the
`particle.
`there is a property of the conjugate which
`differentiates the signal obtained from the particle as
`compared to the signal obtained from the bulk solution.
`Binding pair label—h member of the specific binding
`pair employed for binding its homologous member to
`the particle directly bonded to the particle.
`Signal label—-a member of the signal producing sys-
`tem which is directly or indirectly (through the binding
`of a specific binding pair) bonded to a binding pair
`member or to the particle.
`Binding pair member conjugate or signal label con-
`jugate—the conjugate of the binding pair member with
`a member of the signal producing system (signal label).
`Labeled ligand—the conjugate of the ligand member
`of the specific binding pair with a member of the signal
`producing system, either covalently or noncovalently
`bound. when covalently joined. either joined by abond.
`linking -group, or hub nucleus. Thelabeled ligand may
`have one or more ligands (includes ligand analogs) or
`one or more labels or a plurality of both. the latter being
`referred to as polyfligand analog)-polylabel.
`Labeled receptor—the conjugate of receptor with a
`member of the signal producing system. where the two-
`are bound either covalently or non-covalently, usually
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`4,374,925
`
`7
`covalently by a linking group, where there may be one
`or more receptors bound to the label, but usually one or
`more labels bound to the receptor.
`Macromolecular reagent—a reagent capable of inter-
`acting with a member of the signal producing system to
`modulate the signal and at least in part sterically ex-
`cluded from interacting with a member of the signal
`producing system in the environment of the particle
`conjugate through steric constraints or reduced rates of
`diffusion. The reagent will usually have a minimum
`molecular weight of at least about 20,000, more usually
`at
`least about 40,000 and preferably at
`least about
`100,000. The reagent may naturally have such molecu-
`lar weight or the active compound linked to a hub nu-
`cleus to provide the desired molecular weight.
`METHOD
`
`The subject assay is carried out in an aqueous zone at
`a moderate pH, generally close to optimum assay sensi-
`tivity, without separation of the assay components or
`products. The assay zone for the determination of ana-
`lyte is prepared by employing an appropriate aqueous
`medium, normally buffered,
`the unknown sample,
`which may have been subject to prior treatment, the
`particle conjugate. the binding pair member conjugate.
`all of the materials required for the signal producing
`system for producing a detectible signal. as well as
`members of the specific binding pair or their analogs, as
`required.
`The presence of ligand or its homologous receptor
`(antiligand) in the unknown will affect the partition of
`the signal producing system between the particle or
`solid phase and the bulk solution in the assay medium.
`In carrying out the assay, an aqueous medium will
`normally be employed. Other polar solvents may also
`be included. usually oxygenated organic solvents of
`from 1-6, more usually from 1-4 carbon atoms. includ-
`ing alcohols, ethers and the like. Usually these cosol-
`vents will be present in less than about 40 weight per-
`cent, more usually in less than about 20 weight percent.
`The pH for the medium will usually be in the range of
`about 4-11. more usually in the range of about 5-10, and
`preferably in the range of about 6.5-9.5. The pH is
`chosen so as to maintain a significant level of specific
`binding by the receptor while optimizing signal produc-
`ing efficiency. In some instances, a compromise will be
`made between these two considerations. Various buff-
`ers may be used to achieve the desired pH and maintain
`the pH during the determination. Illustrative buffers
`include borate, phosphate, carbonate, Tris. barbital and
`the like. The particular buffer employed is not critical to
`this invention but in individual assays. one bufier may
`be preferred over another.
`Moderate temperatures are normally employed for
`carrying out the assay and usually constant tempera-
`tures during the period of the measurement. particularly
`for rate determinations. The temperatures for the deter-
`mination will generally range from about 10°-50' C.,
`more usually from about l5'—4-0' C.
`The concentration of analyte which may be assayed
`will generally vary from about l0*‘ to 10- *5 M, more
`usually from about 10*‘ to 10-43 M. Considerations
`such as whether the assay is qualitative, semi-quantita-
`‘live or quantitative, the particular detection technique
`and the concentration of the analytc of interest will
`normally determine the concentration of the other rea-
`gents.
`
`8
`While the concentrations of the various reagents in
`the assay medium will generally be determined by the
`concentration range of interest of the analytc, the final
`concentration of each of the reagents will normally be
`determined empirically to optimize the sensitivity of the
`assay over the range of interest. The total binding sites
`of the members of the specific binding pair which are
`reciprocal to the analyte will be not less than about 0.1
`times the minimum concentration of interest based on
`binding sites of analyte and usually not more than about
`1,000 times the maximum concentration of interest
`based on analyte binding sites, usually about 0.1 to 101}
`times. more usually about (13-10 times the maxirnurn
`concentration of interest. By concentration is intended
`the available concentration, that is. the concentration at
`saturation, and not necessarily the actual concentration
`where members of the specific binding pair may not be
`equally available for binding.
`Depending upon the particular signal producing sys-
`tem, as well as the manner in which the specific binding
`pair members are employed, the amount of the various
`conjugates can be varied quite widely. For example,
`one could have very large excesses of the binding pair
`label
`in the particle conjugate, by first allowing the
`binding pair member conjugate to react with the un-
`known, followed by combining with the particle conju-
`gate. Where a competition mode was employed, in that
`the particle conjugate and the binding pair member
`conjugate are added to the unknown simultaneously.
`large excesses of the binding pair label might reduce the
`sensitivity of the assay. ‘Therefore, as indicated previ-
`ously. by employing various concentrations of the vari-
`ous reagents with analytc at concentrations in the range
`of interest, one would obtain ratios which would Opti-
`mize the assay response.
`The order of addition of the various reagents may
`vary widely, depending upon the particular labels, the
`compound to which the label is conjugated, the nature
`of the conjugates, the nature of the analyte, and the
`relative concentrations of the analyte and reagents.
`Also affecting the order of addition is whether an equi-
`librium mode or rate mode is employed in the determi-
`nation.
`Since with many receptors, the association of the
`specific binding pair members is almost
`irreversible
`during the time period of the assay. one will normally
`avoid combining the particle conjugate with the signal
`label conjugate, prior to the addition of the analyte,
`where the two conjugates are reciprocal members of
`the specific binding pair. By contrast, where the two
`conjugates have the same member of the specific bind-
`ing pair, one could combine them prior to introduction
`of the unknown sample into the assay medium. Regard-
`less of the nature of the analyte, all the reagents can be
`added simultaneously and either a rate or equilibrium
`determination made.
`One or more incubation steps may be involved in
`preparing the assay medium. For example,
`it may be
`desirable to incubate an antigen analyte with labeled
`receptor. In addition, it may be desirable to have a
`second incubation after addition of the particle conju-
`gate. Whether to employ an incubation period and the
`length of the incubation period, will depend to a sub-
`stantial degree on the mode of determination—rate or
`equilibrium—and the rate of binding of the receptor of
`the ligand. Usually,
`incubation steps will vary from
`about 0.5 min to 6 hrs, more usually from about 5 min to
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`

`
`4,374,925
`
`10
`TABLE I-continued.
`
`9
`1 hr. Incubation temperatures will generally range from
`about 4' to 50° C., more usually from about 15' to 37' C.
`After the reagents are combined, the signal will then
`be determined. The method of determination may be
`the observation of electromagnetic radiation, particu-
`larly ultraviolet and visible light, either absorption or
`emission, colorimetric, electrochemical, nephelometric,
`or the like. Desirably, the signal will be read as electro-
`magnetic radiation in the ultraviolet or visible region,
`particularly from about 250 to 750 am, usually from
`-about 350 to 650 um.
`The temperature at which the signal is observed will
`generally range from about 10' to 50' C.. more usually
`from about 15' to 40' C.
`
`5
`
`10
`
`2|)
`
`Standard assay media can be prepared which have
`known amounts of the analyte. The observed signal for
`the standard assay media may then be plotted, so as to
`relate concentration to signal. Once a standard curve
`has been established. a signal may be directly related to
`the concentration of the analyte.
`The time for measuring the signal will vary depend-
`ing on whether a rate or equilibrium mode is used, the
`sensitivity required, the nature of the signal producing
`system and the like. For rate mode the times between
`readings will generally vary from about 5 sec to 6 hrs.
`usually about 10 sec to 1 hr. For the equilibrium mode,
`after a steady state is achieved, a single readig may be
`sufficient or two readings over any convenient time
`interval may suffice.
`The variety of effects which may be achieved by t_he
`particles. allows for great versatility in designing rea-
`gents for the assay. The following table is illustrative of
`the more obvious variations permitted with signal pro-
`ducing systems employing one or more enzymes. The
`list is not intended to be exhaustive, but rather illustra-
`tive of the simpler and more accessible signal producing
`systems and reagent combinations. In addition, it should 40
`be appreciated, that different combinations will be pre-
`ferred depending upon the required sensitivity of the
`assay, the nature of the analyte, as well as the source of
`the unknown sample.
`
`TABLE I
`
`45
`
`Signal
`binding
`Producing
`Pair
`Particle
`‘Member
`System
`
`Analtyel
`Conjugate‘
`Conjugate’
`Reagents‘
`Ag
`P-Ag
`Ab-En:
`H. b or c
`Ag
`P—Ab
`Ab-En:
`s, b or c
`Ag
`P-Ab
`Ag-En:
`a, b or c
`Ag
`P-Ag
`t-\b-Ens:-Enzz
`a
`Ag
`F-ab
`Ab-E1321-Enzz
`a
`Ag
`P—Ab
`Ag-Eur]-Ens:
`a
`Ag
`P-Ag
`Ab-EM}. Ab-Enz;
`a
`Ag
`P-Ab
`Ab-Elli}, Ab-Em;
`a
`Ag
`P-Ab
`As-Enz|. Ag-Enz1
`a
`Ag
`P-Ag-Enzi
`Ab-Ens:
`:1
`Ag
`P-Ab-Enz]
`Ab-Etlzz
`:1
`Ag
`P-Ab-Enz]
`Ag-Enz;
`a
`Ag
`P-Ag
`Ab-F
`e, f
`Ag
`P-Ab
`db-F
`e. I’
`Ag
`‘P-Ag-F
`Ab-En:
`d
`Ag
`P-Ab-F
`Ab-En:
`d
`Ag
`P-Ab-F
`Ag-En:
`d
`Ag
`P-Ag
`Ab-F
`e. 1’
`Ab
`P—Ab
`Ab]:
`c. f
`as
`P-Ab-F
`Ag-En:
`d
`Ab
`P-Ag
`Ab-Ens
`s. b or c
`
`55
`
`65
`
`Signal
`Producing
`System
`Reagents‘
`I. b or c
`
`Binding
`Pair
`Member
`Conjugate’
`Ag-En:
`
`Particle
`Conjugate‘
`P-Ab
`
`Analtyel
`Ab
`'As—lisInIl
`.Ab—rocepInr, usually polyvnleol
`11’-ilg particle oonjuglled wit