`
`[19]
`
`[11]
`
`4,220,450
`
`[45] Sep. 2, 1980
`Maggie
`
`
`
`the determination of an analyte which is a member of an
`ilnmtinological 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 chemilumi-
`nescent aource 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 chetniluminescence.
`By observing the light emitted from the essay medium,
`either
`from the chemiltuninescent
`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 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
`amounts of the reagents, so as to substantially optimize
`the sensitivity of the assay.
`
`[S4] CHEMICALLY INDUCED FLUORESCENCE
`IMMUNOASSAY
`
`['!5}
`
`Inventor:
`
`Edward T. Maggie, Redwood City.
`Calif.
`'
`
`[73] Assignee:
`
`Syva Company. Palo Alto, Calif.
`
`[2l] Appl. No.: 893,910
`
`[22] Filed:
`
`Apr. 5, 1973
`
`[51]
`
`Int. CL?
`
`[52] U.S. Cl.
`
`[58] Field of Search
`
`GIJIN 33/16; GOIN 31/I4;
`CO9l{ 11/00
`23f23i| B; 424/8;
`424/12; 435/7; 435/8
`23/230 13; 424/8, 12;
`195/1035 A. 103.5 1.4435/7. S
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`....
`
`195/103.5 L
`195/103.5 A
`I95/103.5 L
`. 195/103.5 A
`. 195/103.5 A
`23/230 B
`. I95/103.5 A
`23/2303
`
`
`
`3.5:-5,312
`3,550,241
`3352,15?
`3.9965345
`3.998.943
`3.999393
`4.043.371
`4,104,029
`
`Chappelle
`-m9'n
`Chnppolle
`s/1912
`Rubenstein ..
`12/197:1
`12/1976 Ullman
`12/1976 Ullrnan
`8/191-'6
`Szczeanialt
`8/I97?
`Blakemnre
`3/1978 Maier
`
`.Pn'mmy Examt'ner—Sidney Marantz
`Attorney. Agent, or Ft'r:n—I-lertrnm I. Rowland
`
`[57]
`
`ABSTRACT
`
`A competitive protein binding method is provided for
`
`32 (Jaime, No Drawings
`
`SANOFI V. GENENTECH
`SANOFI v. GENE(cid:49)TECH(cid:3)
`IPR2015—01624
`IPR2015-01624
`EXHIBIT 2058
`EXHIBIT 2058
`
`
`
`1
`
`4,220,450
`
`CHEMICALLY 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 distinguish 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 25
`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 35
`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 -to
`equipment, ease of automation, and interaction with
`ligands, are additional considerations, which do not
`exhaust the various concerns in developing a useful
`assay.
`
`30
`
`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. 3,709,368 is exemplary of a radioimmu- S0
`noassay. U.S. Pat. No. 3,960,834-.is exemplary of a spin
`immunoassay. U.S. Pat. No. 3,654,090 and German
`Auslengungsschrift No. 2,223,335 are exemplary of
`enzyme imntunoassays. Articles of interest include an
`article by Ludwig Brand and James R. Gohlke, Annual
`Review of Biochemistry. 41. 843-368 (1972) and Stryer,
`Science. 162, 526 (1968). Smith, FEBS Letters 77, 25,
`(1972-'} describes a fluorescent
`immunoassay. where
`thyroxine is bound to a lluorescer and quenches the
`lluorescer, the quenching being reversed by binding of 60
`antibody to thyroxine. See also, Ullman et al. J. Biol.
`Chem. 251. 4172 (1976).
`An excellent review of chemiluminescence may be
`found in McCapra, Quarterly Reviews 20, 485 (1966).
`SUMMARY OF THE INVENTION
`
`65
`
`55
`
`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 chemiltnninescence 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 n 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 entit-
`ted light and the amount ofanalyte 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 oompont 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
`chemilurninescer in the excited state, the chemilumi-
`nescer may transfer its energy to the quencher without
`collision and without emitting radiation. The qtlencher_
`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-
`live 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.
`Liga.nd—any compound for which a receptor natu-
`rally exists or can be prepared.
`Ligand ana]og—a modified ligand which can com-
`pate with the analogous ligand for receptor, the modifi-
`cation providing means to join to a label or to a hub
`nucleus.-
`_ Polyfligand ana1og)—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——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.
`(13) 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 chexniluniinescer 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
`;:a(:lo:;i1I1gatod to the enzyme to form a poly{'liga.nd analog)
`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 “antiligand". The
`receptor-antiligand-and its reciprocal liqand form an
`immunological pair.
`
`ll}
`
`20
`
`25
`
`3D
`
`35
`
`45
`
`55
`--
`
`60
`
`65
`
`4,220,450
`
`4
`Polyfligand 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 polyfligand analog)-polylabel),
`and as appropriate ligand or antiligsnd.
`The presence of antiligand or ligand in combination
`with a predetermined amount of nntiligand 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:
`conjugated to ligand as
`(I)
`chemilutuinescer
`chernilurninescer labeled ligand and quencher con-
`jugated to receptor as quencher labeled antiligand;
`(2) quencher conjugated toligand as quencher la-
`beled ligand and chemilurniuescer conjugated to
`receptor as chemilurninescer labeled antiligand;
`and
`
`(3) chemiluminescer conjugated to receptor as
`chemiluminescer labeled antiligand and quencher
`conjugated to receptor as quencher labeled antih-
`gand.
`to ligand as
`conjugated
`(4)
`chemilmninesoer
`chemiluniinescer labeled ligand and quencher con-
`jugated to ligand as quencher labeled ligand.
`With the first two combinations. when the reagents
`are combined. the qucher 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(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,
`'l'ollowed'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 polyfligand
`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.
`'
`
`
`
`4,220,450
`
`IS
`
`25
`
`35
`
`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
`chemiluminescer and quencher together into quenching
`distance of each other. The addition of ligand reduces
`the amount of chemilurninescer 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 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 cbemiluminescer will be transferred to the
`quencher, which may then lluoresce. emitting light of a
`higher wavelength than the energy absorbed. Depend-
`ing upon the quantum efficiency of emission of the
`chemilurninescer. the efficiency of energy transfer from
`the chemiluminescer to the quencher. and the quantum
`efliciency 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 ZOCKJ molecular weight. With these
`haptens a substantially reduced chenrilmniitcecence can
`be achieved i.e. quenching without quencher bonded to 49
`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 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
`from one to six, more usually from one to four carbon
`atoms. including alcohols, elhers 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. 3" to 9. Various bull'-
`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 cluring the period of the assay will be employed.
`
`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"‘-5 M, more
`usually from about 10-5 to 10-13 M. Stated another
`way. the concentration ranges of interest will generally
`be from about lU*3 to 1D‘” 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 oonctration 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
`20 require different concentration ratios for the dilferent
`reagents.
`_
`Nonnally. 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 I to 3 times the maximum concen-
`tration of interest based on binding sites.
`For polyepitopic ligand receptor analytes, the equiv-
`aient 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 limes 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 10-2 times the minimum concentra-
`tion of interest and usually in the range ofabout equal to
`the minimum concentration of interest and not exceed-
`ing about the II1t!|J£il‘.t1‘l.tI'r1 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 moncepitopic
`ligand receptor analytes, when employing labeled li-
`gand (Includes poly(ligs.nd analog)-label), the concen-
`tration of labeled ligand based on binding sites will
`usually be not less than 10-‘ 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.
`When polyfligand analog) is employed with labeled
`antiligund, the concentration ofpolyfligand 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 res-
`
`45
`
`50
`
`55
`
`65
`
`
`
`4,220,450
`
`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 chlurninescence 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. Altematively. 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 I6 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«
`da.rd{s) treated in substantially the same marmer, 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 he as small as about 1 it],
`more usually being about 25 ill, 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 iustriunents 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 polyfligsnd analog)-
`label); labeled antiligand, ligand; antilignnd; and addi-
`tional components as required for the chemilumiines-
`cence source.
`
`Analyte The ligand analytes of this invention are
`characterized by being monoepitopic or polyepitopic.
`The polyepitcpic 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
`
`the
`about 10,000. In the po1y(arnino acid) category.
`poly(arnino 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:
`
`10
`
`I5
`
`25
`
`30
`
`35
`
`protamines
`l.'1.l5lO1'tCS
`aiburuins
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins. e.g. sornatotropin,
`prolactin. insulin, pepsin
`_
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealburnin
`Albumin
`cu-Lipoprotein
`cu-Acid glycoprotein
`a1-Antitrypsin
`a1-Glycoprotein
`Transcortin
`4.6 S-Postalbumin
`Tryptophan-poor
`cu,-glycoprotein
`d1X-Glycoprotein
`Tliyroxin-binding globulin
`Inter—n-trypsin-inhibitor
`Go-globulin
`(Gc 1-1)
`(Gr: 2-1)
`(Gc 2-2)
`Haptoglobin
`(Hp 1-1)
`(HP 2-1)
`,
`(up 2-2)
`Ceruloplasmin
`Cholinesterase
`dz-Lipoprotein(s)
`ct;-Macroglobulin
`a;HS-glycoprotein
`Zn-ct:-glycoprotein
`cg-Neurarnino-glycoprotein
`Erythropoietin
`,8-lipoprotein
`60 Transferrin
`Hemopexin
`Fibrinogen
`Plasrninogen
`B1-glycoprotein I
`B1-glycoprotein ll
`Immunoglobulin G
`(IgG) or yG-globulin
`Mol. formula:
`
`45
`
`50
`
`55
`
`65
`
`
`
`9
`
`4,220,450
`
`10
`(adrenocortiootropic hormone)
`Thyfotfopin
`Follicle-stimulating hormone
`Luteinizing hormone
`(interstitial cell-stimulating hormone)
`Luteomammotropic hormone
`(luteotropin. prolactin)
`G ad t
`-
`onh o ropm ad
`I
`(C gnome son on-opm)
`Tissue Hormones
`«
`
`An -cumin I and H
`Ema‘ ki
`.
`3’
`’“’‘
`Human placental Iactogen
`Peptide Hormones from the Neurohypoph}-sis
`on
`.
`.
`_
`Vasopressm
`Releasmg factors (RF)
`CRF. LRF, TRF, Somatotropin-RF, GRF, FSH-
`RF, PIF,
`‘
`_
`Other polymeric materials of mtert are mucopo1y-
`saocharides and polysaccharides.
`Illustrative anti enic
`1 sacclierides d 'ved fro
`_
`_
`g
`130 y
`en
`mtoroorgamsms are as follows:
`
`11'!
`
`5
`
`10
`
`as
`
`15
`
`
`
`.
`.
`.
`Hemosenaunn Found In
`Polysaccheride
`
`Polysaocharide
`Polysacchetide
`Crude extract
`
`30
`
`.
`.
`.
`Species of Microorganisms
`Srrrpnxuccus pwgemr
`fig-.:se.I-1'c m;Int::‘?I::W
`35 Neismnra gonanfiaece
`Coryuebacrerium dfpmhenhe
`.-lcrtnobacilfus mailer‘;
`Acrfuabacmu: whirmod
`Fluncisefla Irrfarenrir
`filsfwnffc p¢:m'.r
`40 Pcrtmrefln putts
`Parrcurofln mnflccda
`e-««m~«« '
`
`It-epanqfmn I"£fi:rl'l‘
`45 Veillonelta _
`ELgs:e‘lot|1nx
`.2
`:2 nwnacrrtogena
`_
`Chromobuctenum
`Mycobecrerfum tuberculosis
`
`5“
`
`Lifilopolysaccharide
`Pu “Echmda
`Polysaccharide
`Ca
`hr
`ti
`«-=f=-‘=
`mom“
`Polysu.colI.en'de
`Iipopolysaccharide
`Polysncchurde
`Polyaacchande
`Llpopolyuochndde
`5l.l]J.'I¢ extract of 90%
`1’h“"°l °"'“‘°‘°d
`mycubacmi-I and polar-
`saccharide fraction of
`Po] saoclreride
`«mils and tuberculin
`Polkharide
`Lipopolysaoehnride.
`Pol
`lurid
`h1 :
`
`as me:
`Kldflm
`Kfebsfellc ckifae
`55 sarmmrra Iyplnosra
`sarmmira aypJ.r—mm'um;
`::.:f_um
`Sfiigeffa djoe.-Ir¢n'ce
`Skigeflaflexnerf
`60 Sltlgefle :HN'I'fl£l‘
`:;:krf=i1o":I:$bicam
`
`smmaeba rm-remiss
`Crude extract
`
`Polysnooiam-id:
`
`Crude. polyseocharide
`
`65
`
`_
`_
`_
`_
`The microorganisms which ‘are assayed may be In-
`tact, lysed, ground or otherw1_se fragmented, and the
`resulting COm[JDSlt!OI1 or portion, e.g. by extraction,
`assayed. Microorganisms of interest include:
`
`hit: 01' 7212
`Immunoglobulin A (IgA)
`or 7A-globulin
`M01 f0l'm“13=
`(~'-12"2)" 01' (‘{27\2)"
`Immunogmbulm M ‘
`(IBM) 0* 7M-s1°bu1w
`Moi. formula:
`am‘1)! of (NM),
`lmmunoglobulin D(IgD)
`or 1-‘D-Globulin (7121)
`M01. formula:
`(am) or (am)
`Irnmunoglobulin E (IgE'.)
`or 'yE-Globulin(°yE)
`M 1. r
`ul
`:
`azxczangt (1232)
`gee If and ')'flight chains
`mp ement actors:
`Crl
`C41q
`C.”
`C.“
`Clz
`C3
`B1A
`MD
`C4
`U5
`U6
`C"!
`cps
`Cfg
`Important blood clotting factors include:
`'
`'
`
`
`
`31,0013 CLO‘1"I'ING Fgcrons
`lngflmfional dflignafion
`Nam:
`I
`Fihinulcfl
`I?
`Pwlhromifi-I
`°
`'""°""’i"
`
`VII
`vs?
`
`*
`P‘u.'-flnwflin .
` Pf]::;:l°b"“" (Aug)
`plum. flnomboplnfin
`confluent (ETC)
`5tum_PrDwl, 1-mm’,
`mmpmnmmb-In "I
`Plasma tlaromboplutin
`antecedent (PTA)
`XII
`Hagermum faooor
`
`sun
`Hana-moron; rector
`
`X
`
`XI
`
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`- (Parathmmmw)
`Thyrocalcitonin
`Insujin
`Giucagon
`_
`_
`R3333“
`Erythropoietun
`Melanotropin
`(me[3n0cyte_sdmu]ating hormone; intermedin)
`somatotmpin
`(growth homwne)
`Corticotropin
`
`
`
`11
`
`Corynebacteria
`
`Corynebacterium dr'p:hen'oe
`Pneumococci
`
`Dipfocaocus pneumonia:
`
`Streptooocci
`
`Streptococcus pyogenes
`Streptococcus safhunu
`
`Staphylococci
`
`Staphylococcus aurens
`Staphylococcus albus
`
`Neisscriac
`
`Neisseria meningitidis ‘
`Neisseria gontlrrheae
`Enterobactcriaciae
`
`Eicbarickin ooh’
`.-lumbar.-tar aenngeuu
`Kftbflella punmania:
`
`} Th: oolifonu bacteria
`
`Safmaneffa lyplmu
`Salmonella chafunesufl
`Salmonella Iypkimunim
`
`The Salmunetlnl:
`
`Skigufia dyseumrce
`smgetra scfl.-m2|'z.|‘i
`Sfiigeffo ombiaomda
`Slsajmfa fleuleri
`Sfigulfo boydo‘
`skigelia Sonnet"
`
`
`The Shigellae
`
`Other tnteric bacilli
`
`Pmteux mfgurts
`Proteus mfmbilis
`J Proteus spccini
`
`Pmmu‘ morgoui
`
`Pseudomonas aerugfnasa
`Alcahjgenes fiiecalis
`Vibrio chalerae
`
`Hemophilus-Bordcteila group
`Hemaphilus fnfluenzae,
`H. duoreyi
`H. hemophilus‘
`H’. aegypricus
`H. purafufluenaae
`Enrderelfa pemmis
`
`Pasteur:-llae
`
`Pasrenrefla pesris
`Pasleureffa tulareuswls
`
`Brncella mefftensis
`Brucella abarms
`Bmcello suis
`
`Brucellac
`
`Aerobic Spore-forming Bacilli
`Bacillus anrhmcrlr
`Bacillus mbtilis
`Bacillus megarerlum
`Bacillus cereus
`
`4,220,450
`
`12
`
`Anaerobic Spore-farming Bacilli
`C!o.s1rz'd:'um batulin um
`Closrridium teramf
`Closrridium perfi-ingens
`Cfasrridfum rmvyi
`CIo.'.':r:'dium spericum
`Cfaslridfum hiftabvrkum
`Cfoarrfdium Iertium
`Clafirrfdium bifermenmm
`Clastrfdium Jporagenes
`
`Mycabacteria
`Myoobacrerfum rubercufosk: hamfnis
`Mycobacrerfum bowie
`Mjmabacrerfurn avium
`Mycabacrerfum Ieprae
`Mycobacrerfum pamrubercufasis
`
`Actinomycetes (fungus-like bacteria]
`Actinamyoas isr'aeh':'
`Actinomyces bovis
`Actfnomyces naeslundii
`Nocardia astemides
`Nocardfa brasilienstlr
`
`The Spirochetes
`Treponema paflidum
`Trepanema perrenue
`Trepanema carweum
`Borrelfa recurremis
`Leptaxpfra icrerohemorrhagfoe
`Leprospim canicufa
`Spirillum minus
`Srreprobacillns monfhfiarmis
`
`Mycoplasmas
`
`Mycoplasma pneumonfae
`
`Other pathogens
`Lis-term monoeytagenes
`Erysfpe!0_n‘m'x' rkusioparkfae
`Streptobac-"mus 'r'r:om‘!:_'formi.t
`Donvnnla granulomafls
`Bartonefla bacilhformis
`
`Rickettsiae (bacteria-like parasites)
`Ricketrsia pr-owazekii
`Rickentsia macaw"
`Rfckettsia rickerrsii
`Rfcketrsia comm’
`Rfckemfo austrab‘