United States Patent
`Tom et al.
`
`[19]
`
`[54] CONCENTRATING ZONE METHOD [N
`HETEfi0GENE()[js IMMUNOASSAYS
`
`[75]
`
`Inventors: Henry H. Tom. II-a Honda; Gerald L.
`RGWIEL Cupemnm both of calm
`53?“! Cflflllfllllb’. P310 Alto, Calif.
`[73] Assignee:
`[2i] Appl- No: 176.177
`[22] Filed:
`Aug. ‘I, 1980
`-[51]
`Int. Cl; ..................... GIJIN 33/s4;Go1N 33/I6;
`C120 I/70
`[52 U'.s.C1. ........................................... 435/12435/5;
`435/805; 435/810; 424/1; 422/56
`1
`[53]
`Field of Search ....................... 435/5, 7, 133, 305,
`435/810; 422/55, 56, 61; 23/230 B. 230 R;
`424/1’ 8’ 12
`_
`References Cm"!
`U. S. PATENT DOCUMENTS
`3.3111140
`5x197-1 Bauer ct al.
`......................... .. 422/56
`4_05l_463 12/I977 Lang‘: at al_
`'
`
`4.094.647
`6/1978 Deulsch er al. .
`4,l6B.l46
`9/1979 Grubb-ct al.
`435/7
`
`[561
`
`[113
`[45]
`
`4,366,241
`Dec. 28, 1982
`
`.................... 435/‘!
`4,190,496 2/1980 Rubenstein et al.
`4,205,058 S/I980 Wagner .................. ..
`
`4.226.978 10/1980 Bogvvslaski at al.
`4,233.40: 11/1930 Maggie el: al. .......................... 435/'1
`Primary Examiner-—Tl2omas G Wiseman
`Attorney, Agem‘. or F£rm—-Bertram I. Rowland
`[57]
`ABSTRACT
`Method and apparatus are provided for performing
`i'.““‘““°"““‘3"‘ “‘“'-"”°Y"‘3 “ d°"i-“'3 "°"."P“‘i“5 3 "?’“'
`nvely smjll tesi :99: relferredrto 1: a1l1°S1m£:1unosorb1r1g-
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`liquid receiving relatimishtp w1tl_1 said Immunosorbmg
`z°".°' The ‘“‘”."““°‘°‘.""'{’_5 ‘.0335 “'."1“d°“ 3 '“"“?’?’ °‘
`3“ 'b“{‘J'““"1‘°'°3‘°a' Pa" (
`'“‘P 3433"“ 31"’ a““''3*“‘'
`— un
`to a support.
`A signal producing system is employed in conjunction
`with said device having as one component a signal label
`bound to a mip. The signal producing system provides
`f°"P'°d“°“°" °f“d°“°“b"‘”i3““”"‘h‘*i‘"m““°5°""
`ing zone in relation to the amount of analyte in a sample.
`
`34 Claims, 5 Drawing Figures
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2048
`
`Genentech Exhibit 2048
`
`IPR2016-00710
`
`

`
` Dec. 28, 1982
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`1
`
`4,366,241
`
`CONCENTRATING ZONE METHOD IN
`I-IETEROGENEOUS IMMUNOASSAYS
`
`BACKGROUND OF THE INVENTION
`l. Field of the Invention
`
`The ability to employ naturally occurring receptors
`or antibodies directed to specific compounds in assaying
`for the presence of a compound of interest has created
`a burgeoning immunoassay business.
`In each of the
`assays, a homologous pair, usually an immunological
`pair, involving a ligand and a receptor (antiligand) is
`involved, wherein one of the members of the immuno-
`logical pair (mip) is labeled with a label which provides
`a detectible signal. The immunoassay methodology
`results in a distribution of the signal label between signal
`label bound in a complex of the mips and unbound sig-
`nal label. The differentiation between bound and un-
`bound signal label can be as a result of physical separa-
`tion of bound from unbound signal label or modulation
`of the detectible signal between bound and unbound
`signal label.
`For the most part, immunoassays have been directed
`to quantitative determination of a wide variety of com-
`pounds of interest, particularly drugs, in clinical labora-
`tories requiring relatively sophisticated equipment and
`careful technique. Immunoassays have found less exten-
`sive commercial application where semi-quantitative or
`qualitative results would be acceptable and where the
`determination would involve.non-laboratory personnel,
`such as in a home or a medical practitioner’s office.
`Even in the clinical laboratory, simple and rapid screen-
`ing tests employing inexperienced personnel could
`sever to provide substantial economies.
`In developing an immunoassay, there are many con-
`siderations. One consideration is to provide substantial
`dilferentiation between the observed signal resulting
`from signal label when bound as compared to unbound.
`Another consideration is to minimize interference from
`endogenous materials in the sample suspected of con-
`taining the compound of interest. A further consider-
`ation is the ease with which the observed signal can be
`detected and serve to differentiate between concentra-
`tions in the concentration range of interest-. Other fac-
`- tors include the ease of preparation of the reagents, the
`accuracy with which samples and reagent solutions
`must be prepared and measured, the storage stability of
`the reagents, the number of steps required in the proto-
`col, and the proficiency and accuracy with which each
`of the steps must be performed. Therefore, in develop-
`ing an assay which can have application with untrained
`personnel, such as assays to be perfonned in the home,
`in forensic medicine, by medical practitioners, or the
`like, the observed result should be minimally affected
`by variations in the manner in which the protocol is
`carried out or provide for simple techniques for per-
`forming the various steps.
`2. Description of the Prior Art
`U.S. Pat. No. 4,l68,l46 describes an immunoassay
`test strip. U.S. Pat. Nos. 3,990,850 and 4,055,394 de-
`scribe diagnostic test cards. A wide variety of patents
`and patent applications provide an extensive literature
`of different techniques for producing detectible signals
`in immunoassays. The following list is merely illustra-
`tive of some of these techniques which can find applica-
`tion in this invention. The following is a list of United
`
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`2
`States patents and patent applications and a general
`statement of the type of label involved:
`U.S. Pat. Nos.
`3,646,346, Radioactive Label;
`3,654,090, 3,?'91,932 and 3,817,838, Enzyme Labels;
`3,996,345, Fluorescer-Quencher Labels; 4,062,733, Ra-
`dioactive Label; 4.061959, Fluorescer or Enzyme La-
`bel; 4,l04,029, Chemiluminescent Label; and 4,160,645,
`Non-Enzymatic Catalyst Label. See U.S. Pat. Nos.
`3,966.39? for an electrophoretic technique employing
`an antibody zone and 4,120,945 for an RIA where la-
`beled analyte is
`initially bound to a solid support
`through antibody. U.S. application Ser. No. 893,650,
`filed Apr. 5, 1978, U.S. Pat. No. 4,233,402, employs
`enzyme pair labels; 893,910, filed Apr. 5, 19?8, U.S. Pat.
`No. 4,720,450, chemically indticed fluorescent
`label;
`and 61,099, filed Aug. 26, 1979, U.S. Pat. No. 4,237,300,
`enzyme anionic charge labels.
`SUMMARY OF THE INVENTION
`
`Novel non-chromatographic assay devices and meth-
`ods employing such devices are described for the deter-
`mination of members of an immunological pair (mip).
`The device has an immunosorbing zone to which a mip
`is fixed against diffusive movement. The itt1munosorb-
`ing zone serves as the entry for the sample and reagent
`solutions.
`
`In liquid-receiving relationship, either directly or
`indirectly with the immunosorbing zone and normally
`extending transversely therefrom is a liquid absorbing
`zone, which serves to draw liquid through the im-
`munosorbing zone, store liquid and may serve to con-
`trol the rate at which the liquid is drawn through the
`immunosorbing zone.
`Employed in the method in conjunction with the
`device is a signal producing system which has a signal
`label member conjugated to a mip. The immunosorbing
`zone may include one or more members of the signal
`producing system which are bound to the zone in a
`manner to permit or inhibit diffusive movement of the
`signal producing system component.
`In accordance
`with the method protocol, the amount of signal label
`bound in a detection zone in the immunosorbing zone is
`related to the amount of analyte in the sample.
`The method involves contacting the assay device
`with the liquid sample to which may have been added
`one or more components of the signal producing sys-
`tem; followed by contact with one or more successive
`solutions which contain any remaining components of
`the signal producing system and serve to wash the im-
`munosorbing zone free of non-specifically bound signal
`label. The signal producing system provides for a de-
`tectible signal in the irnmunosorbing zone which can be
`compared to a signal level based on a standard having a
`known amount of analyte.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a plan view of an assay device;
`FIG. 2 is an enlarged cross-sectional view of the
`assay device of FIG. 1, taken along line 2-2;
`H0. 3 is a plan view of an assay device having a
`sample test strip and a standard test strip;
`FIG. 4 is a cross-sectional view of the assay device of
`FIG. 3 along lines 4-4.
`FIG 5 is a partial cross-sectional elevation of an
`alternate embodiment of the subject invention.
`
`

`
`3
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`4,366,241
`
`4
`of the other molecule. The members of the immunologi-
`cal pair are referred to as ligand and receptor (antih-
`gand) and members of a specific pair are referred to as
`homologous.
`(a) Ligand—any organic compound for which a re-
`ceptor naturally exists or can be prepared;
`(b) Receptor (antiligand)—any macromolecular com-
`pound or composition capable of recognizing (having
`an enhanced binding affinity to) a particular spatial and
`polar organization of a molecule, i.e. epitopic or deter-
`minant site. Illustrative receptors include naturally oc-
`curring receptors, e.g. thyroxine binding globulin, anti-
`bodies, enzymes, Fab fragments, lectins, and the like.
`The term antibody is employed in this case as illustra-
`tive of and to more generally denote receptor;
`(c) Antireceptor—-in some situations, a receptor can
`serve a dual function of binding to a ligand and serving
`as a ligand to a receptor (antireceptor), such that the
`ligand and the antireceptor, which cannot bind directly
`to each other, are joined by the receptor to provide for
`an immunological
`linkage. Antireceptors may fre-
`quently be -antibodies, protein A, rheumatoid factor,
`Clq. or the like.
`Ligand analog—a modified ligand which can com-
`pete with the analogous ligand for a receptor, the modi-
`fication of the ligand providing means to join the ligand
`analog to another molecule; or where the ligand has a
`functionality which is used for bonding directly to an‘-
`other molecule, the ligand portion of the conjugate will
`be referred to as ligand analog. The ligand analog will
`normally differ from the ligand by more than replace-
`ment ofa hydrogen with a bond, which links the ligand
`analog to another molecule, e.g. label or hub. When a
`plurality of ligand analogs are joined together, particu-
`larly by bonding to a central nucleus, e. g. hub, the re-
`sulting aggregation will be referred to as a poly(ligand-
`analog).
`Assay device—the assay device has at least one bibu-
`lous layer, normally having two or more layers, which
`will be relatively free of non-specific adsorptivity for
`the materials of interest. The device has as one element
`a relatively small immunosorbing zone having a mip
`non-diffusively fixed in the zone. The mip remains sub-
`stantially immobilized on a bibulous solid support dur-
`ing the course of the assay; and as a second element a
`reservoir zone, either directly or indirectly in liquid-
`receiving relationship with the immunosorbing zone.
`(a) Irnrnunosorbing zone—a bibulous solid film, layer
`or sheet to which a mip is non-diffusively bound; the
`irnmunosorbing zone has a relatively small fluid capac-
`ity as compared to the total assay device capacity. One
`or more members of the signal producing system may
`be bound directly or indirectly to the immunosorbing
`zone. The immunosorbing zone has specific binding
`capability for the homologous mip.
`Within the immunosorbing zone may be one or more
`zones in tandem or overlapping. Included within the
`immurtosorbing zone will be a detection zone, which
`may be the same or different from the zone to which the
`mip is bound.
`(b) Liquid absorbing zone-a bibulous solid material
`either directly or indirectly in liquid receiving relation-
`ship with the immunosorbing zone and acting as a reser-
`voir or storage zone capable of storing a substantially
`greater liquid volume than the immunosorbing zone.
`The zone acts as a pump to pull liquid through and out
`of the immunosorbing zone.
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`Method and apparatus are provided for performing
`immunoassays. The method and apparatus are devised
`so as to provide for a narrow zone through which rela-
`tively large amounts of fluid pass with relatively uni-
`form flow. The zone serves to concentrate both a small
`amount of analyte in a sample, as well as reagents for
`producing a detectible signal, while inhibiting the local-
`ization of non-specifically bound materials which
`would result in production of background signal unre-
`lated to the amount of analyte in the medium. Specifi-
`cally, assay devices are provided which serve to receive
`a liquid sample and liquid solutions, transmit the solu-
`tions through an irnrnunosorbing zone to which a mem-
`ber of an immunological pair (mip) is bound and to
`draw the liquid and non-specifically bound materials
`into a storage zone or reservoir. The reagent solutions
`provide for production of a detectible signal in the im-
`munosorbing zone which may be compared to a stan-
`dard related to a specific amount of analyte in a sample.
`The immunoassay method is adaptable to a wide
`variety of reagent combinations which have previously
`been employed in other immunoassays, both homogene-
`ous and heterogeneous. The conditions under which
`these other assays have been carried out will normally
`be applicable in the subject method. Thus, the devices
`of the subject invention allow for a simplicity of proto-
`col as compared to prior art methods, while providing
`for qualitative or quantitative results analogous to prior
`art methods. By appropriate choice of the components
`for producing a" detectible signal, the detectible signal
`may be observed visually or by means of various appa-
`ratuses, such as spectrophotometers, lluorometers, scin-
`tillation counters, etc.
`The analyte to be determined will be a mip. The
`specificity of the homologous mips provides a means for
`discriminating between the analyte of interest and other
`materials which may be in the sample. Thus. by appro-
`priate choice of the mip in the immunosorbing zone, one
`can provide for specific binding of a component to the
`irnmunosorbing zone which results in production of a
`detectible signal. The amount of such component in the
`immunosorbing zone can be related to the amount of 45
`analyte in the sample.
`In addition to the assay device, there will be em-
`ployed one or more reagents which will comprise the
`signal producing system. The key reagent in the signal
`producing system is the one which serves as a signal
`label and is conjugated to a mip. The choice of protocol
`will determine whether an increase or decrease in the
`amount of the conjugate of signal label and mip which
`is specifically bound in the imrnunosorbing zone deter-
`mines the amount of analyte in the assay medium.
`DEFINITIONS
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`SI)
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`Analyte-—the compound or composition to be mea-
`sured, which is a mip and may be a ligand, which is
`mono- or polyepitopic, that is, having one or plurality
`of determinant sites, haptenic and antigenic, a single
`compound or plurality of compounds which share at
`least one common epitopic or determinant site; or a
`receptor.
`Mip—a member of an immunological pair, consisting
`of two different molecules, where one of the molecules
`has an area on the surface or in a cavity which specifi-
`cally binds to a particular spatial and polar organization
`
`65
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`

`
`5
`Signal producing system—the signal producing sys-
`tem may have one or more components. at least one
`component being conjugated to a mip. The signal pro-_
`ducing system produces a measurable signal which is
`detectihle by external means, usually the measurement
`of electromagnetic radiation, which signal will be pro-
`duced in a de_tection zone in the imrnunosorbing zone._
`For the most part, the signal producing system will
`involve radioactive substances, enzymes and chromo-
`genic substrates, and chrornophores, where chromo-
`phores include dyes which absorb light in the ultravio-
`let or visible
`region. phosphors,
`lluorescers, and
`chemiluminescers. While for the most part the signals
`can be absorption or emission of electromagnetic radia-
`tion, usually in the ultraviolet or visible range, other
`detectible signals will also find application in particular
`situations.
`Signal generator—the compound which provides for
`production of the detectible signal. The signal generator
`may be produced during the assay or be present initially
`and produce the signal without undergoing any chemi-
`cal change. Where a chemical reaction is involved, the
`compound undergoing the reaction to produce the sig-
`nal is referred to as the signal generator precursor.
`Label—the label may be any molecule conjugated to
`another molecule or support and may be arbitrarily
`chosen. In the subject invention, the labels will be the
`mip bound to the assay device as a support and members
`of the signal producing system whether bound to the
`mip or to the assay device as a support.
`Mip-support conjugatt-.—in the immunosorbing zone,
`the mip bound to the assay device support, which may
`be bound covalently or non-covalently, directly or indi-
`rectly. The mip is bound substantially permanently to
`the support, so as not to migrate during the traverse of 35
`the sample and reagent solutions through the im-
`rnunosorbing zone. The mip serves to binds its homolo-
`gous member to the irnmunosorbing zone.
`Signal label-mip conjugate——a member of the signal
`producing system, which is directly or
`indirectly
`bonded to a mip, which is or becomes bound to the
`immunosorbing zone to provide for production of a
`detectible signal in the detection zone.
`Signal
`label-support conjugate-—a member of the
`signal producing system bound, covalently or noncova-
`lently, directly or indirectly,
`to the immunosorbing
`zone which acts in conjunction with the signal. label-
`mip conjugate to produce a signal in the detection zone.
`METHOD
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`SO
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`4,366,241.
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`6
`placed by the analyte; (2) present with the sample and
`competes or acts in concert with the analyte to become
`bound to the mip-support conjugate; or (3) may be
`added subsequently.
`After contacting the sample, the imrnunosorbing zone
`will be contacted with any remaining members of the
`signal producing system, and as appropriate, wash solu-
`tions, where a cletectible signal will be developed at the
`detection zone in relation to the amount of analyte in
`the medium. In most protocols, the reagent solutions
`will also serve as wash solutions to minimize the num-
`ber of steps in the protocol.
`The subject method is distinguished from prior art
`methods which involve diffusion of solutes diffusing to
`and away from a layer immersed in a liquid. Thus the
`layer encounters a continuously changing solution com-'
`position as solute becomes bound to the layer or dis-
`solves into the liquid. In the subject invention, the mip
`containing layer in contact with the solution continu-
`ously contacts substantially the same solution composi-
`tion as the solution diffuses through the layer. Thus, the
`concentrations of solutes in the solution in the mip con-
`taining layer remain relatively constant during contact
`of the immunosorbing zone with a solution.
`The structure of the assay device with the im-
`munosorbing zone acting as the entry port can be de-
`signed in a variety of ways. The assay device except for
`the irnrnunosorbing zone must be isolated, normally by
`a non-permeable barrier, so that
`the solution is re-
`stricted to being absorbed through the immunosorbing
`zone prior to being absorbed by the liquid absorbing
`zone. The irnmunosorbing zone is exposed by an open-
`ing inthe non-permeable barrier. With this construc-
`tion, the assay device can be conveniently immersed in
`a solution.
`
`Alternatively, the solutions may be applied to the
`immunosorbing zone in a horizontal position, either
`dropwise, as a slowly flowing stream, or in a container
`surrounding the immunosorbing zone.
`"The sample may or may not be subject to prior treat-
`ment, but usually will not be treated. When prior treat-
`ment is employed, buffer with or without one or more
`components of the signal producing system or other
`reagents may be included. _When the sample solution is
`ready for sampling, the immunosorbing zone of the
`assay device is contacted with the sample and absorbs at
`least a portion of the sample, so that a substantial pro-
`portion of the sample solution enters through and tra-
`verses the immunosorbing zone and extends into the
`storage zone. With the subject device, the volumes of
`the solutions normally need not be premeasured. The
`amount of solution absorbed can be controlled by the
`distance the liquid traverse the liquid absorbing zone.
`In this manner, a relatively large sample volume may
`pass through the immunosorbing zone, resulting in sub-
`stantial concentration of the analyte in the limited vol-
`ume of the immunosorbing zone. Limited only by the
`capacity of the liquid absorbing zone and the binding
`constants of the homologous mips, lower concentra-
`tions of analyte can be detected by permitting larger
`volumes of sample to be absorbed and transferred
`through the imrnunosorbing zone.
`The analyte of interest may be present in a wide vari-
`ety of environments. Analytes of interest include drugs,
`hormones, macromolecules,
`and microorganisms,
`which may be found in physiological fluids, such as
`blood—whole serum or plasma—urine, cerebral spinal
`fluid, ocular lens liquid, and saliva; synthetic chemicals;
`
`The subject method employs an assay device in con-
`junction with a sample solution and usually one or more
`reagent or wash solutions. The method involves con-
`tacting the imrnunosorbing zone with a sample solution,
`where the liquid holding capacity of the imrnunosorb-
`ing zone is substantially smaller than the volume of the
`sample, as well as the portion of the sample which tra-
`verses the immunosorbing zone.
`The immunosorbing zone has mip fixed to a solid
`support, where the rnip is inhibited from diffusing from
`the zone. The analyte in the sample solution passing
`through the immunosorbing zone binds to the mip of
`the mip-support conjugate and is concentrated in the
`immunosorbing zone.
`The signal label-mip conjugate may be involved in a
`number of ways: (I) initially present in the irnmunosorb—
`ing zone non—covalent.ly bound through immunological
`complexation to the mip-support conjugate and dis-
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`4,366,241
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`The various combinations can involve two basic pro-
`pollutants in water and air; trace compounds, toxins and
`tocols. Protocol 2 involves combining the sample and
`microorganisms in food. e.g. milk, meat, poultry and
`mip components of the signal producing system and
`fish; or the like. The fact is that any organic substance
`contacting the assay device with the resulting solution.
`which can serve as a ligand or any receptor for which a
`For some applications, the assay device strip would
`ligand may be obtained can be determined, so long as
`then be contacted with a wash solution that optionally
`such analyte can be introduced into a liquid system in a
`would contain the other components of the signal pro-
`form in which it can bind to the homologous mip.
`ducing system to ensure the complete removal of non-
`Depending upon the protocol, one or more compo-
`specific binding mip. Alternatively, in protocol
`1 the
`nents of the signal producing system may be present
`assay device could be contacted with the sample, fol-
`with the analyte in the sample solution. A solution hav-
`lowed by contact with the mip component of the signal
`ing the signal label-mip conjugate may be followed by
`producing system, followed optionally by a wash solu-
`at least one additional solution, to ensure the removal of
`tion which if employed would usually contain the other
`non-specific binding of the signal label-mip conjugate in
`components of the signal producing system. Addition-
`the immunosorbing zone and/or introduce remaining
`ally, with l and 7 the L-mip could be initially bound to
`components of the signal producing system. Where
`the mip-support conjugate, so that upon immersion in
`more than one component is included in the signal pro-
`the sample the L-mip would be displaced by the hapten
`ducing system, conveniently the signal label-mip conju-
`analyte. No further steps would be necessary where the
`gate solution can be sorbed in the immunosorbing zone
`signal label is a radioactive label or fluorescer.
`prior to the addition of the remaining members of the
`For combinations 2 and 8 one would combine the
`signal producing system in a subsequent reagent solu-
`sample with the combined signal label-mip and other
`tion. The subsequent reagent solution then serves as a
`mip and contact the assay device with the resulting
`wash to remove non-specifically bound signal label-mip
`solution.
`conjugate. Alternatively, nothing is added to the sample
`Depending on the nature of the signal label and signal
`which is contacted first, and then followed by one or
`producing system, one would observe the signal, by
`more additional solutions containing the signal label-
`irradiating with light for a fluorescer and observing the
`mip conjugate and the other components of the signal
`level of fluorescence; providing for a catalyst system to
`producing system.
`produce a dye,
`fluorescer, or chemiluminescence,
`A wide variety of protocols will be available, the
`where the dye could be observed visually or in a spec-
`particular configuration being subject to: the nature of
`D trophotometer and the fluorescer could be observed
`the analyte, i.e., whether it is a mono- or polyepitopic
`visually or in a fluorometer; or in the case of chemilumi-
`ligand or a receptor; the nature of the signal label-mip
`nescence or a radioactive label, by employing a radia-
`conjugate; and the nature and number of components in
`tion counter. Where the appropriate equipment is not
`the signal producing system. Therefore, a number of
`available, it will normally be desirable to have a chro-
`illustrative examples of protocols will be given, which
`mophore produced which results in a visible color.
`are not exhaustive, but are rather embodiments of pro-
`Where sophisticated equipment is involved. any of the
`tocols which serve to indicate the numerous opportuni-
`techniques is applicable.
`ties for variation.
`After each combination of reagents, an incubation
`The following Table 1 indicates the various combina-
`step may be involved. Incubation steps involving com-
`tions of analytes and reagents which may be employed
`'
`’
`'
`f
`16 h
`.
`.
`.
`.
`.
`.
`.
`.
`In the subject invention to provide a signal in the Im- 4'0 bmmg solutions can vary mm about 5 Sec to
`is or
`more, generally varying from about 1 min to 1 hr, more
`munosorbing zone related to the amount of analyte.
`usually from about 5 min to" 30 min. By contrast, the
`
`TABLE I
`time during which the assay device is contacted with
`each solution will depend upon the volume of the solu-
`Mig In Siggal Producing System
`tion to be sorbed and the rate at which it diffuses into
`Mip
`Signs]
`Support
`Label
`Other
`Preferred
`the liquid absorbing zone. This time will be controlled
`
`Anulyte
`Conjugate
`Mip
`Mip
`Protocols
`in accordance with the concentration of the mip in the
`1. H
`Ab};
`Ix!-I
`L2
`solution. the concentration of mip in the area of the
`2. H
`Ab};
`L-H
`2
`immunosorbing zone, the binding constant of the ho-
`3. H
`H
`L-Ab}:
`2
`mologous mips, the rate of production of signal, and the
`4. Ag
`Abgg
`1..-Ag
`1.2
`like. The manner in which the time for diffusion of the
`5. Ag
`Abgg
`L-Ab
`1
`solutions through the immunosorbing zone may be con-
`6. Ag
`Ag
`L.As,,,,
`2
`7. Ab};
`H
`L-Abg
`1.2
`trolled will involve the oomposition, construction, size
`it. Any
`I-I
`Lang
`2
`and shape of the immunosorbing and liquid absorbing
`9. Ab“
`H
`L4-Ab,§,5
`l
`zones, the temperature,
`the solvent, and the like. In
`10. Ab};
`H
`1..-H
`I
`view of the wide variety of opportunities for use of the
`1:. Ab;,-
`Am,
`L-H
`2
`12. Ab
`Ag
`L-{kbgg
`L1
`subject method and devices, no particular time range
`13. Ab
`Ag
`L—Ag
`I
`ascribed to the subject metod would be meaningful.
`14. Ah
`Ag
`L-Rbgb
`I
`For the most part, the various solution cornbinings.
`15. Abgg
`Abgg
`‘L-Ag
`2
`contacting of solutions with the assay device, and read-
`Aims
`l6. Ag
`Allgg
`L-Ab,“
`I
`
`
`Ab};L~Ab,¢gAb};17. H l
`
`
`ings will be carried out at a temperature in the range of
`about 0'’ to 50° C., more usually in the range of about
`Protocol 1: Contact sample and assay device prior to Contact with other pans or
`signal producing system.
`15° to 40° C. For the combining of the various reagents,
`Protocol 2: Combine sample with mip cmnponcnts of signal producing system prior
`to or simultaneously with contact with assay device.
`as well as the incubation steps. temperatures will gener-
`H—morioepllopic ligand
`ally range from about 15" to 50’ C., frequently 20° to 35"
`Ag—polyepitopic ligand
`C., and conveniently ambient temperatures. Depending
`Ab—neceptor. normally an antibody: subscript indicates homologous n-lip
`!..—1aheI
`upon whether the measurement
`is qualitative, semi-
`quantitative or quantitative,
`the temperature may be
`
`Ab};
`
`H
`
`45
`
`50
`
`55
`
`60
`
`65
`
`

`
`9
`controlled or uncontrolled, conveniently employing
`ambient
`temperatures. Frequently, when employing
`ambient temperatures, one or more control strips may
`be employed for comparison with the observed signal
`from the sample strip.
`'
`The solutions which are employed will normally be
`buffered at a pH in the range of about 5 to 1!, more
`usually 5 to 10, and preferably about 6 to 9. The pH is
`chosen so as to maintain a significant level of specific
`binding by the mips while optimizing signal producing
`efficiency. Obviously, various pl-I's may be used with
`different solutions. Various buffers may be used to
`achieve the desired pH and maintain the desired pl-I
`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.
`The concentration of analyte which may be assayed
`will generally vary from about 10-4 to 10*”, more
`usually from about 10-5 to 10-33 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 various reagents will vary
`widely depending upon which protocols are employed,
`the nature of the analyte, the mip in the immunosorbing
`zone, 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
`sensitivity 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 antih-
`gand-support conjugate and as signal label-antiligand
`conjugate, without seriously affecting the sensitivity of
`the assay, provided that the assay device is first con-
`tacted with the sample, followed by contact with the
`signal producing system solution, desirably followed by
`a wash solution. Where antiligand is the analyte and the
`protocol involves the combination of the analyte and
`catalyst-antiligand conjugate, prior to contacting the
`antigen-support conjugate, the sensitivity of the assay
`will be related to the ratio of the analyte and catalyst-
`antiligand concentration.
`In addition to the considerations involving the proto-
`col, the concentration of the reagents will depend on
`the binding constant of the antiligand, or the binding
`constant profile where an antiserum is used, as well as
`the required sensitivity of the assay. Desirably, when
`feasible, the concentration of the signal label-mip will be
`sufficiently low to minimize non-specific binding or
`occlusion within the immunosorbing zone, particularly
`the detection zone.
`In view of the wide differences in ratios resulting
`from variations in the properties and nature of the vari-
`ous reagents, the molar ratios can only be very broadly
`stated. For example, based on the analyte range of inter-
`est, the number of molecules of signal label-mip conju-
`gate drawn into the immunosorbing zone will usually be
`not less than about 0.] times the minimum number of
`moles of analyte based on binding sites and not more
`than about 1,000 times the maximum moles of analyte
`based on binding sites drawn into the immunosorbing
`zone, more usually from about 0.1 to 100 times the
`moles of analyte based on binding sites. So far as the mip
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4-0