United States Patent
`
`:19]
`
`Litman et al.
`
`[541
`
`[75]
`
`[73]
`
`[21]
`
`[22]
`
`[51]
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`[51]
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`[53]
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`[56]
`
`MACROMOLECULAR ENVIRONMENT
`CONTROL IN SPECIFIC RECEPTOR
`ASSAYS
`
`inventors: David J. Lilman, Palo Alto; Zvi
`Harel. Stanford; Edwin F. Ullrnan,
`Atherton, all of Calif.
`
`Assignee:
`
`Syvn Company, Palo Alto, Calif.
`
`App]. No.: 964.099
`Filed:
`Nov. 24, 1978
`
`Int. CL1 ..................... .. 3129 1/66; c12N 11/02;
`CIZN ll/10
`435/7; 23/230 B;
`11.5. C1.
`424/12; 435/177; 435/173; 435/310; 435/6
`Field of Search
`23/230 B; 424/1. 15,
`:2; 435/5, 1, 310, in, 173
`424/3.
`References Cited
`
`U.S. PATENT DOCUMENTS
`2/ I914
`Schuurs et al.
`........................ .. 435/T
`10/19??
`Baker et al.
`.... ..
`..
`
`ll/19??
`Johnson
`1/ i9'.i'S
`
`3.".-'91 .9 32
`4.05 2.010
`4,059,685
`11,0619 59
`
`[11]
`
`[45]
`
`4,275,149
`
`Jun. 23, 1981
`
`4,134,792
`4,193,983
`
`........... .. 435/?
`Boguslaski et al.
`1/l9".-'9
`3/1980 Ullman et al.
`............... .. 23/230 B X
`
`OTHER PUBLICATIONS
`
`Wingard, et al., Appiied Bioriiemimy and Bioengineering,
`vol. 1, Academic Press, NY. (1976), pp. 135-133.
`
`Primary Examiner—Thomas G. Wiseman
`Attorney. Agent, or Firm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Method and compositions are provided for performing
`protein binding assays involving a homologous pair
`consisting 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 microenviron-
`merits which allow for discrimination between the label
`associated with the particle—in a discontinuous phase-
`—and the label in the continuous phase.
`Various conjugates and particles are provided which
`find use in the subject method.
`
`46 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech
`IPR2016-00710
`IPR2016-00710
`Genentech Exhibit 2052
`
`Genentech Exhibit 2052
`
`

`
`1
`
`4,275,149
`
`MACROMOLECULAR ENVIRONMENT
`CONTROL IN SPECIFIC RECEPTOR ASSAYS
`
`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 like. One class of
`methods commonly referred to as immunoassays 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 compoundand
`its receptor form a homologous pair. referred to as
`ligand and receptor, where the 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 immunoassays 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 use in the heterogeneous meth-
`ods are radiolabels. enzymes, and fluorescent molecules.
`An alternative to physical separation is to bind one of
`the members ofthe 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 complexed 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 peaks. 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
`monoepitopic ligands. the opportunity exists to allow
`for
`receptors which are labeled differently to be
`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
`
`5
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`-I5
`
`50
`
`SS
`
`65
`
`2
`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
`fluorescing at a wavelength of an energy which is ac-
`cepted by the other chromophore. which acts as a
`quencher. Also, by employing two different enzymes.
`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
`compiex 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 immunoassays 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, t2? (19'i'6) Academic Press, report the
`kinetic and diffusion effects on the immobilization of
`enzymes. U.S. Pat. No. 3,817,337 describes a homoge-
`neous enzyme immunoassay. U.S. Pat. No. 3,996,345
`describes a homogeneous immunoassay employing two
`chromophores related by being ,a fluorescer and a
`quencher. Copertding application Ser. No. 893,650, filed
`Apr. 5, l9'?8. now U.S. Pat. No. 4.233.402, describes a
`technique employing a plurality of enzymes. where the
`substrate of one enzyme is the product of the other
`enzyme. Copending application Ser. No. 815,636, U.S.
`Pat. No. 4.160.645, filed July 14, I977, now U.S. Pat. No.
`4,160,645, describes a homogeneous immunoassay em-
`ployiug a non-enzymatic catalyst as a label. Co-pending
`application Ser. No. 906,514. now U.S. Pat. No.
`4,193,983. filed May I6, I978, describes a labeled liquid
`discontinuous phase for use in immunoassays. Applica-
`tion Ser. No. 667.996, abandoned, filed Mar. I8, 1976,
`describes a homogeneous immunoassay employing as a
`label an enzyme substrate. See also U.S. Pat. No.
`3,853,937, which discloses particles to which are conju-
`gated radioactive and fluorescent labels and antibodies.
`See also U.S. Pat. No. 4,001,400.
`
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for the de-
`termination of an analyte which is a member of a spe-
`
`

`
`4,275,149
`
`3
`cific binding pair—ligand 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 5
`enhancement or diminution of the signal as a result of
`association. The method employs a substantially uni-
`formly dispersed discontinuous phase of discrete solid
`(includes solvent swelled) particles (beads) in an aque-
`ous assay medium. The particles are labeled with one of It]
`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 15
`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 20
`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. 25
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`4
`pair. By appropriate choice of specific binding pair
`conjugates.
`the amount of signal producing member
`bound to the particle can be related to 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 as any additional
`reagents and determines the signal from the assay me-
`dium. By comparing the observed signal with a signal
`obtained from an assay medium having a known 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 categories: (l) 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
`bull: 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 pair, but also a member of the signal producing‘
`system.
`The second effect is a physical effect as a result of the
`chemical nature of the particle. The physical "effect can
`be observed as pl-I, 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 different 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. fluorescer.
`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
`from the particle. Alternatively, phosphorescent
`la-
`beled (includes embedded) particles could be employed
`or particles having labels capable of energy transfer to a
`chromogen.
`
`35
`
`A method is provided for determining low concen-
`trations of organic compounds in a wide variety of 30
`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
`discontinous 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 phase.
`The particles are large discrete solid beads providing
`an environment for a label which may be distinguished 40
`from the environment of the bulk solution, preferably
`po'rous, 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 analyte in the assay me-
`dium.
`
`45
`
`50
`
`55
`
`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-
`ing. Thus receptor as an analyte allows for a number of 65
`alternative conjugates. In addition, one of themembers
`of the signal producing system will be bound or become
`bound to the reciprocal member of the specific binding
`
`

`
`4,275,149
`
`5
`In performing the subject method, therewill be at
`least two reagents: the particle conjugate; and the spe-
`cific binding pair member conjugate. These conjugates
`will vary depending upon the nature ofthe analytc. the
`nature ofthe 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-
`'lEl'¥l.
`
`DEFINITIONS
`
`Analyte—the compound or composition to be mea-
`sured. which may be a ligand. which is mono— 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 different 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 lantiligand).
`Ligand—any organic compound for which a recep-
`tor naturally exists or can be prepared.
`Receptor (antiligand‘,I—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.
`Poly(ligand-analog)—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. hydroxy. 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 10 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 phase}—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 convex surface. preferably being porous and
`having channels or indentations, which can be widely
`varied as to the size of molecule or assembly which is
`excluded. defining an environment different from the
`medium in which the particles are dispersed. The parti-
`cles will be readily dispersible in an aqueous medium,
`and either polyfunctionalized or capable of polyfunc-
`
`6
`tionalization for linking to other molecules. Depending
`on the signal producing system, the particles may be
`substantially transparent to light in a substantial wave-
`length range between 300 and 800 nm, 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 electromagnetic radiation.
`and depending on the system employed, the level of
`signal will vary to the extent the signal producing sys-
`tem is in the environment of the solid phase particles.
`For the most part. the signal producing system will
`involve enzymes and chromophores, 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, electrochemical changes, thermal changes. neph-
`elometric changes. and the like may also find applica-
`tion.
`
`I.abel—-—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--a 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 a bond.
`linking group. or hub nucleus: The labeled 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 poly(Iigani:l 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
`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.
`
`10
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`

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`4,275,149
`
`7
`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 4-1 1, more usually in the range ofabout 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 particle buffer employed is not critical to
`this invention but in individual assays, one buffer 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 l0"—50" C.,
`more usually from about 15"-40“ C.
`The concentration of analyte which may be assayed
`will generally vary from about 10—4to 10-15 M, more
`usually from about 10-5 to 10-13 M. Considerations
`such as whether the assay is qualitative, semi-qualitative
`or quantitative, the particular detection technique and
`the concentration of the analyte of interest will nor-
`mally determine the concentration of the other rea~
`gents.
`While the concentrations of the various reagents in
`the assay medium will generally be determined by the
`concentration range of interest and of the analyte, the
`final concentration of each of the reagents will normally
`
`8
`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 inter-
`est based on analyte binding sites, usually about 0.1 to
`100 times, more usually about 0.3-10 times the maxi-
`mum concentration of interest. By concentration is
`intended the available concentration, that is. the con-
`centration 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 analyte 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.
`the association of the
`Since with many receptors,
`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 to
`the ligand. Usually,
`incubation steps will vary from
`about 0.5 min to 6 hrs, more usually from about 5 min to
`I 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-
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`S0
`
`55
`
`60
`
`65
`
`

`
`4,275,149
`
`9
`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 run. usually from
`about 350 to 650 nm.
`
`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.
`
`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 l hr. For the equilibrium mode.
`after a steady state is achieved. a single reading may be
`sufficient or two readings over any convenient time
`interval may suffice.
`The variety of effects which may be achieved by the
`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
`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
`
`Signal
`Binding
`Producing
`Pair
`Particle
`Member
`System
`
`Analtyci Conjugate:
`Conjugate-3
`Reagents‘
`Ag
`P-Ag
`Ab-En:
`3. b or c
`Ag
`P-Ab
`Ab-En:
`a. b or c
`Ag
`P-Ab
`Ag-Enz
`a. b or c
`Ag
`P-Ag
`Ah-Enzi-Eat;
`a
`Ag
`P-Ab
`Ab-Enzi-Enzz
`a
`Ag
`P-Ab
`Ag-Enz]-Enzz
`rt
`Ag
`P-Ag _
`Ah-Enz]. Ab-Enzg
`3
`Ag
`P-Ab
`Ab-E-'.nz|. Ab-Enz:
`:1
`Ag
`P—Ab
`Ag-Enzt. Ag-Enz:
`:1
`Ag
`P-Ag-Enz:
`Ab-Enz;
`a
`Ag
`P-Ab-Enzt
`Ab-EH21
`a
`Ag
`‘ P-Ab-Enz;
`Ag-Enz;
`at
`A3
`P-Ag
`Ab-F
`e. l'
`A;
`E’-Ab
`Ab-F
`2. 1'
`Ag
`I’-Ag-F
`Ab-En:
`d
`Ag
`l‘-Ab-F
`Ab-En:
`d
`Ag
`P-Ab-F
`Ag-En:
`d
`Ag
`P-Ag
`Al»!-‘
`e. f
`Ab
`P-Ab
`Ab-F
`e. |'
`Ab
`P-Ah-F
`Ag-En:
`d
`Ab
`P-Ag
`Ab-En:
`it. b or c
`
`I0
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`4-0
`
`41-5
`
`50
`
`55
`
`65
`
`Rather than unduly extending the table. the situation
`with antibody or receptor as an analyte is illustrated in
`comparison to the situation with antigen as analyte.
`Generally, where antibody is the analyte, in each of the
`illustrative examples, one need merely replace the sym-
`bol for antigen with antibody and the symbol for anti-
`body with antigen.
`The ligand may be mono- or polyepitopic. In most
`situations this difference will not affect the mann