United States Patent [191
`4,391,904
`[11]
`
`[45] "' Jul. 5, 1983
`Litman et al.
`
`4,163,146 9/I919 Grubb et al.
`4-.lB4,920
`l/1980 Blixtetal. ..
`4,208,479 6/ 1980 Zuk et al.
`.
`4,230.79? 10/ 1930 Boguslaski et al.
`4,233,402 11/1980 Maggio et al.
`......
`4.299316 ll/1981 Litman et al.
`..
`
`
`
`435/'7
`435/305
`435/?
`435/810
`.. 23/230 B
`435/7
`
`
`
`Primary Examiner-—Thomas G. Wisernan
`Attorney. Agent. or Fi'rm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`An assay method and compositions are provided for
`detennining the presence ofan analyte in a sample. The
`analyte is a member of an immunological pair (mip) of
`irnmunogens——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 producing 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 conjunction 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.
`
`9 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2044
`Genentech Exhibit 2044
`
`IPR2016-00710
`
`[54] TEST STRIP KITS IN IMMUNOASSAYS AND
`COMPOSITIONS TI-IEREIN
`
`[75]
`
`Inventors: David J. I..itInan, Cupertino; Edwin F.
`Ullman, Atherton, both of Calif.
`
`[13] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[ * ] Notice:
`
`The portion of the term of this patent
`subsequent to Nov. 10, 1998, has
`been disclaimed.
`
`[21] App]. No.: 255,022
`[22] Filed:
`Apr. 17, 1981
`
`Related US. Application Data
`
`[63]
`
`Continuation-in-part of Ser. No. 106,620, Dec. 26.
`l9T9, Pat. No. 4.299.916.
`
`[51]
`
`Int. 01.3 ................... .. GIJIN 33/54; oom 21/oo;
`com 1/43; G01N 21/06
`[52] us. Cl. ...................................... .. 435/7; 435/153;
`435/805; 435/310; 436/536; 436/537; 422/55;
`.
`422/56
`[53] Field ofsearcir ................... .. 424/1, 3, 12; 435/7,
`435/188. 174, 178, I79, 180, 181, 810. 805;
`23/230 B, 332; 422/55. 56
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`....................... .. 435/?
`3,350,152 11/ 1914 Schuurs et al.
`4,059,401 ll/19?? Hochstrasser et al.
`.... .. 435/?
`4,067,959
`1/ 1918 B0]:
`..................
`. 23/230 B
`4,071,315
`1/ l9'r’8 Chateau . . . . . .. .
`.. . . .. 435/7
`4,094.64?
`6/1918 Deutsch et al.
`435/?
`
`
`
`

`
`4,391,904
`
`I 1
`TEST STRIP KITS IN IMMUNOAISSAYS AND
`COMPOSITIONS THEREIN
`
`REFERENCE TO RELATED APPLICATIONS
`
`This application is a continuation—in-part of applica-
`tion Ser. No. 106,620, filed Dec. 26, l979, now U.S. Pat.
`No. 4,299,916.
`'
`--
`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 analyte 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. No. 2,636,244. Patents
`disclosing a variety of methods involving separations of
`bound and unbound antigen include U.S. Pat. Nos. Re.
`29,169;
`3,949,064;
`3,934,533;
`3,985,867;
`4,020,151;
`4,039,652;
`4,067,959‘;
`4,108,972;
`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 com.
`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
`lignd or their functional equivalent for the purposes of
`this invention. In addition to-the surface, apsignal pro-
`ducing system is provided which has as one member a
`catalyst, normally an enzyme, which is conjugated to a
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`mip. Depending upon theamount 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 a signal
`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 catalyst-labeled-mip,
`various expedients 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 of or 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 compositions 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 oompound 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 inven-
`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 a
`mip; and a solute which undergoes a reaction with a
`
`

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`4,391,904
`
`3
`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
`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 quautitate
`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, not requiring a sepa-
`ration of analyte from the remainder of the sample,
`although the latter may be desirable. This provides
`substantial advantages 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 of a signal at the surface.
`The signal generating compound associated with the
`surface will be of a suflicient depth or on in the surface
`to provide a measurable signal.
`the concentration
`For a large number of analytes,
`range of interest will fall between 100 pg to one pg per
`ml. For many anaiytes, 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. Immunoassays are predi-
`cated on detecting the cornplexation 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-
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`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-
`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 effects. For example, where a fluorescent label
`is employed in a heterogeneous system, .e.g. dipstick,
`after combining all of the reagents with the dipstick, the
`dipstick must be removed and carefully washed to re-
`move any tluorescer which is non-specifically bound.
`Furthermore, the number of fluorescers 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
`polyepitopic, 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
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`5
`analogous mips are either ligands or receptors, which '
`are differentiated in some manner, e.g. labeling.
`Ligand—any organic compound for which a recep-
`tor naturally exists or can be prepared..
`Receptor (antiligand)—-any compound or composi-
`tion capable of recognizing a particular spatial and polar
`organization of a molecule i.e. epitopic or determinant
`site. Illustrative receptors include naturally occurring
`receptors, e.g. thyroxine binding globulin, antibodies,
`enzymes, Fab fragments. lectins, nucleic acids and the
`like.
`
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`
`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 e.g. hydroxy, amino, tnercapto, ethylenic,
`etc. as sites for linking. The hub nucleus is normally
`water soluble or at least dispersible and will usually be
`at least about 35,000 daltons, but generally not exceed-
`ing about 600,000 daltons. 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 um? and generally greater,
`often at least about 1 mmz, frequently from about 0.5
`cm? to 10 cm3, usually being on a support when less then
`about 0.5 cm3; 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. Desirably,
`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 system—the 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 member, which leads directly or indi-
`rectly to 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 be enzymatic or non-
`enzymatic, preferably enzymatic. Whther 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
`J
`receptor.)
`"
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`6
`The solute can be any compound which is capable of
`undergoing a reaction catalyzed by a catalytic member
`of the signal producing system, which reaction results
`either directly or indirectly in modulating the formation
`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 of insol-
`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 detectible signal.
`The signal generating compound will be associated
`with the surface due to its insolubility, 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 mip-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 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-mip 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 1-4 carbon atoms, includ-
`ing alcohols, ethers and the like. Usually these concl-
`vents will be present in less than about 40 weight per-
`cent, more usually in less than about 20 weight percent.
`
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`4,391,904
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`‘7
`The pH for the medium will usually be in the range of
`about 4-11, more usually in the range of about 5-10, and
`preferably in the range of about 6.5-9.5. The pH is
`chosen so as to maintain a significant level of specific
`binding by the receptor while optimizing signal produc-
`ing efficiency. In some instances, a compromise will be
`made between these two considerations. Various buff-
`ers may be used to achieve the desired pH and maintain
`the pH during the determination. Illustrative buffers
`include borate, phosphate, carbonate, Tris, barbital and
`the like. The particular buffer employed is not critical to
`this invention but in individual assays, one buffer may
`be preferred over another.
`Moderate temperatures are normally employed for
`carrying out the assay. Constant temperatures during
`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 10°—50° C., more
`usually from about 15“-45“ C.
`The concentration of analyte which may be assayed
`will generally vary from about 10*‘ to 10-15 M, more
`usually from about l0-5 to 10-” 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.
`The concentrations of the various reagents will vary
`widely depending upon which protocols are employed,
`the nature of the 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
`mip ratios.
`By way of illustration, if the analyte is a polyepitopic
`antigen, one could have excesses of antiligand as antili-
`gand-bound-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 combination of the analyte and catalyst-
`bound-antiligand prior to contacting the antigen-bound-
`surface, the sensitivity of the assay will be related to the
`ratios of the analyte and catalysvbound-antiligand con-
`centration.
`In addition to the considerations involving the proto-
`col, the concentration of the reagents will depend on
`the binding constant of the antiligand, the binding con-
`stant profile for a particular antisera, as well as the
`required sensitivity 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 substantial 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 concentrations will be determined empirically.
`When the sample is combined with the homologous
`catalyst-bound-mip, generally the total binding site con-
`centration of the catalyst-bound-rnip 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 concentra-
`tion of interest based on analyte binding sites, usually
`
`8
`about 0.1 to 100 times, more usually about 0.3—l0 times
`the maximum concentration of interest. When the ana-
`lyte is preadsorbed to the mip-bound-surface, the con-
`centration of catalyst-bound—mip will depend on the
`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—bountl-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 quantitation desired, the
`accuracy with which one must distinguish the lowest
`concentration of interest and the like.
`In most situations, the ratio of concentration in the
`void volume of catalyst-bound-mip unbound to the
`surface to catalyst-bound-mip bound to the surface
`should be not greater than about 100 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 analyte.
`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 analyte 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. There-
`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-mip,
`followed by introduction of the surface into the assay
`medium. For example, with a ligand analyte, enzyme-
`bound-antiligand 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 separating or washing the surface.
`Where a receptor is the analyte, instead of having a
`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 example,
`with lgE, one could bind the allergen or antigen to the
`surface and bind the catalyst to anti-IgE. In this way,
`the IgE acts as a bridge between two mips which in
`themselves cannot interact. In referring to a mip, this
`situation should be considered a special case which 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-bound-mip and the cata-
`lyst-bound-mip are different members eg. one is ligand
`and one is antiligand. the two will be brought together
`prior to or substantially concomitant with combination
`with the surface. The catalyst-bound-mip and solute
`will preferably be combined as a single reagent, except
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`when the solute is the substrate of the catalyst-bound-
`mip. Frequently, the surface and sample will be com-
`bined prior or nearly concomitant with the addition of
`the other reagents.
`5
`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 rnip, and the other bound to the
`surface. One catalyst, preferably the surfac"e-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
`visible 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 of a poly(ligand analog) is
`required to provide the bridging. When the analyte is a
`hapten, one will nonnally add the hapten containing
`sample to the receptor. When the catalyst-bound-mip is
`the receptor, the mip bound to