--
`United States Patent ml
`[1 1]
`Litman et al.
`
`145]
`
`4,299,916
`
`Nov. 10, 1981
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`[543
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`[75]
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`PREFERENTIAL SIGNAL PRODUCITON ON
`A SURFACE IN IMMUNOASSAYS
`
`Inventors:
`
`David J. Litman. Palo Alto; Edwin F.
`Ullnian, Atherton, both of Calif.
`
`Assignee:
`
`Syva Company, Palo Alto. Calif.
`
`Appl. No.: 106,620
`
`Filed:
`
`Dec. 26. 19??
`
`Int. Cl.-‘
`
`GOIN 33/54; CIZQ 1/68;
`GOIN l/43
`435/6; 435/'r';
`U.S. Cl.
`435/805; 435/810; 422/56; 23/230 13; 424/12
`Field of Search
`435/6, 7. 178. 179,
`435/810, 805; 23/230 B; 424/8, 12; 252/408 R;
`422/56
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`3,850,752 ll/I974
`4.1359,-l»0'l' 11/ 191'?
`4.06'l'.959
`1/ l 973
`4,071,315
`l/I913
`4,094.64?
`t‘.-/I978
`4.163.146
`9/19?‘?
`4. l34,92(}
`1/ [980
`4,208,479
`6/1930
`
`B01: ...................... ..
`Chateau ....... ..
`Deutsch et al.
`Cirubb et al.
`Blixt et al.
`.... ..
`Zuk et al.
`
`
`
`..
`
`Primary Exr2miner—Thomas G. Wise-man
`Attorney. Agent. or Firm-—-Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`An assay method and compositions are provided for
`determining the presence ofan analyte in a sample. The
`analyte is a member of an immunological pair {mip} of
`immunogens-——ligand and receptor. The method has
`two basic elements: a solid surface to which one of the
`members of the immunological pair is bonded and a
`signal producing system, which includes a catalytic
`member bonded to a mip, which signal prod Ltcing sys-
`tem results in a measurable signal on said solid surface
`related to the amount of analyte in the medium. The
`signal generating compound is produced without sepa-
`ration of the catalyst labeled mip bound to the solid
`surface from the catalyst labeled mip free in solution.
`In a preferred embodiment, an enzyme is bonded to a
`mip which acts in con] unction with a solute to produce
`a signal generating product which binds preferentially
`to the solid surface when the enzyme is bound to the
`surface. resulting in a signal which is readily differenti-
`ated from signal generating compound produced by the
`catalyst and solute in the bulk solution.
`
`43 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2040
`
`Genentech Exhibit 2040
`
`IPR2016-00710
`
`

`
`I
`
`4,299,916
`
`PREFERENTIAL SIGNAL PRODUCTION ON A
`SURFACE IN IMMUNOASSAYS
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`There is continuing interest in developing new. sim-
`pler and more rapid techniques to measure the presence
`of an analyte in a sample suspected of containing an
`analyte. The analytc may be any of a wide variety of
`materials, such as drugs. naturally occurring physiolog-
`ical compounds. pollutants, fine chemicals, contami-
`nants, or the like. In many cases, speed is important for
`the measurement. particularly with certain physiologi-
`cally active compounds.
`In other situations, conve-
`nience can be a major consideration.
`One convenient
`technique which has found wide
`application is the use of a "dip stick.“ Having a solid rod
`or film which can be clipped in a sample and then subse-
`quently processed to produce a signal based on the
`amount of analyte in the original sample can provide
`many conveniences. There is ample instrumentation to
`measure a signal, such as light absorption or fluores-
`cence, of a compound bound to a solid surface. Also.
`the dip stick allows for convenient handling, transfers,
`separations, and the like.
`In developing an assay, it is desirable that there be a
`minimum number of steps and transfers in performing
`the assay, as well as a minimum number of separate
`reagents. Therefore. while a dip stick adds a conve-
`nience to separations, the separations in themselves are
`undesirable. Furthermore, the fewer the reagents that
`have to be packaged, added, and formulated, the fewer
`the errors which will be introduced into the assay and
`the greater economies and convenience of the assay.
`It is therefore desirable to develop new assay meth-
`ods, particularly employing rigid solid surfaces which
`may or may not be separated from the assay medium for
`measurement, where the signal may be developed with-
`out concern as to the presence of reagents in the solu-
`tion affecting the observed signal on the solid surface.
`2. Brief Description of the Prior Art
`Patents concerned with various immobilized reagents
`in different types of test strips include U.S. Pat. Nos.
`3,993,451; 4,038,485; 4,046,514; 4,129,417; 4,133,639;
`and 4,160,008, and Ger. Offen. 2,636,244. Patents dis-
`closing a variety of methods involving separations of
`bound and unbound antigen include U.S. Pat. Nos. Re.
`29,169;
`3,949,064;
`3,984,533;
`3,985,867;
`4,020,151;
`4.039.652;
`4.067.959‘;
`4-,l08.9?2:
`4.145.406;
`and
`4,168,146‘.
`(‘Patents of particular interest)
`
`SUMMARY OF THE INVENTION
`
`A method is provided employing a relatively rigid
`insoluble. preferably bibulous, surface to which is con-
`jugated a member of an immunological pair (abbrevi-
`ated as “mip") the immunological pair consisting of
`ligand and a receptor which specifically binds to the
`ligand or their functional equivalent for the purposes of
`this invention. In addition to the surface, a signal pro-
`ducing system is provided which has as one member a
`catalyst, normally an enzyme, which is conjugated to a
`mip. Depending upon the amount of analyte present,
`the catalyst labeled mip will be partitioned between the
`bulk solution of the assay medium and the surface. The
`signal producing system provides a signal generating
`compound at
`the surface which generates it signal
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`which is not significantly affected by any signal generat-
`ing compound produced or present in the bulk solution.
`Therefore,
`the signal generating compound may be
`generated in the assay medium in the presence of un-
`bound catalyst labeled mip. When the only catalyst in
`the signal producing system is the catalystdabeled-mip.
`various eitpedients can be employed to enhance the
`difference in the rate of formation of the signal generat-
`ing compound at the surface as compared to the bulk
`solution, e.g. enhancing the catalyst turnover rate at the
`surface. In addition to enhance the simplicity of this
`protocol, the last of the components of the signal gener-
`ating system will be added at about the time ofor before
`the addition of the catalyst bound to the mip.
`Compositions are provided for performing the assay
`comprising combinations of the surface and various
`reagents in relative amounts for optimizing the sensitiv-
`ity and accuracy of the assay.
`The subject assay provides for a convenient method
`for detecting and measuring a wide variety of analytes
`in a simple, efficient, reproducible manner. which can
`employ visual inspection or conventional equipment for
`measuring a spectrophotometric property of a product
`bound to a surface.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`In accordance with the subject invention, an assay
`method and composition are provided for measuring a
`wide variety of analytes, where the analyte is a member
`of an immunological pair (mip), the pair consisting of a
`ligand and a receptor (antiligand) which specifically
`binds to the ligand, or their functional equivalent for the
`purposes of the assay. The assay method has two essen-
`tial elements: a surface to which is conjugated a mip:
`and a signal producing system which results in a signal
`generating compound associated with the surface, pro-
`ducing a detectible signal in an amount related to the
`amount of analyte in the assay medium. Preferably, the
`signal producing system will effect a two or more step
`conversion involving one or more compounds to pro-
`duce, block or destroy the signal generating compound,
`where the rate of change in the concentration of the
`signal generating compound is related to the average
`distance between two molecules on the surface. The
`molecules may be the same or different. The immuno-
`logical binding at the surface allows for localized en-
`hanced concentrations of compounds of the signal pro-
`ducing system at the surface. Also. one may employ a
`scavenger as a third component which acts to inhibit
`the operation at the signal producing system in the bulk
`solution by scavenging an intermediate. catalyst or sig-
`nal generating compound in the bulk solution.
`The surface may be any convenient structure which
`substantially retains its form and may be separable from
`or part of the container. The manner of binding of the
`mip to the surface is not a critical aspect of this in ven-
`tion, so long as a sufficient amount of the mip is exposed
`to allow for binding to its homologous partner.
`The signal producing system has at least two mem-
`bers: A catalyst, normally an enzyme, conjugated to :4
`mip; and a solute which undergoes a reaction with :1
`substance bound to the surface. and thereby directly or
`indirectly enhances or inhibits the production of a de-
`tectible signal. The association of a member of the signal
`producing system with the surface may be as a result of
`insolubilization, complexation with a compound on the
`
`

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`4,299,916
`
`3
`surface or interaction. including reaction. with a com-
`pound on the surface.
`Where an intermediate material is produced by the
`signal producing system in soluble form. both in the
`bulk solution and at the surface. a scavenger can advan-
`tageously be employed. so as to substantially minimize
`the interaction of the intermediate material produced in
`the bulk solution with the surface.
`A wide variety of different systems may be employed
`for altering the degree of production of the product at
`the surface as compared to the bulk solution and for
`inhibiting intermediates or product produced in or mi-
`grating into the bulk solution from interacting with the
`surface. Depending upon the particular protocols. vari-
`ous additions.
`incubation steps, and reagents will be
`employed.
`By providing for the production of a detectible signal
`generating material on the surface that is related to the
`amount of analyte in a sample. one can relate the signal
`level detected from the surface to the amount of analyte
`in the solution. By employing standards having known
`amounts of analyte under the same or substantially the
`same conditions as with an unknown, one can quantitate
`the detected signal level with the amount of analyte in
`the sample.
`In accordance with the subject invention, the method
`is performed without requiring a separation of bound
`and unbound catalyst-bound-mip. nor requiring a sepa-
`ration of analyte from the remainder of the sample,
`although the latter may be desirable. This provides
`substantial ad vantages in the convenience of the proto-
`col and in avoiding the difficulties in achieving a clean
`separation.
`The subject invention achieves a precise, specific and
`sensitive technique for detecting and measuring ligands
`and ligand receptors. The method provides for the pref-
`erential production, inhibition of production or destruc-
`tion of a compound at a rigid surface, which compound
`is involved with the generation ofa signal at the surface.
`The signal generating compound associated with the
`surface will be of a sufficient depth on or in the surface
`to provide a measurable signal.
`the concentration
`For a large number of analytes.
`range ofinterest will fall between 100 pg to one pg per
`ml. For many analytes, the concentration range of inter-
`est will vary from about two-fold to 100-fold so that a
`quantitative determination will require the ability to
`distinguish small differences in the concentration of the
`analyte in the assay medium. lmmunoassays are predi-
`cated on detecting the complexation between ligand
`and receptor. where one or both may be labeled. The
`lower the concentration of the analyte. the fewer the
`number of complexes which are formed. Therefore. in
`order to be able to accurately determine the number of
`labeled complexes which are formed. either the label
`must provide a signal which can be efficiently counted
`at an extremely low level of events. e.g. radioactive
`emission. or the complex must permit amplification or
`multiplication. e.g. fluorescence or a catalyzed reaction.
`When employing an amplification system. many
`problems are encountered. One serious problem is sig-
`nal resulting from other than labeled complexes, namely
`background. Background signal can result from materi-
`als in the sample; labeled contaminants when labeling
`the member of the immunological pair. and unbound
`labeled member. In developing an assay, the signal gen-
`erated by labeled complexes must not be obscured by
`the signal from the background and must be substan-
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`tially greater than the background signal. Therefore any
`amplification achieved by the signal generating system
`must be primarily,
`if not solely. associated with the
`labeled complex rather than with background label.
`In many assay techniques a clean separation of la-
`beled immune complex and background label
`is
`re-
`quired. where careful attention must be given to non-
`specific ellects. For example. where a fluorescent label
`is employed in a heterogeneous system, eg. dipstick.
`after combining all ofthe reagents with the dipstick. the
`dipstick must be removed and carefully washed to re-
`move any fluorescer which is non-specifically bound.
`Furthermore. the number offluorescers involved with a
`complex is limited to the number which can be conve-
`niently conjugated to a member of an immunological
`pair. although further amplification can be obtained by
`employing a second labeled receptor which binds to a
`first receptor which binds to a ligand analyte. This step
`requires an additional reagent. another addition and a
`careful separation to avoid non-specific interactions.
`The subject invention obviates or minimizes many of
`the shortcomings of other methods. For each complex a
`plurality of signal generating events are achieved by
`employing a catalyst. The catalyst
`is partitioned be-
`tween the bulk solution and a surface in proportion to
`the amount of analyte in the assay medium. The produc-
`tion of signal generating product resulting from the
`catalyzed reaction at the surface is substantially inde-
`pendent of concurrent production of signal generating
`product. if any. produced in the bulk solution. Thus. the
`assay operates with the catalyst present
`in the bulk
`solution during the time the modulation of the amount
`of signal generating compound at the surface is occur-
`ring. The need for separating the surface from the bulk
`solution, whether careful or not,
`for measuring the
`signal is avoided in the subject invention. although the
`separation may be preferable.
`Furthermore. in the subject invention, the signal gen-
`erating compound can be of substantial depth on or in
`the surface. The presence of the catalyst at the surface
`allows for the deposition or conversion of a large num-
`ber of signal generating compounds to provide a strong
`signal. This is of great importance when the measure-
`ment is visual inspection. particularly where the signal
`generation involves the‘ absorption of light.
`Before further describing the invention. a number of
`terms will be defined.
`
`DEFINITIONS
`
`Analyte—the compound or composition to be mea-
`sured, which may be a ligand. which is mono- or
`polyepilopic. usually antigenic or haptenic. a single or
`plurality of compounds which share at least one com-
`mon epitopic or determinant 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). These will be referred
`to in the subject application as members of an immuno-
`logical pair, abbreviated as “mip". Homologous or
`complementary mips are ligand and receptor. while
`analogous mips are either ligands or receptors. which
`are differentiated in some manner. eg. labeling.
`Ligand—any organic compound for which a recep-
`tor naturally exists or can be prepared.
`
`

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`5
`Receptor (antiligand)—any compound or composi-
`tion capable of recognizing a particular spatial and polar
`organization of a molecule i.e. epitopic or determinant
`site. Illustrative receptors include naturally occurring
`receptors, cg. thyroxine binding globulin, antibodies,
`enzymes, Fab fragments, lectins nucleic acids 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 usually 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. but need not.
`Polyfligand-analog)-a plurality of ligands or ligand
`analogs covalently joined together, normally to a hub
`nucleus. The hub nucleus is a polyfunctional material,
`normally polymeric, usually having a plurality of func-
`tional groups :2. g. hydroxy, amino, mercapto, cthylenic,
`etc. as sites for linking. The hub nucleus is normally
`water soluble or at least dispersiblc and will usually be
`at least about 35,000 daltons, but generally not exceed-
`ing about 6{l0,000 rlaltons. Illustrative hub nuclei in-
`clude polysaccharides. polypeptides, including proteins.
`nucleic acids, ion exchange resins and the like.
`Surface-—the surface will be non-dispersed and of a
`dimension of at least about 1 pm? and generally greater.
`often at least about l mmz. frequently from about 0.5
`cm? to 10 CTII2, usually being on a support when less than
`about 0.5 crnl; and may be of any material which is
`insoluble in water and provides the necessary properties
`for binding of a mip and a detectible signal generating
`compound to provide a desired signal level. Dcsirably.
`the surface will be gelatinous, permeable, porous or
`have a rough or irregular structure. which may include
`channels or indentations, generally having a substantial
`void volume as compared to total volume. Depending
`upon the nature of the detectible signal generating com-
`pound, the surface will be adsorbent or non-adsorbent,
`preferably being weakly or non-adsorbent. The surface
`may be transparent or opaque, a single material or a
`plurality of materials. mixtures or laminates. A wide
`variety of materials and shapes may be employed. The
`surface will be capable of substantially retaining its
`integrity under the conditions of the assay so that sub-
`stances which are bound to the surface will remain
`bound to the surface and not diffuse into solution.
`Signal producing systern—thc signal producing sys-
`tem has at least two members: (1) a catalytic member;
`and (2) a solute, which undergoes a reaction catalyzed
`by the catalytic mcmher. which leads directly or indi-
`rectly lo a product on or in the surface which provides
`a detectible signal. Desirably. a third compound will be
`present which provides for enhanced rate of change of
`the signal generating compound at the surface as com-
`pared to the bulk solution. This can be as a result of the
`component being bound to the surface or interacting
`with another member of the signal producing system.
`The catalytic: member may he enzymatic or non-
`enzymatic. preferably enzymatic. Whether one or more
`than one enzyme is employed, there will be at least one
`enzyme bound to a mip. {An enzyme acting as a catalyst
`should be distinguished from an enzyme acting as a
`receptor.)
`The solute can be any compound which is capable of
`undergoing a reaction catalyzed by a catalytic member
`of the signal producing syslem, which reaction results
`either directly or indirectly in modulating the formation
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`of a detectible signal generating compound associated
`with the surface. The association of the signal generat-
`ing compound to the surface may be as a result ofinsol-
`ubilization of the product produced when solute under-
`goes the catalyzed reaction, complexation of the prod-
`uct with a compound on the surface or reaction or
`interaction of a compound on the surface with the prod-
`uct of the catalyzed reaction.
`The signal generating compound will Provide an
`electromagnetic signal. e.g. a spectrophotometric or
`visible, electrochemical or electronic dctectible signal.
`The signal generating compound will be associated
`with the surface due to its insoiubility, or covalent or
`non-covalent binding to the surface. The observed de-
`tectible signal from the surface will be related to the
`amount of catalyst bound to the surface through the
`binding of the catalyst-bound-mips to the rnip-bound-
`surface.
`Various techniques and combinations of reagents
`may be employed to enhance the production of the
`detectible signal at the surface, while minimizing inter-
`ference from materials in the bulk solution.
`Label—the label may be any molecule conjugated to
`another molecule where each of the molecules has had
`or can have had a prior discrete existence. For the most
`part, labels will be compounds conjugated to a mip. In
`referring to a catalyst conjugated to an antiligand, the
`reagent will be referred to as a catalyst—bound-antili—
`gand, while for a ligand conjugated to a surface. the
`reagent will be referred to as a ligand-bound surface.
`Method
`
`The subject assay is carried out in an aqueous zone or
`medium, where the final assay medium may be the re-
`sult of prior individual additions of reagents or combi-
`nations of reagents and incubations. prior separations
`involving removal of the surface from an aqueous me-
`dium and transfer to a different aqueous medium having
`one or more reagents. or combinations thereof. The
`subject method, however, does not require a separation
`of catalyst-bound-rnip which is unbound from that
`which is bound to its homologous partner bound to the
`surface (mip-bound—surface). The medium consists of a
`liquid phase and a non-fluid phase which is the “sur-
`face."
`In carrying out the assay, the mip-bound surface will
`be contacted by the sample, and by the members of the
`signal producing system. and any ancillary materials in
`an aqueous medium, either concurrently or stepwise. to
`provide a detectible signal associated with the surface.
`The detectible signal will be related to the amount of
`the catalyst-bound-mip bound to the surface, which in
`turn will be related to the amount of analyte in the
`sample. Depending upon the nature of the signal pro-
`ducing system and the desired method for detecting the
`signal, the surface may be read in the assay medium or
`will be read separate from the assay medium.
`In carrying out the assay, an aqueous medium will
`nonnally be employed. Other polar solvents may also
`be included, usually oxygenated organic solvents of
`from 1-6, more usually from I-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-l l. 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
`
`

`
`4,299,916
`
`7
`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 pll 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. Constant temperatures duritig
`the period of the measurement are generally required
`only if the assay is performed without comparison with
`a control sample. The temperatures for the determina-
`tion will generally range from about l0°—50” C._. more
`usually front about 15°—45” C.
`The concentration of rinalyte which may he assayed
`will generally vary from about 10-4 to 10-1-‘M, more
`usually from about
`l[l“‘-‘ to IO “M. Considerations
`such as whether the assay is qualitative. semi-quantitw
`live or quantitative. the particular detection technique
`and the concentration of the analyte of interest will
`normally determine the concentration of the other rea-
`gents.
`The concentrations of various reagents will vary
`widely depending upon which protocols are employed,
`the nature ofthc analyte, the mip which is bound to the
`surface and the mip which is bound to the catalyst. the
`required sensitivity of the assay. and the like. In some
`instances, large excesses of one or the other of the mips
`may be employed. while in some protocols the sensitiv-
`ity of the assay will be responsive to variations in the
`rnip ratios.
`By way of illustration. ifthe analyte is a polyepitopic
`antigen. one could have excesses of aritiligand as antili-
`garid~-bouiicl-surface and as catalyst-bound-antiligand.
`without seriously affecting the sensitivity of the assay,
`provided that the surface is first contacted by the sam-
`ple.
`followed by contact with the signal producing
`system. Where antiligand is the sample and the protocol
`involves the conibiriatiori of the analytc and catalyst-
`bound—antiligand prior to contacting the antigen-bounch
`surface. the sensitivity ofthe assay will be related to the
`ratios of the analyte and catalyst-bound—antiligand con-
`ccritralioti.
`In addition to the considerations involving the proto-
`col. the concentration of the reagents will depend on
`the binding constant of the antiligarid. the binding con-
`stant profile for a particular iintisera. as well as the
`required St!l'l.!itll\-'ll_\.- of the assay. Also. when all of the
`signal producing system is present in the liquid phase.
`the catalyst substrates and ancillary reagents should be
`at a concentration which allows for sithstaritial immuno-
`logical pair binding before a large amount of signal
`producing product is formed. Where the sensitivity of
`the assay is concentration related. frequently the partic-
`ular conceiitrations will be determined empirically.
`When the sample is combined with the homologous
`catalyst—bourid-rnip. generally the total binding site con-
`celitrutiori of the t‘atal_v$1—botind-rriip will he not
`less
`than about
`ll.l
`times the minimum concentration of
`interest based on binding sites of unulyte and usually not
`more than about 1.000 tinics the maiiirnuni concentra-
`tion of interest based on analyte binding, sites. usually
`about [It to lot} times. more usually about 0.3—lt) times
`the maximum cunceiitnitioii of interest. When the ana-
`lyte is preaidsorhcd to the rnip—b-ourid-surface. the con-
`cciilration of c:ital_vsI—hound-inip will depend on the
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`desired rate of binding to the surface. the production of
`interfering signal generating compound in the liquid
`phase, the cost of the reagent. etc.
`The concentration of catalyst-bound-mip will be
`chosen so that the amount of catalyst-bound—mip in the
`void volume—liquid immediately adjacent
`to and oc-
`cluded in the surface will not significantly interfere with
`the measurement of the change in concentration of the
`signal generating compound at the surface as a result of
`catalyst-bound—mip bound to the surface. The chosen
`concentration will be affected by the sensitivity of the
`measurement.
`the degree of quarititatiori desired,
`the
`accuracy with which one must distinguish the lowest
`concentration of interest and the like.
`ln most situations. the ratio of concentration in the
`void volume of catalyst-hound-mip unbound to the
`surface to catalyst-bound-mip bound to the surface
`should be not greater than about I00 fold, usually not
`greater than about 10 fold at the maximum concentra-
`tion of interest of the analyte. preferably at the mid-
`range concentration range of interest of the anal ytc.
`The combination of the solid surface with the sample
`may be prior to. concomitant with. or subsequent to
`combining the catalyst-bound-mip with the sample. By
`employing a single unit or entity as the surface. one can
`use the surface to concentrate the analytc in a large
`sample. Also,
`the surface allows for removal of the
`analyte from other materials in the sample which could
`interfere with the determination of the result.
`'I'l1ere-
`fore. a preferred embodiment will be to combine the
`surface with the sample, followed by removal of the
`surface from the sample containing medium and transfer
`to the assay medium.
`Alternatively. one could leave the surface in contact
`with the sample and add the remaining reagents. It
`is
`also feasible, although in some instances not desirable.
`to combine the sample with the catalyst-bound-rriip.
`followed by introduction of the surface into the assay
`medium. For example. with a ligand analyte. enzyme-
`bound-antiligiind and ligand-bound-surface.
`this last
`technique could be effectively used.
`Frequently. the last of the components of the signal
`producing system will be added at about the same time
`as the catalyst-bound-mip. without any intermediate
`step. such as separalingor washing the surface.
`Where a receptor is the analyte. instead of having it
`single immunological pair. one may employ two immu-
`nological pairs, where the receptor acts as the ligand in
`one pair and the receptor in the other. For esarnple.
`with lgli. one could bind the allcrgcii or antigen to the
`surface and bind the catalyst to anti-lgE. In this way,
`the Igl-L acts as at bridge between two rnips which 111
`themselves cannot interact.
`In relerriiig to a mip. this
`situation should be considered a special case whicli is
`intended to be included.
`
`In developing protocols for the method. certain basic
`considerations will govern the order of addition and the
`combinations of reagents. The first consideration is that
`preferably where the surface-bouiid-rnip and the cata-
`lyst-hound-mip are different members eg. one i.- ligand
`and one is aiiiiliganrl, the two will be brought ttigctlicr
`prior to or subsluittially concomitant with coinbnialioii
`with the surlacct. The catul_\'st-brititid-rnip and siiiutc
`will pr:-.-l'er:ibly be comhiiico as it siiigle rcagciit. except
`when the solute is the substrate of the L‘:tl£ll_\_-'\'l-h(\lJlt(l-
`mip.
`l-‘reqiicritly. the F-tIl‘ft|Ct" and sample will be com-
`bined prior or nearly concotnitiiiit with the iitltliiiun of
`the other rcageiits
`
`

`
`4,299,916
`
`10
`formation of signal generating compound will be fol-
`lowed as a rate, observing the change in signal on the
`surface with time. The rate of course will be related to
`the amount of label which binds to the surface. This
`measurement may be made prior to establishing full
`equilibrium between the analyte. catalyst-bound-mip
`and mip-bound-surface. and thus the rate may vary with
`time.
` TABLE I
`
`Protocols
`
`9
`Various protocols will have various degrees of com-
`plexity. In the simpler protocols, there will be two cata-
`lysts involved in the signal producing system. one
`which is bound to a mip, and the other bound to the
`surface. One catalyst, preferably the surface-bound-
`catalyst, reacts with the solute to produce a first prod-
`uct. This first product is acted on by the second catalyst,
`which first product by itself or in combination with
`other reagents produces a second product which prefer-
`entially binds to the surface or interacts with a com-
`pound bound to the surface, when produced adjacent to
`the surface. This can be achieved conveniently by pro-
`ducing a second product which is insoluble. By insolu-
`ble is intended a solubility of less than about 10-3 M.
`The insoluble product may effect changes in electrical
`properties e. g. electrostatic or have spectrophotometric
`properties.
`including absorption in the ultraviolet or
`visiblc wavelength range, chemiluminescence.
`reflec-
`tance and fluorescence. preferably absorption.
`In order to minimize the amount of repetition, a table
`is provided which assembles various illustrative proto-
`cols. While the table is directed to polyepitopic anti-
`gens, haptens can be employed in place of the antigens.
`However, with haptens it will normally not be conve-
`nient to bridge between receptors, and in protocols that
`require bridging. the addition ofa polyfligand analog) is
`required to provide the bridging. When the analyte is a
`hapten, one will normally add the hapten containing
`sample to the receptor. When the catalyst-bound-mip is
`the receptor, the mip bound to the surface is normally
`hapten. When the mip bound to the surface is a recep-
`tor, the mip bound to the catalyst is normally hapten.
`Thus, one will normally saturate a portion of the recep-
`tor binding sites with the hapten analytt: and cause the
`remaining sites to combine with the hapten either conju-
`gated to the surface or to the catalyst.
`The antigen or polyepitopic analyte as a ligand offers
`addition