`[11]
`[19]
`United States Patent
`
` Maggie [451 * Jul. 7, 1981
`
`
`[54] KIT FOR CARRYING OUT CHEMICALLY
`INDUCED FLUORESCENCE
`IMMUNCIASSAY
`_
`_
`Inventor: '1 Tl Maggm Redwood Cm“
`
`P51
`
`[73] Assjgnee:
`_
`[' 1 N03“?
`
`syn c,,mp,_m,_ page A1to_ calm
`_
`_
`The P011109 0f the term Of “"5 P-“ant
`5“b5e‘|“°'-‘it ‘O 339- 2» 997- has been
`di5°1aimed-
`[21] APPL NW 191,935
`
`[22] Filfidi
`
`DEE 10; 1979
`
`Reiated U.S. Applimtion Data
`Continuation ofser. No. 393,910, Apr. 5. 1978. Pat.
`NC“ 4.230I45D_
`
`G‘-“N 33/54; GWN 33/53
`Int C1-3
`Uusu CI. ...................................-
`B;
`23/915: 252/403; 424/3; 424/12; 435/7; 435/33
`435/319
`B} 422/61;
`Of Search ..................-.--u
`424/3» 12; ‘l‘35/7- 310
`Rfimnm Cited
`
`_
`
`[63]
`
`[51]
`
`[56]
`
`U-5‘ PATENT DOCUMENTS
`3/1977
`NiS'l‘|ii
`5/1973
`M3"=1'- 3“
`I/1980 Meier. Jr.
`9/1930
`Bucltler
`
`54-4/237
`-~-- 23/33” B
`424/12 1:
`
`23x23o B x
`
`..
`
`4,011,219
`"'-1°45”-9
`4,131,650
`4,225,435
`
`Attorney. Agent. or Fr'rm—Bertram I. Rowland
`
`[571
`‘”’STR“‘3T
`A com etit'v
`rotein bi di
`thud is
`rov'd ti f
`the detgrminaiign ofan annalyrig izilfich is a irriemiiefof:1:
`immunological pair consisting of ligand and receptor
`for thegiganti. A chunilummescélentdso;1lrce is irnployed
`eornprr
`o one or more in no u mem ers, 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-
`jug-ation. Aquencher 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 chemilumi-
`nescent source so as to inhibit the emission of light by
`‘he °"°',“‘1““““°5°"" 5?“'°"" The “"°“'“ °f a“3,'Y‘“
`present In the assay medium affects the amountof bind-
`mg between the members of the immunological pair
`which results in quenching of the chemiluminescence.
`observing thg
`emiued from the assay medium’
`either
`from the chemiluminescent
`source or
`the
`quencher, the change in light emission in relation to the
`concentration of analyte pfesgnt in the 355337 medium
`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 proviflfid having pfedetgrmined
`amounts of the reagents, so as to substantially optimize
`-
`-
`-
`—
`the ““"'”“t" °f the may‘
`
`Primary Exominer—Sidney Marantz
`
`4 Claims. No Drawings
`
`SANOFI V. GENENTECH
`SANOFI v. GENENTECH
`IPR2015—01624
`IPR2015-01624
`EXHIBIT 2043
`EXHIBIT 2043
`
`
`
`KIT FOR CARRYING OUT CHEMICALLY
`INDUCED FLUORESCENCE IMMUNOASSAY
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This is a continuation of application Ser. No. 393,910.
`filed Apr. 5. 1973 now U.S. Pat. No. 4,220,450.
`
`BACKGROUND OF THE INVENTION
`
`5
`
`10
`
`15
`
`20
`
`25
`
`3-0
`
`35
`
`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 aparticular
`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» 40
`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.
`
`45
`
`50
`
`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. 3,709,363 is exemplary of a radioimmu—
`noassay. U.S. Pat. No. 3,960,334 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. Gohike, Annual
`Review offiiachemfsny. 41, 343-363 (1972) and Stryer,
`Science. 162. S26 (1968). Smith, FEBS Letters 77, 25,
`(1977) describes a fluorescent
`immunoassay. where
`thyroxine is bound to a tluorescer and quenches the
`fluorescer. the quenching being reversed by binding of
`antibody to thyroxine. See also. Ullman et al, J. Biol.
`Chem. 251. 4112 (1976).
`
`55
`
`60
`
`65
`
`1
`
`4,277,437
`
`2
`An excellent review of chemiluminescence may be
`found in Mccapra, Quarterly Reviews 20, 435 (1966).
`SUMMARY OF TI-IE 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 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 ren-
`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-
`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 of the 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.
`Polyfligand 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 ofcompeting 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
`chernilurninescence source.
`
`(a) chemiluminescer label—a 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.
`(in) quencher—-a molecule capable of inhibiting the
`chemiluminescent emission oflight, when within a short
`but non-colliding distance, usually less than about 100
`A, of the chernilurninescer 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 polyfiigand 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 rnonovalent or polyvaleut in receptor sites usu-
`
`ll}
`
`15
`
`10
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,277,437
`
`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
`chemilurninescer labeled reagent and the quencher la-
`beled reagent (inoludes poly(ligand analog)-polylabel).
`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 cherniluminescer reagents. The four
`variations are:
`as
`to ligand
`conjugated
`(1)
`cherniluminescer
`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) chemilumincscer conjugated to receptor
`chemiluminescer labeled antiligand and quencher con-
`jugated to receptor as quencher labeled antiligand.
`(4)
`chemiluminescer
`conjugated
`to ligand as
`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 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 poly(ligand analogj) 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 polyfligand
`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
`assay is for ligand,
`the two label-conjugates are em-
`ployed in conjunction with antiligand which brings the
`chemilurn-inescer 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
`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
`wavelengths which might otherwise have been emitted
`by the chemilurninescer 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 elficiency of energy transfer from
`the chemilurniuescer 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
`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. 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 essay
`will be performed in the same manner, reading the light
`emitted by the chetniluminescer.
`In carrying out the assay, an aqueous medium will
`normally be employed. Other polar solvents may also
`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 3'' 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-
`
`it}
`
`IS
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`S5
`
`65
`
`4,277,437
`
`6
`rures 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*‘ to l[}—'5 M. more
`usually from about 10"“ to 10"” M Stated another
`way, the concentration ranges of interest will generally
`be from about 10-3 to 10- 1" 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 rec-
`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
`require different concentration ratios for the dilferent
`reagents.
`Normally, for mouo- 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 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 10-3 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 maairnum 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 poly(ligand 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 I04
`times the minimum concentration of interest and usually
`in the range of about the minimum concentration of
`interest
`to the maximum concentration of interest.
`When poly(ligand 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
`
`
`
`'7
`the nature of the res-
`rate measurement is involved,
`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 chernilununescence 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
`c-hemiluminescence 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 pt],
`more usually being about 25 _u.l. 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 poly(1iga.nd analog)-
`label); labeled antiligand; ligand; antiligand; and addi-
`tional components as required for the chemilumiines-
`cence source.
`
`Analyte
`
`The ligand analytes of this invention are character-
`ized by being monoepitopic or polyepitoplc. 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.
`
`[0
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,277,437
`
`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{arnino 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_rnolecular
`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
`histones
`albumins
`globulins
`scleropmteins
`phosphoproteins
`mucoproteins
`chromoproieins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropin, prolactln,
`insulin. pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`ct 1-Lipoprotein
`E1-Acid glycoprotein
`:11-Antitrypsin
`0:1-Glycoprotein
`Transcortin
`4.68-Postalbumin
`Tryptophan-poor
`apglycoprotein
`agx-Glycoprotein
`Thyroxin-binding globulin
`Inter-o:-trypsinvinhibitor
`Gc-globulin
`(Gc l-1)
`Gr: 2-1)
`(Ge 2-2)
`Haptoglobin
`(HP 1-1)
`(Hp 2-1)
`(H13 2-2)
`Ceruloplasmin
`Cholinesterase
`
`ag-Lipoprotein(s)
`oz-Macroglobulin
`ct:-HS—glycoprotein
`Zn-az-glycoprotein
`ct;-Neuramino-glycoprotein
`Erythropoietin
`B-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`,8;-glycoprotein I
`Bpglycoprotein II
`
`
`
`9
`
`4,277,437
`
`ll]
`
`Imrnunoglobulin G
`(IgG) or 7G-globulin
`Mo}. formula:
`T2 K3 or 73%;
`Irnrnunoglobulin A (lgA)
`or 'yA-globulin
`Mol. formula:
`(r.tgr¢2}" or (ct-3)t3)*"
`Immunoglobuljn M
`(IgM) or 7M-globulin
`Mol. formula:
`(p.g:c;)5 or (p,3)Lg)5
`Immunoglobulin D (lgD)
`or 7D-Globulin ('yD)
`MOL formula:
`(Ezra) or (8311)
`lrnmunoglobulin E (IgE)
`or 'yE-Globulin ('yE)
`Moi. fonnula:
`(egrcz) or (ezhz)
`Free K and
`li ht chains
`Complememyfacgmrs:
`C’!
`C’ lq
`C'."lr
`C'1s
`C'2
`C'3
`31A
`a;D
`C4
`C'5
`.
`.
`C'6
`Hemosensitln Found in
`C7
`Polysacclwride
`C3
`a:*“:::::"i:'.:
`cs
`Polmcchaiicle
`Nutrserirr ganolgeme
`Important blood clotting. factors include:
`Polysaccharide
`Corynelmcrerlum dljrilrtlieriae
`Crude extract
`Acrfnobacrllus malm‘;
`
`
`
`.
`.
`.
`Spcrnes of Mlcfflflfgflnfims
`35 Suapracoceurpiogener
`
`' BLOOD CLOTTING FACTORS
`
`International designation
`Name
`1
`Fibfillflserl
`11
`Prothrornbin
`111‘
`T!1l'0mbiII
`III
`Tissue thrornboplastin
`V “"51 VI
`P1'03C°B]E|'lfl- |1D':9i£l'3f01'
`globulin
`Proconvertin
`VII
`VIII
`Antihemophilic globulin LIKHG}
`IX
`Christmas factor.
`_
`plasma rhromboplasun
`Wmpflflfll (FTC)
`Stuart-Prowcr factor.
`autoprothrombin III
`Plfifleiii li1:€I(|l1:1_i1l!_!::)i35lID
`xi
`311
`On
`311
`H_as_=mann_I';:Ictor
`
`XI"
`Flbl'1“*5l3b|l1ZI|13 f’-‘CWT
`
`X
`
`_
`_
`Important protein horrnones Include:
`_
`_
`Peptide and Protein Hormones
`.
`Pa(1;:;l13g?1:l‘:'£Hl:Drn;Dne
`01'-"5
`Thyrocaicitunin
`Insulin
`Gluca on
`E’ HXIH
`R I
`_g
`Erythropoietin
`Melanmmpin
`[melanocyte-stimulating
`
`40 Acrfnolsaciffus wlu'regr:on'
`Fmflflflfla 'mm""'m
`Pasreuralla perrls
`Pa.rreurellr: pr-srrs
`Pasteur!-lla mulrorfdn
`45 flnrcella abanrr:
`Haemopliilus rnfluerrzae
`H
`is‘!
`I
`is
`Tfe::!::rn’a":e‘l?;:l“”
`Vgillonellu
`Eryipelothrix
`so LI}:-aria naonocjrrogrmvr
`Chromobacteri um
`Myrnbccrarium rum-rculosrx
`
`55 Klebriellrr aeragerres
`Klebsiella elaame
`Salmnella typlmra
`
`’
`o ysacc
`
`_
`
`c
`
`Polysaccharide
`Capsular antigen
`Crude extract
`Polymwhafide
`Crud
`Polysfaocharide
`Lipnpolysgccharidg
`Polysaccharide
`Polysaccharide
`Lipopolysaceharide
`Saline extract of 90%
`phgnol exu-ggrgd
`mgrcobntgeriin and pair}!-
`saocheri e raction o
`celis and tubercuiin
`Polysacchar-Ede
`Polyaaccharide
`Lipopolysncclraridc.
`Pnlysagchgrjflg
`Folysuccharide
`
`Salmonella rypln'-mrmhm:
`Safmaneffa deg-by
`55 Salmonella pullonrm
`Polysmocharidc
`Slrufg-ella d_}-.r.-mrenire
`Sfirgellaflexnefi
`Crude. polysaccharide
`Slitgella serum’
`Crude extract
`Rickettsiac
`Candida allricans
`Polysuccharide
`
`65 Ermzrmarba lrr'.r1'ol'y1'I'-:0
`Crude extract
`_
`_
`_
`_
`The microorganisms which are assayed may be m-
`tact, lysed. ground or otherwise fragmented. and the
`
`hormone; interrnedin)
`Somatotropin
`(growth hormone)
`Corticotropin
`{adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Ltlteinizing hormone
`(interstitial cell-stimulating hormone)
`Lntoomammotropic hormone
`(luteotropin, prolactin)
`Gonadotropin
`(chorionic gonadotropin)
`.
`Tussue Hormones
`
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`.
`,
`Peptide Hormones from the Neurohypophysts
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF, Somatotropin-RF.
`GRF. FSH-RF, PIF, MIF
`Other polymeric materials of interest are muccpo]y-
`saccharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`5
`
`10
`
`,5
`
`29
`
`35
`
`30
`
`
`
`11
`resulting composition or portion, 2.1;. by extraction,
`assayed. Microorganisms of interest include:
`
`4,277,437
`
`I2
`
`Cfosrridfum Ierrium
`Cfaszridfnm bgfermenraru
`Cfosn-1'dfum sporogerrcs
`
`Mycobactcria
`Myoabocterium tuberculosis homfmls
`Mycobocterium bovix
`Mymbocrerium ovium
`Mycobocrerfum Icprac
`Mycobocterfnm pamruberculosis
`
`Acrfnomyceres [furzgus-like bacteria)
`
`Acrinomyces :1sroeIr':'
`Ac-rinomyces bovfs
`Actirtomyces naesfuodff
`Nocordfo osterofdes
`Nocordfo bros:'!£ens:'s
`
`The Spirochetes
`
`.7)-*eponemo paflfdum
`Treponemo perremte
`Treponemo carareum
`Borrelio mcurremis
`
`Leptospiro ictemhemorrhaglae
`Leptospim confcofa
`Spfrfllum minus
`Streprobacfflus monflifiirmis
`
`Mycoplasrnas
`Mycoplosma pneumonia:
`
`Other pathogens
`Li.-rterio mooocyrogene:
`Erysajoefotfxrix rhus:‘oparIu'ae
`Streprobociiius mom'h_‘fi:rm£9
`Donvanio grormlomatfs
`Bartonelia baciH{f'orm£s
`
`Riokettsiae (bacteria-like parasites)
`Rickerrsfa prowazekif
`Rickettsia mooseri
`Rickerrsfa r1'ckcm¢':'
`Rickerrsio conori
`Rfckertsfo ousrralis
`Rickcrrsfo sibfrfcus
`R:'cken‘.n‘a akori
`Rickertsfa rsutsugamuxhi
`Rfckertsfo bnrnettii
`Rlckeztsio qnimono
`
`Chlamydia {unclassifiable parasites bacterial.’viral)
`Chlamydia agents (naming uncertain)
`
`
`
`Fungi
`Oypracoccus .-Ieafonnons
`Blasramyces dermorfdis
`Hakropfnsmo mp.m."o.I'um
`Caccidfofdes I‘mmir£s
`Phracoccuiaffofdes bmstflemix
`Candida ofbfcans
`Axpergifius fumlgorux
`Mumr cwymbfifer (flhrfdfa wqmbyera}
`Rhimpu: oryzac
`Rhisopus mrrk.-'zus
`Rhfzopus nljgrimns
`Spamfrickum schenkfi
`Forcwcoea pedmsoi
`Fcmsecaea cwrlpacra
`Foruecaca derrnnrflfdis
`Cfudosporium carrfonif
`
`} Phyaornyceles
`
`5
`
`jg
`
`'5
`
`30
`
`25
`
`30
`
`35
`
`45
`
`SO
`
`55
`
`60
`
`65
`
`Corynebacteria
`
`Caryn ebocreriom diprhcrfoe
`Pneurn