4,277,437
`an
`[19]
`United States Patent
`Maggie
`[45]
`* Jul. 7, 1981
`
`
`U51
`
`[54] KIT FOR CARRYING OUT CHEMICALLY
`INDUCED FLUORESCENCE
`IMMUNOASSAY
`Inve==tor=
`Edward T- Maze’-=. Redwood City»
`Cam‘
`Syva Company, Palo Alto, Calif.
`_
`_
`The Porno" Of the term °f “"5 Pale“
`5‘;‘b5°'-_l“°'“ "3 5513- 2v 997’ has be“
`d'5°1a‘med-
`[21] APPL No; 191,935
`_[221
`Fi3¢d=
`1390- 19; 1979
`
`[73] Assignee:
`‘E
`_
`_
`1 N°“°e'
`
`[
`
`Attorney. Agent. or Firm—Bertrarn I. Rowland
`
`[571
`ABSTRACT
`A
`.
`.
`tit.
`.
`.
`.
`a.e°§§E§§niL‘;'E§§‘L‘?L'l §L"Ji§§§ L"§£i3§.Ls£2§$§f3ff§§
`immunological pair consisting of ligand and receptor
`for the ligand. A chemiluminescent source I8 employed
`comprised of one or more individual members, one
`chemiluminescent source member being conjugated to
`one of the members of the immunological pair, so as to
`provide chemiluminescence adjacent to the site of con-
`{jugatitonfiA_quenchv.l=.r moallecule iskognjugfiited to ajemern;
`eroteimrn 0
`1c
`an:
`t
`the irnmunologilclzil Erfir bmld, the qu:|1cheerr:':?l1eciiI5eci's
`brought within quenching distance of the chemilumi-
`nescent source so as to inhibit the emission of light by
`‘he °}'°f“‘1“m‘“e‘°"‘“‘ 5?“““'=- The ‘““°“’" 0f a“a_‘Y'°
`present In the assay medium affects the amount of bind-
`ing_ between the members of the immunological pair
`which results in quenching of the chemllurnmescence.
`By observing the ugh, emitted fmm the may medium,
`either
`from the chemiluminescent
`source or
`the
`hr,th 11
`'
`1'
`11: " '
`It‘
`lth
`2§.‘?;2§.,f,....0§ ‘:,ra$:1;Iie1§;es:]:is::1o$: zigsgyiorrriiegiurs
`can be used to determine the amount of analyte 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
`-
`-
`-
`:§:°sL;';t:it?£i:heOrfe;.g:';:’a5° as to wtistannany °pt"'"z°
`y
`Y‘
`I
`
`Relatetl U.S. Application Data
`Continuation oFSer. No. 393,910, Apr. 5. 1973, Pat.
`Na 4’23o_450_
`_
`In C] 3
`Sl
`l
`I
`---------------------- Gm-N 33/54» G‘-“N 33/53
`t-
`-
`[52] U.S. c1. .................................. .. 422/61; 23/230 B;
`23/915; 253/403: 424/33 424/12; 435/7; 435/3:
`435/810
`[53] Field of Search ........................ 23/230 B; 422/51;
`424/8» 12; 435/7» 310
`Cited
`
`[63]
`
`56
`[
`
`1
`
`Ref
`flames
`US‘ PATENT DOCUMENTS
`auer,
`r.
`424/12 x
`..
`1/1930 Meier, Jr.
`9/ 1950 Buckler ........................ .. 23/230 B X
`
`
`
`fi-lftlylfgg
`.
`,
`4,181,650
`4,225,485
`
`Primary Examfrzer—Sidney Marantz
`
`4 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2043
`
`Genentech Exhibit 2043
`
`IPR2016-00710
`
`

`
`I
`
`4,277,437
`
`KIT FOR CARRYING OUT CHEMICALLY
`INDUCED FLUORESCENCE IMMUNOASSAY
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This is a continuation of application Ser. No. 893,910,
`filed Apr. 5, 1978 now U.S. Pat. No. 4,220,450.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`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 distinguish between labeled
`ligand which is bound to receptor and unbound labeled
`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.
`there are many consider-
`In developing an assay,
`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
`equipment, ease of automation, and interaction with
`ligands, are additional considerations, which do not
`exhaust the various concerns in developing a useful
`assay.
`
`There is therefore a continuing need for new and
`accurate techniques which can be adapted for a wide
`spectrum of dilferent 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 radioirnmu-
`noassay. U.S. Pat. No. 3,960,834 is exemplary of a spin
`immunoassay. U.S. Pat. No. 3,654,090 and German
`Auslegungsschrift No. 2,223,385 are exemplary of en-
`zyme immunoassays. Articles of interest
`include an
`article by Ludwig Brand and James R. Gohlke, Annual
`Review affliochemfsrry, 41. 343-863 (1972) and Stryer,
`Science.
`l62, S26 (1968). Smith, FEBS Letters 77, 25,
`(1977) describes a fluorescent
`immunoassay, where
`thyroxine is bound to a fluorescer and quenches the
`fluorescer. the quenching being reversed by binding of
`antibody to thyroxine. See also, Ullman et al, J. Biol.
`Chem. 251. 4172 (1976).
`
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`An excellent review of chemiluminescence may be
`found in McCapra, Quarterly Reviews 20, 485 (1966).
`SUMMARY OF THE INVENTION
`
`A competitive protein binding assay is provided hav-
`ing as an analyte a member of an immunological pair
`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 ofanalyte 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 analyie is a
`member of an immunological pair which includes li-
`gand 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 reagents 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 ofthe chemi-
`
`

`
`3
`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.
`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.
`Poly(ligand analog)—a plurality of ligand analogs
`joined together convalently, 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—a compound which by
`itselfor 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.
`(b) quencher—a molecule capable of inhibiting the
`chemiluminescent emission of light, when within a short
`but non-colliding distance, usually less than about 100
`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 polyfligand 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-
`
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`4,277,437
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`4
`ally polyvalent e.g. antibodies. For any specific ligand,
`the receptor will be referred to as “antiligand". The
`receptor«antiligand»and its reciprocal
`ligand form an
`immunological pair.
`Poly(ligand analog)-label—a composition in 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 pl-I, 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{ligand analog)-polyiabel),
`and as appropriate ligand or antiligand.
`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 chemiluminescer reagents. The four
`variations are:
`_
`to ligand as
`(1)
`chemiluminescer
`conjugated
`chemiluminescer labeled ligand and quencher conju-
`gated to receptor as quencher labeled antiligand;
`(2) quencher conjugated to ligand as quencher la-
`beled ligand and chemiluminescer conjugated to recep-
`tor as chemiluminescer labeled antiligand; and
`as
`(3) chemiluminescer conjugated to receptor
`chemiluminescer labeled antiligand and quencher con-
`jugated to receptor as quencher labeled antiligand.
`as
`(4)
`chemiluminescer
`conjugated to ligand
`chemiluminescer labeled ligand and quencher conju-
`gated 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 anaiyte,
`either ligand or antiligand. will serve to reduce the
`amount of energy transfer between the chernilumi-'
`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(ligand 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 to increase. 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 poly(ligand
`analog), the presence of monoepitopic ligand will serve
`to diminish quenching. With receptor as analyte,
`in-
`
`

`
`5
`creasing concentrations of receptor will also serve to
`diminish quenching.
`Where the chemiluminescer and the quencher are
`both conjugated to ligand, an assay for either ligand or
`polyvalent antiligand may be performed. Where the 5
`assay is for ligand, the two label-conjugates are em-
`ployed in conjunction with antiligand which brings the
`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
`increasing amounts of antiligand, there will be a de-
`crease of chemiluminescence to a minimum and then an
`increase as the concentration of antiligand increases. If is
`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 20
`from the chemiluminescer to the quencher. The result
`of this transfer will be that light of a single or range of
`wavelengths which might otherwise have been emitted
`by the chemiluminescer will be transferred to the
`quencher, which may then fluoresce, emitting light of a 25
`higher wavelength than the energy absorbed. Depend-
`ing upon the quantum efliciency 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 30
`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
`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 40
`be achieved i.e. quenching without quencher bonded to
`receptor, particularly where the receptor is an anti-
`body. While 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 50
`be employed, usually oxygenated organic solvents of
`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 10, 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-
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`tures during the period of the assay will be employed.
`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 10-4 to 10-15 M, more
`usually from about 10"‘ to 10-'3 M. Stated another
`way, the concentration ranges of interest will generally
`be from about 10-3 to 1044 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 tlte binding constant and binding profile
`of receptors will vary, for example, with antibodies
`from bleed to bleed, each new batch of antibodies may
`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 l to 3. times the maximum concen-
`tration of interest based on binding 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*? 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*?
`times the minimum concentration of interest and usually
`in the range of about the minimurn concentration of
`interest
`to the maximum concentration of interest.
`When poly(1igand' analog) is employed with labeled
`antiligand, the concentration of poly(ligand 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
`
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`4,277,437
`
`7
`rate measurement is involved, the nature of the rea-
`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
`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 16 hrs.
`more usually not exceeding 6 hrs. Usually. incubation
`times will vary from about 0.5 min 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 _l.tl,
`more usually being about 25 pi, 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
`counters, photocells or the like, which are capable of
`measuring light at a single or over a range of wave-
`lengths.
`
`Materials
`
`The primary components in the subject assay for
`analyte, which may or may not be employed in every
`case are: labeled ligand (includes po1y(ligand analog)-
`label); labeled antiligand; ligand; antiligand; and addi-
`tional components as required for the chen1ilumiines-
`cence source.
`
`Analyte
`
`The ligand analytes of this invention are character-
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poly(amino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations of assemblages include bacteria,
`viruses, chromosomes, genes, mitochondria, nuclei, cell
`membranes, and the like.
`
`8
`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
`about 10,000.
`In the poly(amino acid) category,
`the
`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,
`protns 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
`alburnins
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`ct;-Lipoprotein
`ct]-Acid glycoprotein
`ct;-Antitrypsin
`on-Glycoprotein
`Transcortin
`4.68-Postalbumin
`Tryptophan-poor
`:1 1-glycoprotein
`my-Glycoprotein
`Thyroximbinding globulin
`Inter-ct-trypsin-inhibitor
`Gc-globulin
`(Gc l-l)
`Gc 2-1)
`(Ge 2-2)‘
`Haptoglobm
`(Hp 1-1)
`(H13 2-1)
`(HP 2-2)
`Ceruloplasrnin
`Cholinesterase
`ag-Lipoprotein(s)
`ct:-Macroglobulin
`a.2-I-IS-glycoprotein
`Zn-a2-glycoprotein
`or;-Neuramhio-glycoprotein
`Erythropoietin
`,8-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`,8;-glycoprotein I
`[31-glycoprotein II
`
`[0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`SS
`
`60
`
`65
`
`

`
`9
`
`Immunoglobulin G
`(IgG) or 'yG-globulin
`Mol. formula:
`‘yz K2 or ‘yak;
`Immunoglobulin A (EgA)
`or 'yA—globulin
`Mol. formula:
`(a2K2)" or (a2)t2)"
`Immunoglobulin M
`(IgM) or 'yM-globulin
`Mol. formula:
`
`Utzkz)’ or (M27\2)‘
`Immunoglobulin D (IgD)
`or -yD-Globulin ('yD)
`M01. formula:
`(Syn) or (6313)
`Immunoglobulin E (IgE)
`or 'yE-Globulin (yE)
`M01. formula:
`(QK1) or (6;-A2)
`Free K and 7 light chains
`complement factors:
`C’!
`C'1q
`C'1r
`C'1s
`C'2
`03
`51A
`.1213)
`C4
`C'5
`
`-
`
`4,277,437
`
`_
`
`5
`
`19
`
`,5
`
`20
`
`35
`
`30
`
`10
`
`hormone; intermedin)
`Somatotropin
`(growth hormone)
`Corticotropin
`(adrenocorticotropic hormone)
`Thyrotropin
`Fol1icle—stimulating hormone
`Luteinizing hormone
`(interstitial cell-stimulating hormone)
`Luteomammotropic hormone
`(luteotropin, prolactin)
`Gonadotropin
`(chorionic gonadotropin)
`
`Tissue Hormones
`
`(S}ecretin
`astrin
`Angiotensin I and 11
`Bradykinin
`Human placental lactogen
`
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF, Somatotropin-RF,
`GRF, FSH—RF, PIF, MIF
`Other polymeric materials of interest are rnucopo1y-
`saocharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`
`
`Hemosensitin Found in
`Pol
`ccharide
`Polmeeltatide
`Polysaccharide
`Polysacehafide
`Polysaccharide
`Crude extract
`
`Species of Micronrganisrns
`35 Sm: racocrus
`.-mes
`Dmtimccus pfgfiflfoflffle
`Neirserra meniugiridis
`Neifierfa gammeoeae
`Carynebacterfam d:‘p.‘i.tJ'ae.I1'ae
`Acffnobaciflias maHe:','
`
`49 Aerfnobarfflus Infiiternorf
`Fmnwflfla mlwenm
`Pasreuneffa pestis
`Pasteureffa peso":
`Pasteurefle mulrocida
`45 Bruceffe abomrs
`Haemopfiiius influenzae
`Haemaphilas perrrassis
`Trepouema refterf
`Veillonella
`Eryipelothrix
`Listeria monacyrogenes
`50 Chrornobaclerium
`Myoobzacrerfani rublercu-’a.n's
`
`55 Kfebsiefle aemgeries
`Klebsiefla cfaacae
`Salmonella rypfrosa
`
`I
`_
`Il;':fi'::E°c]:::cr:1h:rlde
`Polysaocharide
`Capsular antigen
`Crude extract
`Polysaccharide
`Crude
`.
`Polysaoeharide
`Lipopolysaccharide
`Polysaoeltaride
`Polysaccltaride
`Lipopoiysaccharide
`Saline extract of 90%
`phenol exrmmed
`mycobacteria and poly-
`saocharide fraction of
`cells and tuberculin
`Polysaocharide
`Polysaecharide
`Lipop-olysaccharide.
`Polysaocharide
`Polysaccharide
`
`Salmonella rypIn'-in arittm.‘
`Sirfmaneffa derby
`60 §:lt;::;reg:s::::::em
`Shfigelfaflexnert‘
`Crude. polysaccharide
`Siiigeffa sonnet
`Crude extract
`Rtckeltsiae
`Candida afbitwes
`Folysaccharide
`
`65 Entamaeba }rr‘rra:'y!i'ca
`Crude extract
`
`Polysaccharide
`
`,
`_
`_
`The microorganisms whlch are assayed may be m-
`tact, Iysed, ground or otherwise fragmented. and the
`
`fig
`I
`gig;
`Important blood clotting factors include:
`
`
`
`' BLQQD CL0-[TING 1=AC'roR5
`International designation
`Name
`I
`Fibrinogen
`II
`Prothrombin
`Ila
`Thrombin
`III
`Tissue thromboplastin
`V and VI
`P|'0fi0W]€|'il1. 3063139310‘!
`globulin
`Proconvertin
`Antihemophilic globulin LAHG}
`Christmas factor.
`Plasma lhwmbbplastifl
`component (PTC)
`SW81’!-PIOWCF 530'-01',
`auloprothrombin III
`Plasma thromboplastin
`XI
`antecedent (PTA)
`X11
`Hagematm factor
`
`X1”
`Fibl'1'fl—-flawizlfflfi 73‘-'I0T
`
`VII
`VIII
`IX
`
`X
`
`_
`_
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`(PaP3thT0m0D3)
`Thyrocalcitonin
`Insulin
`G1
`8 anti
`R llmajgon
`Erythropoietin
`Mflanotropin
`{rnelanocyte-stimulating
`
`

`
`11
`resulting composition or portion, e.g. by extraction,
`assayed. Microorganisms of interest include:
`
`4,2T?,437
`
`12
`
`Cfostridfum rertium
`Clostridium brfcrmenrans
`Clostridfum sporogenes
`
`Mycobacteria
`
`Mycobocrerium tuberculosis hominfs
`Mycobocterfum bow":
`Mycobacrerium avirzm
`Mycoboczerfum lepme
`Mycobacterium parotuberculosis
`
`Aczinomyccres {fimgus-like bacteria)
`
`Actinomyces :'sraeI:'1'
`Acrirzomyces bovis
`Acrinomyccs naeslurrdif
`Nocardia asteroids;
`Nocordia brasfliensfs
`
`The Spirochetes
`
`Treponemo paflfdum
`Treponemo perrerzue
`Treporiemo caroteum
`Borrelfo recorremfs
`Leprospim icterohemorrbagfoe
`Leprospfro conicofa
`Spfrilhim minus
`Srrepzobacfflus monilgformo
`
`Myooplasmas
`Mycopfasmo pneum onfae
`
`Other pathogens
`L:':r:er:'o monocyrogenes
`Erysfpelorhrix rhusioporhfoe
`Streprobacfflus mom'I.ff'orm1‘s
`Doovamo grormlomatfs
`Borronella barcilltformfs
`
`Rickettsiae (bacteria-like parasites)
`Ricketfsfo prowazekfi
`Rfckertsfo moosen‘
`Rfickertsfa rfcketrsii
`Rfckertsia canon‘
`Rickertsia australfs
`Ricketrsfa sfbirfcus
`Rickcrrsfo okori
`Ricketrsfa rsursugamusffi
`Rfckerrsio bu:-nem':'
`R:'ckeflsio quintamz
`
`Chlamydia (unclassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`
`Fungi
`Cryproooccus neoformons
`Bfostomyces dermorfdis
`Hisropfnsma captmiatum
`Caccidfofdes immirfs
`Pbrococcidfordes bmsiifensis
`Candida afbicans
`Asperg:'!.i'usfum;‘3_4!u:
`Mucor corymbtfer {Abfldia corymbrfera)
`Riifzopmrx ogazoe
`Rhizqaus nrrmzus
`R.*u'z:opus nigrfcans
`Sporoirichum schenkfi
`Fonsecaea pedrosai
`Fans-amen compacm
`Fonsewcu dermatirfdis
`Cfadaspofium carrfonif
`
`) Phycomycetes
`
`5
`
`10
`
`15
`
`30
`
`25
`
`30
`
`35
`
`45
`
`0
`
`5
`
`55
`
`65
`
`Corynebacteria
`
`Corynebactermm dgorheriae
`Pneumococci
`
`Dipl