United States Patent
`
`[19]
`
`4,220,450
`-[111
`[45] Sep. 2, 1980
`Maggie
`
`
`
`the determination of an analyte which is a member of an
`immunological pair consisting of ligand and receptor
`for the ligand. A chemiluminescent source is employed
`comprised of one or more individual members. one
`chemiluminescent source member being conjugated to
`one of the members of the immunological pair, so as to
`provide chemiluminescence adjacent to the site of con-
`jugation. A quencher molecule is conjugated to a mem-
`ber of the immunological pair. When the members of
`the immunological pair bind, the quencher molecule is
`brought within quenching distance of the chemi.l1m1i-
`nescent source so as to inhibit the emission of light by
`the chemiluminescent source. The amount of analyte
`present in the assay medium affects the amount of bind-
`ing between the members of the immunological pair
`which results in quenching of the cheiniluminescence.
`By observing the light emitted from the assay medium,
`either
`from the chemiltuninwcent
`source of
`the
`quencher, the change in light emission in relation to the
`concentration of analyte present in the assay medium
`can be used to determine the amount of anslyte present
`in the assay medium. By employing standards having
`known amounts of analyte, the amount of analyte in an
`unknown sample can be quantitatively determined.
`Reagent kits can be provided having predetermined
`amounts of the reagents, so as to substantially optimize
`the sensitivity of the assay.
`
`[54] CHEMICALLY INDUCED FLUORESCENCE
`IMMUNOASSAY
`
`[1-'5}
`
`Inventor:
`
`Edward T. Maggie, Redwood City,
`Calif.
`'
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] Appl. No.: 393,910
`
`{Z21 Filed:
`
`Apr. 5. 1978
`
`[51]
`
`[52] U.S. Cl.
`
`Int. CL? ................... .. GIJIN 33/15; GOIN 31/I4;
`C09K ll/00
`23/230 3; 424/3;
`424/12; 435/7; 435/3
`[53] Field of Search .................... 23/230 B; 424-/8, I2;
`195/103.5 A, 103.5 L; 435/7. 3
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`Chappelle
`4/19'.-‘I
`5/ 1972 Chappelle
`12/ l9'l'4
`Rubenstein ..
`12/ 1976 Ullman
`12/ 1976 Ullman
`3/1976
`Szczesniak
`S/1977
`Blakernore
`8/19'I8 Meier
`
`
`
`195/103.5 L
`195/ [(33.5 A
`I95/lO3.5 L
`. l9S/103.5 A
`. 195/103.5 A
`...... 23/230 B
`. 195/I015 A
`23/2303
`
`3,575,812
`3,660,240
`3,852,151
`3,996,345
`3,998,943
`3,999,293
`4,043,812
`4,104,029
`
`Primary Examr'ner—Sid.ney Marantz
`Attorney. Agent, or Fr'rm—Bertram I. Rowland
`
`[37]
`
`ABSTRACT
`
`A competitive protein binding method is provided for
`
`32 Claims, Nu Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2058
`
`Genentech Exhibit 2058
`
`IPR2016-00710
`
`

`
`1
`
`4,220,450
`
`Cl-IEMICALLY INDUCED FLUORESCENCE
`IMMUNOASSAY
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`5
`
`15
`
`20
`
`The clinical diagnostic field has seen a broad expan-
`sion in recent years, both as to the variety of materials
`which may be readily and accurately determined, as
`well as the methods for the determination. One broad
`category of techniques involves the use of an organic
`receptor which is able to specifically bind to a particular
`spatial and polar organization of another molecule. For
`the most part. these compounds are antibodies, which
`are able to distinguish between the compound_ or com-
`position of interest, and other compounds of analogous
`structure. By virtue of the binding of the receptor to a
`labeled ligand, one is able to distingtlish between labeled
`liqand which is bound to receptor and unbound lageled
`ligand.
`The observed effect of binding by the receptor will
`depend upon the label. In some instances, the binding of
`the antibody merely provides for a differentiation in
`molecular weight between bound and unbound labeled"
`ligand. In other instances, the presence of the receptor
`may affect the nature of the signal obtained from the
`label, so that the signal varies with the amount of recep-
`tor bound to labeled ligand. A further variation is that
`the receptor is labeled and the ligand unlabeled. Where
`receptors are labeled with two different labels which
`interact when in close proximity, the amount of ligand
`present affects the degree to which the labels on the
`receptor may interact.
`In developing an assay, there are many consider-
`ations. One consideration is the signal response to
`changes in the concentration of analyte. A second con-
`sideration is the ease with which the protocol for the
`assay may be carried out. A third consideration is the
`variation in interference from sample to sample. Ease of
`preparation and purification of reagents, availability of 40
`equipment, ease of automation, and interaction with
`ligands, are additional considerations, which do not
`exhaust the various concerns in developing a useful
`assay.
`
`25
`
`35
`
`There is therefore a continuing need for new and 45
`accurate techniques which can be adapted for a wide
`spectrum of different ligands or be used in specific cases
`where other methods may not be readily adaptable.
`2. Brief Description of the Prior Art
`U.S. Pat. No. 3,709,868 is exemplary of a radioimrnu- 50
`noaasay. U.S. Pat. No. 3,960,834 is exemplary of a spin
`immunoassay. U.S. Pat. No. 3,654,090 and German
`Auslengungsschrifi No. 2,223,385 are exemplary of
`enzyme immunoassays. Articles of interest include an
`article by Ludwig Brand and James R. Gohlke, Annual
`Review of Biochemistry, 41, 843-868 (l9'i2) and Stryer,
`Science, 162, 526 (1968). Smith, FEBS Letters Ti‘, 25,
`(197?) describes a fluorescent
`immunoassay, where
`thyroxine is bound to a fluorescer and quenches the
`fluorescer, the quenching being reversed by binding of 60
`antibody to thyroxine. See also, Ullman et al, J. Biol.
`-
`Chem. 25}, 4172 (1916).
`An excellent review of chemiluminescence may be
`found in McCapra, Quarterly Reviews 20, 485 (1966).
`SUMMARY OF THE INVENTION
`
`-
`65
`
`$5
`
`A competitive protein binding assay is provided hav-
`ing as an analyte a member of an immunological pair
`
`2
`which consists of ligand and receptor for the ligand.
`The assay is predicated on the presence of the analyte in
`an assay medium affecting the degree to which a chemi-
`luminescence source is quenched by energy transfer to
`a quencher, at relatively long distances. By conjugating
`the chemiluminescence source or where the chemilumi-
`nescence source requires a plurality of components, one
`component of the chemiluminescence source, with a
`member of the immunological pair and conjugating a
`quencher with a member of the immunological pair,
`reagents can be prepared which when combined in the
`assay medium will provide varying degrees of light
`emission, depending upon the amount of analyte present
`in the assay medium.
`In particular, the chemiluminescence source or com-
`ponent thereof and the quencher may be conjugated to
`either the ligand or the receptor and the resulting rea-
`gent combined in an aqueous, normally buffered me-
`dium at a mild temperature, and the amount of light
`emitted determined. By comparison with assay media
`having known amounts of analyte, a quantitative rela-
`tionship can be developed between the quanta of emit-
`ted light and the amount of analyte in the assay medium.
`Kits can be provided, where the reagents are in-
`cluded in premeasured amounts, so that they may be
`used directly or may be readily diluted to assay reagent
`solutions to provide concentrations which substantially
`optimize the sensitivity and performance of the assay.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`In accordance with the subject invention, chemilumi-
`nescence is employed to provide a signal related to the
`amount of analyte in an assay medium. The analyte is a
`member of an immunological pair which includes li-
`qand and receptor. By conjugating the chemilumines-
`cence source or where the source is comprised of more
`than one component, one component of the chemilumi-
`nescence source, with a member of the immunological
`pair and a quencher with a member of the immunologi-
`cal pair, the presence of analyte affects the amount of
`quencher which is within quenching distance of the
`conjugated chemiluminescence source. By combining
`the
`chemiluminescence
`source
`reagent
`and
`the
`quencher reagent where the two labels are on different
`molecules, and additional immunological pair members,
`as required, with the analyte in an assay medium, in-
`cluding any ancillary reagenu‘. necessary for the chemi-
`luminescence, and determining the amount of light
`emitted from the assay medium, at a particular wave-
`length or a range of wavelengths from the assay me-
`dium, in relation to an assay medium having a known
`amount of analyte, the amount of analyte in the sample
`can be determined.
`The method is predicated on the observation that
`when a dye is within a limited distance from a
`chemiluminescer in the excited state,
`the chemilumi-
`nescer may transfer its energy to the quencher without
`collision and without emitting radiation. The quencher_
`may then emit radiation of a higher wavelength than the
`chemiluminescer or may lose the energy by radiation-
`less decay. One can conjugate the member of the chemi-
`luminescence source and the quencher to either ligand
`or receptor, so that when the two conjugates are
`brought
`together
`the amount of quencher within
`quenching distance of the chemiluminescer is affected
`by the amount of analyte present in the assay medium.
`
`

`
`3
`The nature and amount of light emitted from the assay
`medium will therefore be a function of the analyte pres-
`ent in the assay medium. By performing assays with
`known amounts of analyte, one can develop a quanta-
`tive relationship between the amount of analyte in the
`assay medium and the amount of radiation emitted from
`the assay medium at one or more wavelengths.
`Definitions
`
`Analyte--the compound or composition to be mea-
`sured, which may be a ligand which is mono- or
`polyepitopic, antigenic or haptenic, a single or plurality
`of compounds which share at least one common epi-
`topic site or a receptor.
`Ligand—any compound for which a receptor natu-
`rally exists or can be prepared.
`Ligand analog—a modified ligand which can com-
`pete with the analogous ligand for receptor, the modifi-
`cation providing means to join to a label or to a hub
`nucleus.
`I Poly(ligand analog)—a plurality of ligand analogs
`joined together covalently, normally to a hub nucleus,
`to provide a compound having a plurality of epitopic
`sites capable of competing with the analogous ligand for
`receptor.
`Label—either a component of a chemiluminescence
`source or a quencher dye, which form a light emitting
`reciprocal pair, where the quencher dye has a high
`transition probability of absorbing energy from the
`chemiluminescence source.
`(a) chemiluminescer label-Ha compound which by
`itself or in combination with other compounds produces
`a molecule in an electronically excited state, which
`molecule can decay to a lower energy state by the emis-
`sion of light and the total process results in a chemical
`change in one or more of the compounds.
`Cb) quencher—a molecule capable of inhibiting the
`chemiluminescent emission oflight, when within a short
`but non-colliding distance, usually less than about W0
`A, of the chemiluminescer molecule, by accepting the
`energy which would otherwise be emitted as chemilu-
`minescent light. In effect, the quencher need not be the
`nearest neighbor
`to the chemiluminescer
`to effect
`quenching.
`Label-conjugate-—the label, either a compound of the
`chemiluminescence source or the quencher, is bonded,
`either by a bond or linking chain, to a member of the
`immunological pair but not both to the same molecule.
`The conjugate will have at least one label and may have
`a plurality of labels bonded to the member of the immu-
`nological pair or a plurality of such members bonded to
`the label or a plurality of ligands and labels i.e. poly(li-
`gand analog)-polylabel. In particular, where an enzyme
`is the component of the chemiluminescence source em-
`ployed as the label, a plurality of ligand analogs may be
`conjugated to the enzyme to form a poly(ligand analog
`label.
`'
`Receptor—any compound or composition capable of
`recognizing a particular spatial and polar organization
`of a molecule i.e. epitopic site. Illustrative receptors
`include naturally occurring receptors, antibodies, en-
`zymes, lectins, Fab fragments and the like. The receptor
`may be monovalent or polyvalent. in receptor sites usu-
`ally polyvalent e.g. antibodies. For any specific ligand,
`the receptor will be referred to as "antiligan ”. The
`receptor-antiligand-and its reciprocal
`liqand form an
`immunological pair.
`
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`65
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`4,220,450
`
`4
`Polyfligand analog)-label—-a compositionin which a
`plurality of ligand analogs and one or a plurality of
`labels are bonded together whereby the ligand analog
`and label are in juxtaposition, so that when receptor is
`bound to ligand analog, label on the labeled receptor is
`in within quenching distance of the reciprocal label.
`Where an enzyme is part of the chemiluminescence
`source and the ligand is haptenic, a plurality of ligand
`analogs may be bonded to the enzyme. Alternatively, a
`plurality of ligand analogs and one or more labels may
`be conjugated to a water soluble polyfunctionalized hub
`nucleus.
`
`Assay
`
`The subject assay is carried out in an aqueous, nor-
`mally homogeneous, zone normally, but not necessarily
`at a moderate pH, generally close to optimum assay
`sensitivity. The assay zone for the determination of
`analyte is prepared by employing in an appropriate
`assay solution, usually buffered, the unknown sample,
`which may have been subject to prior treatment, the
`chemiluminescer labeled reagent and the quencher la-
`beled reagent (includes poly(1igand analog)"-polylabel),
`and as appropriate ligand or anti|igand.-
`' The presence of antiligand or ligand in combination
`with a predetermined amount of antiligand in the assay
`medium controls the degree to which the quencher
`comes within quenching distance of the chemilumi-
`nescer.
`
`There are four basic variations in the preparation of
`the quencher and cherniluminescer reagents. The four
`variations -are:
`as
`to ligand
`conjugated
`(I)
`cherniluminescer
`chetniluminescer labeled ligand and quencher con-
`jugated to receptor as quencher labeled antiligand;
`(2) quencher conjugated to ligand as quencher la-
`beled ligand and chemiluruinescer conjugated to
`receptor as chemiluminescer labeled antiligand;
`and
`
`as
`(3) chemiluminescer conjugated to receptor
`chemiluminescer labeled antiligand and quencher-
`conjugated to" receptor as quencher labeled antih-
`gand.
`'
`to ligand as
`(4)
`chemiluminescer
`conjugated
`chemiluminescer labeled ligand and quencher con-
`jugated to ligand as quencher labeled ligand.
`'
`With the first two combinations, when the reagents
`are combined, the quencher will be in quenching dis-
`tance of the chemiluminescer. The presence of analyte,
`either ligand or antiligand, will serve to reduce the
`amount of energy transfer between the chemilumi-
`nescer and quencher by diminishing the number of
`quencher molecules within quenching distance of the
`chemiluminescer. In the third combination, a polyepi-
`topic ligand (includes ' po1y(1igand analog)) must be
`added for either antiligand or monoepitopic ligand as
`analyte. Where the ligand is polyepitopic,
`increased
`quenching will be observed as the concentration of the
`polyepitopic ligand increases to a maximum quenching,
`'followed'by decreased quenching as the concentration
`of polyepitopic ligand continues toincrease. Thus, a
`biphasic response is obtained, so that one must know on
`which portion of the curve one is operating in order to
`obtain a discrete result. By contrast, with po1y(ligand
`analog), the presence of monoepitopic ligand will serve
`to diminish quenching. With receptor as analyte, in-
`creasing concentrations 'of‘receptor will also serve to
`diminish quenching.
`
`

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`4,220,450
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`
`5
`Where the chemiluminescer and the quencher are
`both conjugated to ligand, an assay for either ligand or
`polyvalent antiligand may be performed. Where the
`assay is for ligand, the two label-conjugates are em-
`ployed in conjunction with antiligand which brings the 5
`chemiluminescer and quencher together into quenching
`distance of each other. The addition of ligand reduces
`the amount of chemiluminescer and label which are
`within quenching distance For the determination of
`antiligand the two label-conjugates are employed. With 10
`increasing amounts of antiligand, there will be a de-
`crease of chemiluminescence to a
`and then an
`increase as the concentration of antiligand increases. If
`one is uncertain as to which portion of the biphasic
`curve is involved, one or more sample dilutions will
`indicate the particular concentration.
`It should be understood, that in referring to quench-
`ing, all that is intended is that there be transfer of energy
`from the chemiluminescer to the quencher. The result
`of this transfer will be that light of a single or range of 2
`wavelengths which might otherwise have been emitted
`by the cherrtiluminescer will be transferred to the
`quencher, which may then fluoresce, emitting light of a
`higher wavelength than the energy absorbed. Depend-
`ing upon the quantum efficiency of emission of the
`chemiluminescer, the efficiency of energy transfer from
`the chemiluminescer to the quencher, and the quantum
`efficiency of emission of the quencher, as well as the
`wavelength range which is monitored, one may observe
`greater or lesser amounts of light due to the quenching.
`Therefore, when referring to quenching, it is not in-
`tended that there necessarily be a diminution of the
`signal which is observed. In fact, if one is observing the
`light emitted by the quencher,
`increasing quenching 35
`will result in an increasingly large signal.
`A special situation exists with small haptens, those of
`from about 125 to 2000 molecular weight. With these
`haptens a substantially reduced chemiluminescence can
`be achieved i.e. quenching without quencher bonded to
`receptor, particularly where the receptor is an anti-
`body. Wlnle the reduction in signal will not be as great
`as when quencher is conjugated to receptor, a sufficient
`reduction may be achieved to have an acceptable assay.
`Except for using receptor without quencher, the assay
`will be performed in the same manner, reading the light
`emitted by the chemiluminescer.
`In carrying out the assay, an aqueous medium will
`normally be employed. Other polar solvents may also
`be employed, usually oxygenated organic solvents of 50
`from one to six, more usually from one to four carbon
`atoms, including alcohols, ethers and the like. Usually,
`these cosolvents will be present in less than about 40
`weight percent, more usually in less than about 20
`weight percent.
`The pH for the medium will usually be in the range
`from about 5 to 12, more usually in the range from
`about 7 to I0, and when enzymes are employed as part
`of the chemiluminescence source. 7' to 9. Various buff-
`ers may be used to achieve the desired pH and maintain
`the pH during the determination. Illustrative buffers
`include borate, phosphate, carbonate, Tris, barbital and
`the like. The particular buffer employed is not critical to
`this invention, but in individual assays, one buffer may
`be preferred over another.
`Moderate temperatures are normally employed for
`carrying out the assay and usually constant tempera»
`tures during the period of the assay will be employed.
`
`65
`
`6
`The temperatures will normally range from about 10° to
`50° C., more usually from about 15° to 40° C.
`The concentration of analyte which may be assayed
`will generally vary from about 10"‘ to 10-45 M, more
`usually from about
`l0—5 to 10-13 M. Stated another
`way, the concentration ranges of interest will generally
`be from about 104 to l0—” g/ml.
`In addition to the concentration range of analyte of
`interest, considerations such as whether the assay is
`qualitative, semi-quantative or quantitative, the equip-
`ment employed, and the characteristics of the reagents
`will normally determine the concentration of the rea-
`gents. While the concentration of analyte will deter-
`mine the range of concentrations of the other reagents,
`normally to optimize the sensitivity of the assay, indi-
`vidual reagent concentrations will be determined em-
`pirically. Since the binding constant and binding profile
`of receptors will vary, for example, with antibodies
`from bleed to bleed, each new batch of antibodies may
`0 require different concentration ratios for the different
`reagents.
`,
`Normally, for mono- and polyepitopic ligand ana-
`lytes, the concentration of antiligand based on binding
`sites will be about equal to the minimum concentration
`of interest based on binding sites and not more than
`about 50 times the maximum concentration of interest
`based on binding sites, usually about 1 to 10 times, and
`more usually about 1 to 3 times the maximum concen-
`tration of interest based on bindi.ng sites.
`For polyepitopic ligand receptor analytes, the equiv-
`alent ratios of labeled ligand or ligand to receptor ana-
`lyte will generally be in the range of about 0.01 times
`the minimum concentration of interest and not more
`than about 100 times the maximum concentration of
`interest based on binding sites. The labeled receptor
`employed in conjunction with the labeled ligand or
`ligand will generally be present in from about 0.01 to
`100 times the concentration of ligand or labeled ligand
`based on binding sites.
`For polyepitopic ligand analytes, where labeled li-
`gand is employed, the concentration of labeled ligand
`will generally be not less than about 10*‘, more usually
`not less than about 10-3 times the minimum concentra-
`tion of interest and usually in the range of about equal to
`the minimum concentration of interest and not exceed-
`ing about the maximum concentration of interest. The
`ratio of labeled receptor will generally be not less than
`about 0.1 times the concentration of labeled ligand
`based on binding sites and not greater than about 100
`times the concentration of labeled ligand based on bind-
`ing sites.
`For monoepitopic ligand analytes and monoepitopic
`ligand receptor analytes, when employing labeled li-
`gand (includes polyfligand analog)-label), the concen-
`tration of labeled ligand based on binding sites will
`usually be not less than 10-4 times the minimum con-
`centration of interest, more usually not less than 10-1
`times the minimum concentration of interest and usually
`in the range of about the minimum concentration of
`interest
`to the maximum concentration of interest.
`
`45
`
`55
`
`When polyfligand analog) is employed with labeled
`antiligand, the concentration of polyfligand analog) will
`fall within the same ranges as indicated for the labeled
`ligand and the concentration of antiligand has been
`indicated previously.
`The order of addition of the various reagents may
`vary widely, depending upon whether an equilibrium or
`rate measurement is involved, the nature of the rea-
`
`

`
`7
`gents, the rate at which equilibrium is achieved between
`the ligand and antiligand, and the nature of the chemilu-
`minescence source. Where the chemiluminescence
`source has a plurality of components, with one of the
`components being a label, the chemiluminescence can 5
`be initiated at any time by the addition of the other
`components of the chemiluminescence source. In those
`situations where the chemiluminescence source in-
`volves more than one component, the labeled reagents
`and the unknown may be combined simultaneously,
`followed by the addition of the other components of the
`chemiluminescence source. Alternatively, one could
`combine the analyte with the labeled antiligand, fol-
`lowed by the addition of labeled ligand, as appropriate,
`followed by the addition of the remaining components
`of the chemiluminescence source. The various additions
`may be interrupted by incubation. In those instances
`where the chemiluminescence source is a single compo-
`nent, normally the labeled receptor will be combined
`with the analyte, followed by the addition of the labeled
`ligand, as appropriate.
`Depending on the mode employed, equilibrium or
`nonequilibrium, the rate of binding of the antiligand to
`ligand and labeled ligand and the relative concentra-
`tions of the ligand, labeled ligand and labeled antiligand,
`one or more incubation steps may be involved. Nor-
`mally, times between additions may vary from a few
`seconds to many hours, usually not exceeding l6 hrs,
`more usually not exceeding 6 hrs. Usually, incubation
`times will vary from about 0.5 rnin to 1 hr, more usually
`from about 0.15 min to 30 min. Since the ultimate result
`will be dependent upon the result obtained with stan-
`dard(s) treated in substantially the same manner, and
`when possible in the identical manner the particular
`mode and periods of time are not critical, so long as
`significant reproducible differentiations are obtained
`with varying concentrations of analyte.
`Depending upon the choice of assay protocol, the
`equipment employed and the concentration of analyte
`involved, assay volumes may be as small as about 1 pl,
`more usually being about 25 ii], and will usually not
`exceed 5 ml, more usually not exceeding 2 ml.
`The assay measurement will depend upon counting
`the quanta of light emitted from the assay medium.
`Various instruments may be used, such as scintillation 45
`counters, photocells or the like, which are capable of
`measuring light at a single or over a range of wave-
`lengths.
`
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`the
`about 10,000. In the poly(amino acid} category,
`poly(amino acids) of interest will generally be from
`about 5,000 to 5,000,000 molecular weight, more usu-
`ally from about 20.000 to 1,000,000 molecular weight;
`among the-hormones of interest, the molecular weights
`will usually range from about 5,000 to 60,000 molecular
`weight.
`The wide variety of proteins may be considered as to
`the family of proteins having similar structural features,
`proteins having particular biological functions, proteins
`related to specific microorganisms, particularly disease
`causing microorganisms, etc.
`The following are classes of proteins related by struc-
`ture:
`
`protamines-
`histories
`albumins
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropiu,
`prolactin, insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`cu-Lipoprotein
`o;1-Acid glycoprotein
`on-Antitrypsin
`ct:-Glycc-protein
`Transcortin
`4.6 S-Postalbumin
`Tryptophan-poor
`apglycoprctein
`ct 1X-Glycoprotein
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin
`(Gc 1-1)
`(Gr: 2-1)
`(G: 2-2)
`Haptoglobin
`(Hp 1-1)
`(HP 24)
`(HP 2-2)
`Ceruloplasmin
`Cliolinesterase
`
`az-Lipoprotein(s)
`rig-Macroglobulin
`ctgi-IS-glycoprotein
`Zn-ct;-glycoprotein
`az-Neuramino-glycoprotein
`Erythropoietin
`;3-lipoprotein
`Transferrin
`I-lemopexin
`Fibrinogen
`Plasrninogen
`3;-glycoprotein I
`B2-glycoprotein II
`Irntnunoglobulin G
`(IgG) or 7G-globulin
`Mol. formula:
`
`Materials
`
`The primary components in the subject assay for
`analyte, which may or may not be employed in every
`case are: labeled ligand (includes poly(ligand analog)-
`label); labeled antiligand, ligand; antiligand; and addi-
`tional components as required for the chemilun1iines- S5
`cence source.
`
`Ahalyte The ligand analytes of this invention are
`characterized by being monoepitopic or polyepitopic.
`The polyepitopic ligand analytes will normally be
`poly(amino acids) i.e. polypeptides and proteins.
`polysaccharides, nucleic acids, and combinations
`thereof. Such combinations of assemblages include
`bacteria, viruses, chromosomes, genes, mitochondria.
`nuclei, cell membranes, and the like.
`For the most part, the polyepitopic ligand analytes
`employed in the subject invention will have a molecular
`weight of at least about 5,000, more usually at least
`
`60
`
`65
`
`

`
`.
`
`9
`
`4,220,450
`
`(adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stitnulating hormone
`Luteinizing hormone
`(interstitial cell-stimulating hormone)
`Luteomantrnotropic hormone
`(lnteotropin, prolactin)
`Gonadotropin
`(chorionic gonadotropin)
`Tissue Hormones
`
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors
`CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-
`RF, PIF, MIF
`Other polymeric materials of interest are mucopoIy-
`saocharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`
` Species of Microorganisms Helnosensitin Found in
`Snepmcotrm pyoge.-res
`Poiysaccharide
`D-ipfacoccttt pneumaniae
`Polysaccharide
`Neisreria meningitidtls
`Polysaccharide
`Nettie:-it: gonorrisoeae
`Polysaecharide
`Carynebaeterfum diphtheria:
`Polysaccharide
`Acrinabacilius mnlle:‘.'
`Crude extract
`Actfnobdclllus wbltenmfi
`Fmncirella mlarensir
`
`Lipopolysaccharide
`Polysaccluride
`
`fltrtemefla peso":
`Pbstewrefla para‘:
`Ens-reurellc nmftocfda
`Brucefln abamrs
`Haemnphllu: influenzee
`Haemopnila: pertussis
`Ihqaanemn refteri
`Veillonella
`Erysipelothrht
`Line:-la monaqytogene:
`Chromobacterium
`Mycotlucrerfum tubemulaso
`
`Kiebnelta nerogenes
`Kfebsiellc cloaoae
`Salmonella g-photo
`
`Polysacciiaritle
`Capsular antigen
`_ Crude extract
`Polysaccharide
`Crude
`Polysaccharide
`Lipopolysaecharide
`Polysaccharide
`'
`Polysaoeharide
`Lipopolysaocharide
`Saline extract of 90%
`phenol extracted
`mycobacteria and poly-
`saocharide fraction of
`cells and tuberculin
`Polysaoclmride
`Polysaecharide
`Lipopolysaocharide.
`Polysaccharide
`Polysaccharide
`
`Salmonella gwhf-mudum:
`Salnwnella derby
`Salmonella puflarwn
`Skrjgelfo dysenterfae
`S-‘rigella flexnefi
`Sltfgelln sonnet‘
`Crude, polysaccharide
`Crude extract
`Rickettsiae
`Candida eloltnns
`Polysaecharide
`Crude extract
`Entnmaeba fifsrabrrlce
`
`Polysaechnride
`
`The microorganisms which are assayed may be in-
`tact, lysed, ground or otherwise fragmented, and the
`resulting composition or portion. e.g. by extraction,
`assayed. Microorganisms of interest include:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`71K: 01' ‘V212
`Immunoglobulin A (lgA)
`or -yA-globulin
`Mol. formula:
`(¢12I<2)" 01' (a27t2}"
`lmmunoglobulin M
`(IgM) or 'yM-globulin
`Mel. formula:
`
`(J12k'2)5 01' @212)’
`Immunoglobulin D0313)
`or yl)-Globulin (7121)
`Mol. formula:
`(szltz) or (32);)
`Imrnunoglobulin E (IgE)
`or 7E-Globulin ('yE}
`M01. formula:
`(€2K2) 01' (€2l2)
`Free K and 7 light chains
`Complement factors;
`01
`C'lq
`C’lr
`C'l.s
`C2
`03
`131A
`ct2D
`C4
`C5
`C'6
`C’?
`C8
`C9
`
`Important blood clotting factors include:
`BLOOD CLOTTING FACTORS
`International designation
`Name
`I
`Fibrinogen
`I]
`Prothrombin
`Ila
`'I'ln-onabin
`Ill
`Tssue thmmboplustin
`V and VI
`Proaccelerin, accelerator
`globulin
`VII
`Procorwertin
`
`
`
`VI]!
`Ix
`
`X
`
`Antilteanophilic globulin (AHG)
`Christmas factor.
`plasma tllrounbopilstin
`component (FTC)
`Stuart-Prower factor,
`auboprothrombln III
`
`Plasma thromboplastln
`KI
`antecedent (PTA)
`XI!
`Hagemann factor
`
`xn:
`Fibrin-stabilizing factor
`
`so
`
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`(parathromorle) _
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`Erythropoietin
`Mel-anotropin
`(melanocyte-stimulating hormone; intenneclin)
`Somatotropin
`(growth hormone)
`Corticotropin
`
`55
`
`50
`
`55
`
`

`
`ll
`
`Coryncbactcria
`
`Corynebacrerfum dlprherioe
`Pneumococci
`
`Dfphacoccus pneumonia:
`
`Streptococci
`Streptococcus pyogenes
`Streptococcus saffvaros
`
`Staphylococci
`
`Staphylococcus aomrs
`Staphylococcus albus
`Neisscrine
`
`Neisseria meningitidis '
`Neisseria gonorrheae
`
`Enterobacteriaciae
`
`Escherichia colt‘
`Atmhdclor aerngzus
`Kiehiiello pneumonia:
`
`J» The ooliforen bscleria
`
`Safmomffa glplaom
`Safmoneflo ckofcmccufs
`Salmonella mokouunhm
`
`Th: Salmoncilac
`
`S!u‘ga'!a dywltcrfoe
`S!n'3e!u"a xfimfrzii
`
`Skligdfo ombinomrdo
`Sltigwffo flexueri
`Siufigoffa boydir‘
`Shfigeflo Sam-m‘
`
`
`
`The Shigcllae
`
`Other cnteric bacilli
`
`P.-areas mfgoe-is
`Proteus mfrooifis
`} Proteus species
`
`Prareos morgoni
`
`Pseodomonos oeruginoso
`AIcoh'genesfl1ecofis
`Wbrfo choleme
`
`Hemophilus-Bordetella group
`Hemophilos fnfluenzoe,
`H. docngvi
`H. hemopmfos‘
`H. oegypricus
`H. paraiofloenzoe
`Bordetello perru.m's
`
`Pasteurellae
`
`Posreweflo mills
`%reore!Io 1'uloreus:'s
`
`Bmcello mofirensis
`Bmcello oborms
`Bmcello suis
`
`Brucellae
`
`Aerobic Spore-forming Bacilli
`Bociflos onthroctk
`Bacillus subrilis
`Bacillus megoreriom
`Bacillus cereos
`
`4,220,450
`
`12
`
`Anaerobic Spore-forming Bacilli
`Clostridxhm batoifnom
`Cfosrrfdiam mom‘
`Ciosrridfom perfringens
`Closrridfum navy!
`Cl_o.rm'd1'um sperfcum
`Cfostrzflium hisrolyrfcom
`Clostrtifium rertium
`Cfmtfidium btfermenrons
`Clostrfdium sporogenes
`
`10
`
`Mycobacteria
`Mycobacreriom tuberculosis flomfluis
`Mycobocrerfom bovfs
`Mycobocrerfum avium
`Mywobocreriom Ieproe
`Mycobacterfom porotubercuiosis
`
`Actinomycetes (fungus-like bacteria)
`Actfnomyces isroelfi
`Actinomyces bovfs
`Actinomyces naeslundii
`Nocordio osterofdes
`Nocardia brosfffemb
`
`The Spirochetes
`Treponemo paflidom
`Trepooemo pertenoe
`Treponemo coroteum
`Borrcfia recorremis
`Leprospfra fcrerohemorrhogioe
`Leprospir