United States Patent [:91
`
`[111
`
`4,256,834
`
`[4s] Mar. 17, 1981
`Znk et al.
`
`
`
`particulate reagents for determining an analyte which is
`a member of a specific binding pair-ligand and homolo-
`gous receptor. The assay employs as a first reagent, a
`member of said pair bound to an insoluble particle (par
`ticle conjugate); as a second reagent, a label which is
`part of a signal producing system, bound to a member of
`said pair (signal label conjugate); and as a third reagent,
`a signal
`repressor comprising an insoluble particle.
`where the signal repressor is obstructed from interact-
`ing with said label of said signal label conjugate, when
`said signal
`label conjugate is bound to said particle
`conjugate.
`
`In performing the assay, the analyte, the reagents, and
`any ancillary materials are combined in an aqueous
`assay medium and the signal determined as compared to
`an assay medium having a known amount of analyte.
`
`Tl1e repressor greatly enhances the sensitivity and accu-
`racy of the immunoassay in repressing the signal pro-
`duced by labels which are not bound to the particle
`conjugate, thus substantially limiting the observed sig-
`nal to label bound to the particle conjugate. The labels
`which are employed provide a signal which does not
`differ significantly from when the signal label conjugate
`is bound to the particle conjugate or is free in the bulk
`solution. Illustrative labels include chromogens, such as
`fluorescers. chemilumincscers, and the like.
`
`Particular reagents and kits are provided, where the kits
`have predetermined amounts of the various reagents to
`substantially optimize the sensitivity of the assay.
`
`[54] FLUORESCENT SCAVENGER PARTICLE
`IMMUNOASSAY
`
`['l 5]
`
`Inventors: Robert F. Zulr, Mountain View;
`Edwin F. Ulla-nan, Atherton, both of
`Calif.
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] Appl. No.: 28,640
`
`[22} Filed:
`
`Apr. 9, 1979
`
`[51]
`
`[52] U.S. Cl.
`
`Int. Cl.-‘ ..................... .. C12Q 1/66; A6lK 37/00;
`GUIN 31/00;Go1N 23/00
`435/7; 23/230 3;
`424/3; 424/12; 435/5; 435/810
`435/T, 810; 424/l,l.5,
`424/3, 12; 23/230 B
`
`[58] Field of Search
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`424/3
`............... ..
`Bennich et al.
`3,‘l2(},'!60 N19?!
`. 424/S
`4,103.9".-'2 M1973 Dreyer .......... ..
`424/12
`4,115,535
`9/I973 Giaever ............. ..
`424/12
`4,130,462
`l2.r’l9T3
`Ruberlstcin et al.
`435/7
`.
`4,134,792
`l/19?‘)
`Boguslaski et al.
`424/I2
`4.160.645
`T/19?? Ullrnan .............. ..
`23f230 B
`4,l61.5|5
`'1',/1979
`Ullrnan .......... ..
`....... .. 424/1
`4.166.104
`8/I979 Wagner et al.
`.
`4.l93,983 M1980 Ullman et al.
`................... .. 23/230 B
`
`
`
`Primary Examiner—Thomas G. Wiseman
`Attorney. Agent, or Fi'rm—Bertram 1. Rowland
`
`[57]
`
`ABSTRACT
`
`Novel immunoassays are provided employing discrete
`
`23 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2054
`
`Genentech Exhibit 2054
`
`IPR2016-00710
`
`

`
`1
`
`4,256,834
`
`FLUORESCENT SCAVENGER PARTICLE
`IMMUNOASSAY
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The interest in analyzing for trace amounts ofa wide
`variety of organic materials. such as drugs, contami-
`nants, pollutants, and the like, has engendered efforts to
`provide simpler and more accurate techniques for mea-
`suring low concentrations of compounds of interest.
`One group of techniques is referred to as imrnunoassays,
`which are based on the ability to have a compound,
`usually an antibody, which is capable of recognizing or
`specifically binding to a compound having a specific
`spatial and polar organization. The specific binding pair
`may be referred to as ligand and receptor (antiligand}.
`In performing the immunoassays, normally the ligand
`is labeled with a label which provides a detectible signal
`and the labeled ligand is allowed to compete with the
`ligand in the sample for a limited amount of the antih-
`gand. The immunoassay techniques then provide for
`distinguishing between the associated labeled ligand
`and antiligand and dissociated labeled ligand which is
`free in the bulk solution. Distinguishing the associated
`signal label may be achieved by separating the unassoci-
`ated signal label from the associated signal label and
`determining the amount of signal label in either of the
`fractions.
`
`A preferred method is to employ a procedure which
`does not require separation: one distinguishes associated
`signal
`label
`from unassociated signal
`label by there
`being a substantial difference in the level of signal be-
`tween the two. One of the problems with the latter
`technique is the fact that the signal label which is mea-
`sured is not freed from materials present in the assay
`system which may provide a background or cause non-
`specific interference with the signal measurement.
`For many ligands of interest, particularly large mole-
`cules. such as proteins, polysaccharides, nucleic acids.
`and the like. obtaining the ligand in pure form is fre-
`quently difficult, and in some instances impossible. Fur-
`thermore, where the antiligand is an antibody, the anti-
`body is normally isolated as a complex mixture of glob-
`ulins, of which a portion, usually less than 50%, is the
`antibody of interest. Where one is labeling the impure
`ligand or antiligand, a substantial proportion of the label
`will be conjugated to molecules other than the ligand or
`antiligand. These labels will be capable of providing a
`detectible signal, which will act as a background for the
`measurement. That is, these labels will provide a base
`value which will be additive to the value obtained from
`the label bound to the ligand or antiligand. Where one
`is determining a small value between two large values,
`substantial errors and uncertainities are introduced. For
`example, where there are non-specific effects affecting
`the label, the presence of a large amount of label unre-
`lated to ligand or antiligand will greatly increase the
`variability due to the non-specific effects on a sample-
`by-sample basis.
`It is therefore desirable to provide techniques which
`will allow for discrimination between label bound to
`ligand or antiligand participating in the assay and re-
`lated to the amount of analyte in the assay and label
`which is present which is not involved with ligand or
`antiligand. The techniques provided must. therefore, be
`able to discriminate between the label which is provid-
`ing signal related to the amount of analyte in the me-
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`dium and the signal being obtained from label unrelated
`to the amount of analyte in the medium.
`2. Description of the Prior Art
`Engasser and Horvath, Applied Biochem. Bioengi-
`neering, Vol. I, 127 (1976) Academic Press, report the
`kinetic and diffusion effects on the immobilization of
`enzymes. US. Pat. No. 3,817,837 describes a homoge-
`neous enzyme immunoassay. US. Pat. No. 3,996,345
`describes a homogeneous immunoassay employing two
`chromophores related by being a fluorescer and a
`quencher. Copending application Ser. No. 815.636, U.S.
`Pat. No. 4,160,645 filed July 14, 1977, describes a homo-
`geneous immunoassay employing a nonenzymatic cata-
`lyst as a label, while copending application Ser. No.
`815,632, US. Pat. No. 4,208,479, describes means for
`modulating signals in immunoassays. Copending appli-
`cation Ser. No. 906,514, US. Pat. No. 4,193,933, filed
`May [6, 1978. describes a labeled liquid discontinuous
`phase for use in immunoassays. Application Ser. No.
`667,996, abandoned, filed Mar. 18, 1976. describes a
`homogeneous immunoassay employing as a label an
`enzyme substrate. See also U.S. Pat. No. 3,853,987,
`which discloses particles to which are conjugated radi-
`oactive and fluorescent labels and antibodies. See also
`US. Pat. No. 4,001,400. See also copending application
`Ser. No. 964,099, filed Nov. 24, 1978.
`'
`
`SUMMARY OF THE INVENTION
`
`The subject invention provides methods and compo-
`sitions for determining the presence of an organic ana-
`lyte, which is a member of a specific binding pair, i.e.
`ligand and its homologous receptor (antiligand). The
`method is predicated upon the ability to partition a
`chromogenic substance between a phase where the
`chromogenic substance retains its chrornogenic activity
`and a phase where its chromogenic activity is repressed,
`with the degree of partitioning being a function of the
`concentration of analyte in the assay medium.
`The method employs three reagents: (1) a conjugate
`of a member of the specific binding pair with an insolu-
`ble particle (particle conjugate); (2) a label which is a
`member of a signal producing system conjugated to a
`member of said specific binding pair (signal label conju-
`gate); and (3) a signal repressor which is an insoluble
`particle which interacts with said label to repress the
`contribution of the label to signal production.
`In carrying out the assay, the unknown suspected of
`containing the analyte. the above three reagents, and
`any ancillary reagents are combined in an assay medium
`in accordance with a predetermined protocol and the
`signal level determined from the assay medium, either
`by a rate or equilibrium mode. When the signal label
`conjugate binds to the homologous member of the parti-
`cle conjugate, the signal repressor is obstructed from
`interacting with the label associated with the particle
`conjugate. Signal label conjugate in the bulk solution
`interacts with the signal repressor, resulting in inhibi-
`tion of signal production from such signal label. The
`number of signal label conjugates able to bind to the
`particle conjugate will be a function of the number of
`analyte molecules in the assay medium. Thus, the ob-
`served signal level will be a function of the number of
`analyte molecules in the assay medium. By comparing
`the observed signal level with an unknown amount of
`analyte with the signal level obtained with at least one
`assay medium having a known analyte concentration,
`
`

`
`4,256,834
`
`3
`the analyte concentration of the unknown can be quali-
`tatively or quantitatively determined.
`Reagent compositions and kits are provided, where
`the kits have predetermined amounts of compositions
`and ancillary materials to substantially optimize the 5
`sensitivity and reliability of the assay.
`DESCRIPTION OF ‘THE SPECIFIC
`EMBODIMENTS
`
`4
`labels which. are un-
`by the signal repressor. Signal
`bound, adventitiously bound to compounds other than
`members of the specific binding pair. or bound to a
`member of the specific binding pair which is not bound
`to the particle conjugate, will interact with the signal
`repressor and their contribution to production ofa sig-
`nal substantially inhibited.
`In this manner. relatively
`impure mixtures of a member of the specific binding
`pair may be labeled without concern about introducing
`a large background signal level. since the signals from
`these labels will be substantially repressed by the signal
`repressor.
`Both the particle conjugate and the signal repressor
`will be discrete. insoluble particles. The nature of the
`particles is chosen, so that the signal repressor is ob-
`structed from interacting with signal
`label which is
`bound to the particle conjugate through noncovalent
`binding with the reciprocal member ofthe specific bind-
`ing pair.
`The analyte will be a member of a specific binding
`pair, consisting of ligand and its homologous receptor.
`The insoluble particles employed for the particle conju-
`gate will be bound. directly or indirectly, covalently or
`noncovalently,
`to one of the members of the specific
`binding pair. There is an exception where a receptor is
`the anal yte. Because of the dual nature of the receptor.
`two specific binding pair couples may be employed. For
`example. a ligand recognized by the receptor analyte
`may be conjugated to the particle to form particle con-
`jugate. Receptor for the receptor analyte (antireceptor)
`may be conjugated to a label to form the signal label
`conjugate. In this way. one has the alternative to use
`either a single specific binding pair or two specific bind-
`ing pairs. employing the duality of properties of the
`receptor.
`In carrying out the subject method, one combines in
`an appropriate assay medium, the analyte containing
`sample, the particle conjugate, the signal label conju-
`gate and the signal repressor, as well as any additional
`reagents and determines the signal from the assay me-
`dium. By comparing the observed signal with the signal
`obtained from an assay medium having a known amount
`of analyte. one can qualitatively. semi-quantitatively. or
`quantitatively determine the analyte of interest. The
`significant factor in the subject assay. is that one em-
`ploys two particulate species, which by virtue of their
`physical structure sterically inhibit interaction between
`a molecule on one particle and the second particle or
`molecule bound to the second particle. The second
`particle is able to interact with label unassociated with
`the first particle and free in the bulk solution to substan-
`tially inhibit the production of a signal by such signal
`labels.
`
`DEFINITIONS
`
`Analyte——the compound or composition to be mea-
`sured. which may he 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 (antiligand).
`I..igand—any organic compound for which a recep-
`tor naturully exists or can be prepared.
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`A sensitive, accurate simple assay method is provided
`for determining low concentrations ofa wide variety of
`organic materials. The materials of interest
`include
`those having physiological activity, drugs or naturally
`occurring compounds present
`in physiological fluids.
`disease related materials, contaminants. pollutants, and
`the like.
`The method does not employ a separation step, main-
`taining 21 substantially uniform dispersion and also mini-
`mizes background interference. The method affords a
`reduction in signal from materials or labels which are
`unrelated to the signal
`label bound, directly or indi-
`rectly, with ligand receptor in the medium. Also, the
`subject method provides substantial advantages with
`analytes which normally exist in impure form arid are
`not readily amenable to purification.
`is nor-
`it
`In preparing reagents for immunoassays,
`mally required to label either the ligand or its homolo-
`gous receptor. The homologous receptor, particularly
`when antibody, is normally a mixture of specific and
`non-specific immunoglobulins. With many antigens, the
`low concentrations of the antigens make their purifica-
`tion or concentration tedious, inefficient and expensive.
`Therefore, frequently. when labeling a member of the
`specific binding pair, one labels the impure mixture.
`Labeling of the impure mixture creates a number of 35
`problems. One problem is that there will be a substantial
`amount of adventitious label unrelated to the assay
`reagents. The second problem is the measurement of the
`signal label associated with the ligand or antiligand in
`the presence of a large amount of adventitious label. A
`third problem is non-specific interactions of the adventi-
`tious signal
`label which can cause sample-to-sample
`variation unrelated to the amount of analyte.
`The subject method alleviates or cures these prob-
`lems by a unique combination of reagents. The first
`reagent has a member of the specific binding pair bound
`to a particle. By employing high molecular weight par-
`ticles, one can insure that one can bond the specific
`bonding pair member as well as impurities to the parti-
`cles, with each particle conjugate being an active rea-
`gent in the assay.
`The second reagent is a signal label conjugate, where
`a member of the specific binding pair is labeled with a
`member of a signal producing system. The member of
`the specific binding pair may be pure or impure. In the
`assay, the amount of the labeled member of the specific
`binding pair which binds to the particle conjugate will
`be related to the amount ofanalyte in the medium. Only
`labels which are bound to a member of the specific
`binding pair can become bound to the particle conju-
`gate.
`As a third reagent, included in the assay medium. is a
`signal repressor which interacts with the signal label to
`significantly inhibit the production of :1 signal by the
`label. The signal repressor is an insoluble particle which
`is obstructed from interacting with label which is bound
`to the particle conjugate. Therefore. only label which is
`free in the bulk solution will be significantly inhibited
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`4,256,834
`
`5
`Receptor (antiligand}——any compound or composi-
`tion capable of recognizing a particular spatial and polar
`organization of a molecule i.e. determinant or 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 analog}-—a plurality of ligands or ligand
`analogs joined together covalently, normally to a hub
`nucleus. The hub nucleus is a pnlyfunctional material.
`normally polymeric. usually having a plurality of func-
`tional groups e. g. hydroxy, amino, mercapto. cthylenic,
`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 may be the
`same as those indicated for the particle.
`Particle {insoluble phase)—the particle conjugated
`with a member of the specific binding pair is a discrete
`particle. which may be swelled or remain unswelled by
`the liquid phase, may be solid or liquid, and composed
`of a wide variety of both hydrophobic and hydrophilic
`materials. Depending upon the role of the particle. the
`particles may be solid. hollow or porous, having a sub-
`stantially smooth or irregular surface, having a primar-
`ily concave or convex surface, for some applications,
`preferably being porous. that is, having channels. frac-
`tures or indentations, which can be widely varied as to
`the size of molecule or assembly which is excluded or
`whose rate of diffusion is substantially affected. For
`other applications, the particle will desirably be solid,
`having a smooth or irregular surface. The particles may
`be stable discrete liquid particles, such as dispersed
`surfactant stabilized oil particles, liposomes, or the like.
`The particles will be readily dispersible in an aqueous
`medium, and for the particle conjugate and in some
`instances the signal repressor be polyfunctionalized or
`capable of polyfunctionalization for binding, covalently
`or non-covalently, to other molecules. The particles of
`the particle conjugate will be preferably substantially
`transparent to light at wavelengths used for detection of
`a signal produced by the signal producing system, par-
`ticularly in the range between 300 and 800 nm, prefera-
`bly through the range.
`Signal producing system—the reactants and products
`involved in producing a measureable signal, which var-
`ies with the amount ofanalyte in the assay medium. The
`signal producing system may have one or more compo-
`nents. at least one component being conjugated to a
`specific binding pair member not covalently bonded to
`a particle. The signal producing system produces a
`measurable signal which is detectible by external means,
`the measurement of electromagnetic radiation, with the
`level of signal varying to the extent the signal label
`conjugate is in the environment of the solid phase parti-
`cles. The signal prcducing system will contain a chro-
`mophore. where chrornophores include dyes which
`absorb light in the ultraviolet or visible region, phos-
`
`6
`light emitting
`fluorescers, chemiluminescers,
`phors,
`energy acceptors, chromogenic enzyme substrates and
`the like. With fluorescers. the lluorescer may be excited
`by light or by an energy donor molecule, while with
`chemiluminescers, the excitation will be the result of a
`chemical reaction.
`Label—-the label is a molecule which has a particular
`function and is conjugated to another molecule or mate-
`rial. In the subject invention, the labels will be the spe-
`cific binding pair molecule that is conjugated to the
`particle (particle conjugate) 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. Signal
`labels bound to the
`particle through the specific binding pair will be influ-
`enced by and in sufficient proximity to the particle
`surface, so that a signal repressor particle which would
`otherwise be able to interact with the label will be ob-
`structed from interacting with the label. Thus, the sig-
`nal repressor will be prevented from interacting with
`the signal label bound to the particle and affecting the
`signal emanating from the signal
`label. The particle
`conjugate will preferably be relatively nonadsorptive to
`minimize non-specific binding of proteins.
`Binding pair label—a member of the specific binding
`pair bound. directly or indirectly. to the particle.
`Signal label—a member of the signal producing sys-
`tem bound to a binding pair member or to the particle.
`Signal label conjugate—the conjugate of the binding
`pair member with a member of the signal producing
`system (signal label).
`Labeled ligand—thc 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(ligand 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, there being one or more
`labels bound to the receptor or o_ne or more receptors
`bound to a label.
`
`insoluble, optionally
`Signal Repressor—a water
`swellable, particle. which may be solid, hollow or po-
`rous, may have a smooth or irregular surface, generally
`of indeterminate molecular weight, may be crosslinked
`or noncrosslinked, frequently crosslinked, may be natu-
`rally occurring or synthetic. absorptive in the wave-
`length range of interest, subject or not subject to non-
`specific protein binding, polyfunctionalized or non-
`functionalized for linking. and conjugated or unconju-
`gatcd to specific compounds which interact with the
`signal label
`to diminish the signal produced by such
`label. The particle may or may not have an affinity or
`adsorptive capacity for signal label bound to a binding
`pair member and signal
`label bound to non-specific
`impurities, where present. but when having no affinity
`for signal label.
`it will be conjugated to a compound
`having affinity for signal label or signal label conjugate.
`The signal repressor may express its effect by virtue of
`the inherent properties of the particle or by virtue of
`particular functionalities or compounds conjugated to
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`7
`the particle. The signal repressor acts to inhibit
`emission of light produced by signal
`label
`that
`juxtaposition or bound to the signal repressor.
`METHOD
`
`the
`is in
`
`4,256,834
`
`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 signal label conj ugate. the signal
`repressor, 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 artalyte—ligand or its homologous
`receptor (antiligand)—in the unknown sample will af-
`fect the partition of the signal label conjugate between
`the separate phase of the particle conjugate. where the
`signal label is protected from the signal repressor. and
`the bulk solution ofthe assay medium. where the signal
`label is subject to the signal repressor. Therefore, the
`observed signal will be related to the amount of anal yte
`in the sample.
`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
`to 6. more usually from I
`to 4 carbon atoms,
`including alcohols. ethers and the like. Usually these
`cosolvents will be present in less than about 40 weight
`percent, more usually in less than about 20 weight per-
`cent.
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`The pH for the medium will usually be in the range of 35
`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 particular 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°—S0° C..
`more usualy from about l5°—40” C.
`The concentration of anal yte which may be assayed
`will generally vary from about l0-‘to 10-‘ '5 M. more
`usually from about
`lt}"" to 10-'3 M. Considerations
`such as whether the assay is qualitative, semi-quantita-
`tive or quantitative. the particular detection technique
`and the concentration of the analyte of interest will
`normally 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 of the analyte, 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 '
`
`60
`
`65
`
`8
`reciprocal to the analyte will be not less than about 0.1
`times the minimum concentration of interest based on
`an-alyte binding sites and usually not more than about
`L000 times the maximum concentration of interest
`based on analytc binding sites, usually about 0.] to [00
`times, more usually about 0.3-10 times the maximum
`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
`signal label conjugate containing the same binding =pair
`member to react with the unknown, followed by com-
`bining with the particle conjugate. Where a competition
`mode is employed, in that the particle conjugate and the
`signal
`label conjugate contain the reciprocal binding
`pair members. large excesses of‘ the binding pair label
`might reduce the sensitivity of the assay. By employing
`various concentrations of‘ the various reagents with
`analyte at concentrations in the range of interest with a
`particular protocol, one can determine ratios which
`optimize 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. With fluorescers, normally an equilibriummode
`is employed.
`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 theztwo
`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. .
`A primary consideration of the subject assay is the
`role of the signal repressor in inhibiting the production
`of a detectible signal from signal
`label which is'not"
`bound to the particle conjugate. All protocols must take
`this role into account. Readings of the detectible-signal
`will not be meaningful until the signal repressor is sub-
`stantially at equilibrium in relation to the signal label in
`the bulk solution. Furthermore. a competition between
`the particle conjugate and signal repressor is undesir-
`able. In the light of these considerations. protocols will
`normally allow for substantially-complete equilibration
`between the particle conjugate, analyte and signal label
`conjugate, followed by the addition ofthe signal repres-
`sor. The first reading would then be taken after the
`signal reprcssor has had sufficicnt time to interact with
`the signal label in the bulk solution.
`part of the signal
`Where an enzyme is involved
`producingsystem a rate mode could be employed. Rate
`modes have the advantage of"subtracting out inherent
`interferences. such as light scattering. signal production
`
`

`
`9
`from substances unrelated to the label, equipment varia-
`tions and the like.
`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 will frequently be desirable to
`have a second incubation after addition of the particle
`conjugate and 3 third incubation after addition of the
`signal repressor. Whether to employ an incubation per-
`iod and the length of the incubation period, will depend
`to a substantial degree on the rate of binding of the
`receptor to the ligand and the signal label to the signal
`repressor. Usually.
`incubation steps will vary from
`about 0.5 min to 6 hrs, more usually from about 5 min to
`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 is
`the
`observation of electromagnetic radiation, particularly
`ultraviolet and visible light, either absorption or emis-
`sion. The signal will usually be read as electromagnetic
`radiation in the ultraviolet or visible region, particularly
`from about 250 to 750 nm, 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 I5” 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 r