United States Patent [191
`
`[11]
`
`4,203,479
`
`
`
`Zak et al. Jun. 17, 1980 [451
`
`
`
`[54] LABEL MODIFIED IMMUNOASSAYS
`[75]
`Inventors: Robert F. Zuk, Mountain View;
`Ellwatd To M338“; Redwflfid City»
`boll? 0fC9—1if-
`
`Activity by Human IgG,” Clin. Chem., vol. 23, No. 8,
`(1977) 913- 1335“338'
`Primary Exam£ner—Tl1omas G. Wiseman
`Attorney, Agent, or Firm—Bertram I. Rowland
`
`Syva Company, Palo Alto, Calif.
`[73] Assignee:
`[7-ll APPL N0-= 315,532
`[22] Filed.
`Jul_ 14' 1977
`
`[51]
`
`[56]
`
`4,036,946
`
`____‘_ 424/3
`
`AB5TRAcr
`{57]
`Methods and reagents are provided for irntnunoassays
`employing as reagents a labelled receptor, where the
`label is capable of providing a detectable signal, and
`modifying reagents’ Capable of modifying the signal
`Int. Cl} ..................... G01N 31/14; GOIN 21/22;
`obtained from the label. When ligand (ligand analog for
`G01” 33/15
`[52] U‘S° Cl‘ """"""""""""""""""“ /283:" monoepitopic ligands) is present in the assay medium, a
`[581 Field of Search
`195/103 5 A, ‘I03 5 R
`complex is formed which inhibits interaction between
`_
`.
`the label modifying reagent and the label. By measuring
`195/99’ 127’ 424/I2’ 3’ 23/230 B
`the signal obtained in the presence of a known amount
`Refeffillces Cited
`of ligand in the assay medium and comparing that signal
`U_3_ pATENT DOCUMENTS
`with the signal obtained with an unknown sample sus-
`pected of containing ligand, one can qualitatively or
`quantitatively determine the amount of ligand in the
`unknown. For detenninating antifligand) the assay is
`carried out
`in substantially the same way, except a
`source of ligand or ligand analog must be provided.
`
`
`
`311%: 3118]"
`-_,/197-__. Klememm
`
`
`
`OTHER PUBLICATIONS
`Wei. et a.l., “Preparation of a Phospholipase C-Antihl.l-
`man IgG Conjugate, and Inhibition of its Enzymatic
`
`3? Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2057
`
`Genentech Exhibit 2057
`
`IPR2016-00710
`
`

`
`1
`
`4,208,479
`
`LABEL MODIFIED IMMUNOASSAYS
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`There is an expanding interest in the ability to deter-
`mine or monitor small amounts of organic antigenic or
`haptenic materials. Frequently, the concentrations of
`interest are micromolar or less. Various techniques have
`been developed which are capable of isolating and de- 10
`tecting a specific compound, despite the presence of
`myriad other compounds of similar and different struc-
`ture.
`
`One group of techniques referred to as competitive
`protein binding assays or imrnunoassays depend for
`their specificity on the use of a receptor, normally an
`antibody, which is specific for a compound ofa particu-
`lar spatial and polar organization. For these assays it is
`normally necessary to produce antibodies by injecting
`antigens or hapten conjugated antigens into a vertebrate
`to induce the formation of antibodies which may then
`be harvested. The bleeds which are obtained can be
`usually purified to separate a globulin concentrate from
`other proteinaceous materials. To further purify the
`globulin concentrate to separate the antibodies of inter-
`est from other globulins is only dillicultly achieved.
`While affinity chromatography will provide for some
`concentration of the desired antibodies, the procedure is
`nonnally slow and frequently results in substantial loss
`of the desired antibodies as well as reduction in the
`binding constant. That is, those antibodies in the com-
`position which have the strongest binding, frequently
`cannot be removed from the column. Therefore, most
`methods have avoided labeling antibodies, since either
`the antibodies had to be purified to concentrate the
`antibodies of interest or a large amount of label was
`introduced associated with proteins which were not
`involved in the assay. This normally results in a large
`background signal which inevitably rednces the sensi-
`tivity of the assay, unless a physical separation of the
`extraneous label is incorporated into the procedure.
`The alternative has been to label ligand. While label-
`ing of ligand is feasible where the ligand is a simple
`hapten or the antigens are available in substantially pure
`form, in those situations where the antigen is only diffi-
`cultly purifiable, exists in only small amounts, or is
`labile, labeling of antigens is not feasible for a commer-
`cial process.
`An assay is therefore desirable which avoids the
`problems of purification and isolation of both the anti-
`bodies of interest and the ligand of interest. In addition,
`the assay should provide for minimizing introduction of
`label into the assay medium which produces a signal
`which interferes with or is additive with the signal
`which is measured.
`2. Brief Description of the Prior Art
`Radioimmunoassay is described in two articles by
`Murphy, J. Clin. Endocr. 2'.-', 973 (I967); ibid 28, 343
`(I968). U.S. Pat. No. 3,317,837 teaches a homogeneous
`enzyme
`immunoassay. US. Pat. Nos.
`3,654,090,
`3,791,932, 3,350,752 and 3,839,153 teach hetereogene-
`ous enzyme immunoassays. In the agenda for the Ninth
`Annual Symposium on Advanced Analytical Concepts
`for the Clinical Laboratory, to be held March 1'.-' and 18,
`19?? at the Oakridge National Laboratory, a paper
`entitled “Phospholipase C-Labeled Antihuman lgG:
`Inhibition of Enzyme Activity by human IgG,” to be
`presented by R. Wei and S. Riebe is reported. U.S. Pat.
`
`IS
`
`25
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`55
`
`65
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`2
`Nos. 3,935,074 and 3,998,943 disclose immunoassay
`techniques involving steric inhibition between two dif-
`ferent receptors for dillerent epitopic sites. U.S- Pat.
`No. 3,996,345 teaches the use of a common receptor, a
`portion of which is bound to a lluorescer and the re-
`maining portion bound to quencher, whereby the pres-
`ence of ligand brings the receptors together so as to
`allow for quenching of fluorescence. Carrico, et al,
`Anal. Biochem. 72 271 (1976) and Schrocler, et al, ibid
`72 283 (l9';‘6) teach competitive protein binding assays
`where a label is bonded to a hapten with the label being
`subject to enzymatic transformation to produce a signal.
`Antibody bound to the hapten inhibits the approach of
`enzyme to the label.
`
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for use in
`immunoassays for the accurate detennination of a mem-
`ber of an immunological pair i.e.
`ligands and ligand
`receptors, at concentrations down to l pg/ml or less.
`With polyepitopic ligands labeled receptors are em-
`ployed, where the label is capable of modification by
`modifying reagents. Upon combining polyepitopic li-
`gand, (polyfligand analog) with haptens) labeled recep-
`tor and the modifying reagent(s), a complex is formed
`between the ligand and the labeled receptor which
`inhibits the modification of the label by the modifying
`reagent(s). The label outside the complex is modified, so
`that the observed signal is from the unmodified label in
`the complex and any residual signal from the modified
`label. By comparing the results to known standards, the
`concentration of ligand can be determined.
`When a monoepitopic ligand is involved, a plurality
`of ligand analogs will be conjugated to a hub nucleus to
`provide a poly(ligand analog). The polyfligand analog)
`will be included with the other reagents, so that a com-
`petition exists between the monoepitopic ligand and the
`poly(ligand analog) for the labeled receptor. The la-
`beled receptor which binds to the polyfligand analog)
`will form a complex which inhibits the modification of
`the label, while free labeled receptor and labeled recep-
`tor bound to the ligand will be modified. By employing
`known standards, the amount of rnonoepitopic ligand
`may be determined.
`For receptors, the assay is carried out in substantially
`the same way, except that a source of ligand or ligand
`analog must be provided.
`The compositions can be provided as kits, whereby
`measured amounts of the labeled antibody, and, where
`required, ligand or polyfligand analog), and modifying
`reagent(s) are provided, particularly as dry powders or
`concentrated solutions which can be reconstituted as
`reagent solutions for use in the immunoassays.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Method and compositions are provided for sensitive
`irnmunoassays, where reagents can be prepared without
`cumbersome and difficult purilications and/or isola-
`tions of antigens and antibodies. The method will nor-
`mally involve bringing together an analyte and a la-
`beled receptor (with monoepitopic analytes, a po1y(li-
`gand analog) will be employed in addition), so as to
`form a complex which sterically inhibits the approach
`of macromolecules to the label. The label is capable of
`providing a distinctive signal by being exposed to an
`agent e.g. electromagnetic radiation, usually light, or
`
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`

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`4,208,479
`
`3
`chemical reagents. After an appropriate time, a modify-
`ing agent is added which is capable of interacting with
`the label and modifying. preferably reducing, its distinc-
`tive signal. The agent interacts with the label and the
`signal. from the assay medium is measured. By compari-
`son of the determined signal to signals from assay media
`having known amounts of analyte, the concentration of
`analyte in an unknown sample may be determined.
`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 a plurality of ligand
`analogs in a single molecule.
`Polyfligand analog)—a plurality of ligand analogs
`joined together, 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--—a compound or composition capable of pro-
`viding a detectable signal in conjunction with physical
`activation (or excitation) or chemical reagents and ca-
`pable of being modified, so that the particular signal is
`diminished or increased.
`Receptor-—Any compound or composition capable
`of recognizing a particular spatial and polar organiza-
`tion of a molecule i.e. an epitopic site. Illustrative recep-
`tors include naturally occurring receptors, antibodi,
`enzymes or fragments thereofthat contain a binding site
`e.g. Fab, and the like. For any specific ligand. the recep-
`tor will be referred to as antifligand). The receptor
`anti(ligand) and its reciprocal ligand form an immuno-
`logical pair.
`Labeled Receptor—-receptor having at least one label
`covalently bonded to it and retaining at least one bind-
`ing site.
`Modifier-«—~a macromolecule capable of physically or
`chemically interacting with the label to reduce the sig-
`nal produced by the label.
`Complex—a combination of at least one labeled re-
`ceptor and one polyepitopic ligand (includes poly(li-
`gand analog)), normally in the assay medium there
`being on the average at least two of one of the compo-
`nents in each of the complexes and frequently a total of
`four or more of the components bound together.
`ASSAY
`
`The subject assay is carried out in an aqueous, nor-
`mally homogeneous, zone at a moderate pH, generally
`close to optimum label detection. The assay zone for the
`determination of analyte is prepared by employing an
`appropriately buffered aqueous solution, the unknown
`sample, which may have been subject to prior treat-
`ment,
`labeled receptor, modifier, and as appropriate
`polyfligand analog) and ancillary reagents for reacting
`with the label to produce the detectable signal. For
`determination of anti(Iigand) in the sample, ligand or
`po_ly(ligand analog) will normally be added. The assay
`zone will normally be homogeneous.
`In carrying out the assay an aqueous medium will
`normally be employed. Other polar solvents may also
`be employed, usually oxygenated organic solvents of
`
`4»
`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 35
`weight percent, more usually in less than about 10
`weight percent.
`The pH for the medium will usually be in the range
`from about 5 to 10, more usually in the range from
`about 6 to 9. Various buffers may be used to achieve the
`desired pH and maintain the pH during the determina-
`tion. Xllustrative buffers include borate, phosphate, car-
`bonate. Tris, barbital and the like. The particular buffer
`employed is not critical to this invention, but in individ-
`ual assays, one buffer may be preferred over another.
`Moderate temperatures are normally employed for
`carrying out the assay and usually constant tempera-
`tures during the period of the assay will be employed.
`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 l0—4 to [045 M, more
`usually from about 10*“ to 10-13 M. Considerations
`such as whether the assay is qualitative, sen1i-quantita-
`tive or quantitative, the particular detection technique
`and the concentration of the analyte of interest will
`normally determine the concentration of the other rea-
`gents.
`Receptor (antiligand) may be a mixture of labeled and
`unlabeled receptor, generally having from about 5 to
`100% of the receptor as labeled receptor. The propor-
`tion of unlabeled receptor will depend on the nature of
`the label, the manner of preparation, the sensitivity of
`the label detection system and the like. For example,
`with a fluorescer label, there may be substantial self-
`quenching when all of the receptor is labeled, so that it
`is desirable to introduce a significant amount of unla-
`beled receptor in the assay medium.
`Normally, for polyepitopic ligand analytes the oon- _
`centration of total antiligand based on binding sites will
`be about 1-50 times either the minimum or maximum
`concentration of interest based on epitopic sites, usually
`about 1-10 times and more usually 1-3 times the maxi-
`mum concentration of interest. For monoepitopic li-
`gand analytes and receptor analytes, based on binding
`sites, the respective concentrations of polyfligand ana-
`log) and labeled antiligand will have concentrations
`about equal to the minimum concentration of interest,
`normally not exceeding the maximum concentration of
`interest. generally not less than 10-‘, more usually not
`less than lD—3of the minimum concentration of interest.
`Concentration ranges of interest will generally vary
`from about 10-3 to 104‘ g/ml. For monoepitopic ana-
`lytes and receptor analytes the concentration of total
`antiligand other than analyte will usually be up to fifty
`times the concentration of polyligand analog or ligand,
`more usually up to 10 times, most usually up to three
`times.
`The concentration of modifier will vary widely de-
`pending on the nature of the modifier, its effectiveness
`in modifying the signal, its side reactions and the like.
`Normally, large excesses of modifier can be used to
`insure that the rate of modification is rapid and the
`concentration of modifier is not limiting. Therefore.
`when the modifier is not a catalyst at least stoichiomet-
`ric concentrations of modifier will be used and molar
`excesses of 100 or more may be employed. Where the
`modifier is an enzyme, concentrations of at least 10‘?
`times Km will usually be employed. For other catalysts,
`the catalytic concentration will generally range from
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`4,208,479
`
`5
`about t0-3 to 10-5 times the minimum or maximum
`concentration of interest.
`The order of addition may vary widely. Normally,
`the unknown sample and labeled receptor will be com-
`bined in an appropriate medium before the introduction
`of the modifier. When the modifier reversibly modifies
`the label, the modifier and labeled receptor may be
`premixed. Depending on the nature of the ancillary
`reagents, if any, they may be added initially or with or
`subsequent to the addition of the modifier. After com-
`bining the unknown with the labeled receptor and, as
`appropriate,
`ligand or polyfligand analog),
`the assay
`medium may be incubated for a sufficient time to form
`complexes.
`The times between the various additions for the assay
`components and for the immunological reactions which
`are involved may vary widely, depending upon the
`particular compounds involved, the mode of addition,
`the concentrations involved, the binding constants of
`the receptors, and the like. Normally, times between
`additions may vary from a few seconds to many hours,
`usually not exceeding twelve hours, and more usually
`not exceeding six hours. After adding each component
`to the assay mixture, different incubation periods before
`adding the next component or taking the measurement
`will be involved. Since the ultimate results will be de-
`pendent upon the results obtained with standard(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 be as small as about 1 14.1,
`more usually being at least 25 ,u.l, and will usually not
`exceed 5 ml, more usually not exceeding about 2 ml.
`In particular situations, the subject method allows for
`the simultaneous determination of two or more ana-
`lytes, usually not more than about five analytes. By
`employing labels which give substantially non-interfer-
`ing signals, each different label can be conjugated to a
`receptor for a different analyte. This embodiment is
`readily illustrated with fluorescers. One employs fluo-
`rescers which fluoresce by emitting light at different
`wavelengths. Therefore, a particular wavelength of
`emitted light would be associated with a particular
`analyte. The assay would be performed in the normal
`way for each analyte, except that all the reagents and
`sample would be included in one assay medium. The
`assay medium would be irradiated with light of wave
`lengths which correspond to the absorption bands of
`the different fluorescers and the amount of fluorescence
`from each of the fluorescers determined. By appropriate
`calculations the contribution to the emission spectrum
`of each of the lluorescers could be determined.
`The same technique could be applied with other la-
`bels, but for the most part not as conveniently. With
`enzyme labels, the different enzymes would have to
`have substrates and products which were not interfer-
`ing and could be independently detected. The problem
`of interference between labels and their associated sys-
`tems will vary to lesser or greater degrees depending
`upon the particular label and its modifiers.
`In determining antifligand),
`the procedure is the
`same, with the exception indicated previously, but the
`observed result may be an increase or decrease in the
`signal depending on the relative proportions of the
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`6
`various components. That is. the antifligand) may dis-
`place labeled antifligand) from the complex or enhance
`complex fomiation. Preferably, a protocol is employed
`where antifligand) will displace labeled antifligand).
`In a preferred embodiment, the modifier is anti(label),
`that is, a receptor which specifically binds to the label,
`affecting the label in a variety of ways.
`One effect is to inhibit the interaction of a chemical
`with the label. For example, with an enzyme label,
`antibodies to enzyme can be prepared which sterically
`or allosterically inhibit the enzyme. Those enzymes
`which become bound by anti(enzyme) will be deacti-
`vated. Another technique is to employ a label which
`chemically reacts with the modifier to change the
`chemical nature of the modifier. For example, with a
`redox reaction, binding of antiflabel) to one of the reac-
`tants in the redox reaction will inhibit the approach of
`the other rmctant to the label. If the label is an enzyme
`substrate, binding by antilabel will inhibit the enzyme
`catalyzed reaction. Finally as a further illustration, an-
`ti('label) may change the environment of the label, so as
`to change the physical characteristics of the label. With
`a fluorescent label, antiflluorescer) will change the light
`absorption and/or emission characteristics of the fluo-
`rescer when bound to the fluorescer. Thus, by irradiat-
`ing the solution with light within the absorption band of
`unbound lluorescer, free fluorescer will lluoresce, at a
`different wave length or efficiency than the bound fluo-
`resoer, allowing free and bound fluorescer to be distin-
`guished.
`The use of an anti(label) as the modifier has many
`advantages. It is specific for the label and will generally
`not be subject to interference from materials normally
`encountered in samples to be assayed. Furthermore,
`with the enzyme, the redox and the fluorescer labels,
`amplification can be achieved in that a single unbound
`label can be used to cause a plurality of rneasureable
`events. Another preferred embodiment is a fluorescent
`enzyme substrate which is quenched upon interaction
`with an enzyme.
`The measurement of the detectable signal from the
`label will vary widely depending upon the nature of the
`label. The measurement will normally involve measur-
`ing electromagnetic radiation at a particular wave-
`length or narrow band of wavelengths in various
`ranges, such as radiofrequency, ultraviolet, visible, etc.,
`although other measurements may be made e.g. electri-
`cal or microcalorimetric. For electromagnetic measure-
`ments, the absorption or emission of radiation will be
`involved.
`Depending upon the nature of the label, various tech-
`niques may be employed. For the most part, the tech-
`niques employed will involve the absorption or emis-
`sion of electromagnetic radiation. Such techniques may
`involve fluorescence, chemiluminescence, ultraviolet or
`visible light absorption, electron spin resonance, and the
`like.
`
`MATERIALS
`
`The primary components in the subject assay for
`analyte are: the analyte; the labelled receptor; the modi-
`fier; any ancillary reagents for the label; and, as appro-
`priate polyfligand analog). In addition, in the assay for
`anti(1igand),
`ligand or polyfligand analog) will be
`added. The different labels allow for great variety in
`protocols and methods of measurement, although cer-
`tain techniques will be vastly superior to other tech-
`niques. Of particular significance are those techniques
`
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`4,208,479
`
`7
`which allow for amplification, that is, where a single
`event results in the occurrence of a plurality of events.
`Within this category are redox reactions and transfer
`reactions, which are enzymatically mediated and result
`in a variety of products which may be detected in a
`number of different ways.
`ANALYTE
`
`The ligand analytes of this invention are character-
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`po1y(arnino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations or assemblages include bacteria,
`viruses, chromosomes, genes, mitochondria, nuclei, cell
`membranes, cell walls, 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 l,000, usually 5,000, more usu-
`ally at least about 10,000. In the poly(amino acid) cate-
`gory, the poly(amino acids) of interest will generally be
`from about 5,000 to 5,000,000 molecular weight, more
`usually 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:
`
`(HP 2-2)
`Ceruloplasmin
`Cholinesterase
`:1;-Lipoprotein(s)
`ct;-Macroglobulin
`0.;-HS-glycoprotein
`Zn-ag-glycoprotein
`ag-Neuramino-glycoprotein
`Erythropoietin
`13-lipoprotein
`Transferrin
`I-lemopexin
`Fibrinogen
`Plasrninogen
`B3-glycoprotein I
`B2-glycoproteiu II
`Imrnunoglobulin G
`(IgG) or 'yG-globulin
`Mol. formula:
`'l"2K2 01' ’}'2?'2
`Imrnunoglobulin A (IgA)
`or 7A-globulin
`M01. formula:
`(a2K2)" or (a2'r2)"
`[mmunoglobulin M
`(IgM) or 7M-globulin
`Mol. formula:
`(,u2K2)5 01‘ (J12')'2)5
`Immunoglobulin D (IgD)
`or 'yD-Globulin (-yD)
`Mol. formula:
`
`5
`
`10
`
`I5
`
`20
`
`25
`
`30
`
`32K!) Of (3z'r2)
`lmmunoglobulin E [[gE)
`or -yE~G1obulin ('yE)
`M01. formula:
`(E-2K2) or (sale)
`Free K and ~,- light chains
`Complement factors:
`C1
`C’ lq
`C11-
`C15
`0'2
`c'3
`_3,A
`Q20
`(,"4
`05
`C5
`C"?
`cs
`U9
`Important blood clotting factors include:
`TABLE V"
`BLOOD CLOTFING FACTORS
`International designation
`Name
`I
`Fibrinogen
`1'
`P'°‘*“‘°{“'°i"
`gtigztstldlijiiliombophstin
`Proeecelcrin, accelerator
`globulin
`_
`Procouwenin‘
`$:::;:iI:[)fl;g:rg1obul1n (AHG)
`plasma tiiromhogilastin
`component (PTC)
`:::::,:t‘.*;::*:,'..:.:°;:’."
`Plasma thrornboplastin
`
`V and Vi
`
`VII
`mil
`
`"
`X!
`
`
`
`35
`
`40
`
`protamines
`histones
`"“’“““_“5
`globulms
`scleroproteins
`phosphoproteins
`mucoprotems
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropin, prolactin, 45
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`an-Lipoprotein
`an-Acid glycoprotein
`‘1l‘Anl“1'YP5i1"_
`%1§i§§§i‘2.Z°’°'“
`4_5S_P0st3]bu_rnjn
`T1-ypt0phan_poor
`at-glycoprotein
`a1X—Glycoprotein
`Thyroxiu-binfiiliig $l(,)bufi'n
`IT“-er'Cl'lrYP5131'l|1l11l31l01'
`Ge-globulin
`(GO 1.1)
`(Ge 2_1)
`(G3 2'2)_
`I-laptoglobm
`<Hv 1-“
`(HP 2-1)
`
`50
`
`55
`
`60
`
`65
`
`

`
`4-,208,4-79
`
`10
`-continued
`I-{cmosensitin Found in
`
`
`
`Species of Microorganisms
`
`.
`
`‘
`9
`TABLE VII-continued
`BLOOD CLOTFING FACTORS
`
`International designation
`
`Name
`antecedent (PTA)
`X1]
`Hagemann fame:
`
`XIII
`Fibrin-stabilizing factor
`
`'
`
`_
`_
`Important protein hormones Include:
`_
`‘
`Peptldfi and Protein Hormones
`.
`Pal-athyrmd hormone
`(P31'3lh°_Tm9"°)
`Thyrocalcltonm
`lnsufin
`wagon
`‘
`‘
`Relax!”
`Erythropoletm
`Melanotropin
`melt“-we tbsfimnlatin
`Elormoneyintennedin) 3
`'.
`Somatotropin
`(growth hormone)
`C01-ficotrgpj;-1
`(adrenocorticotropic hormone)
`Thymtl-.;,Pi_n
`Fo]1j;;]e.5fimu]afing hormone
`Lutemlzmg hormone
`(interstitial cell-stimulating
`hormone)
`Luteomammotropic hormone
`(luteotropin, prolactin)
`Genadon-opin
`(chorionic gonadotropin)
`
`Tlssue H°““°”E5
`
`Secrgtin
`Gastrin
`.
`.
`Angiotensm I and II
`Bradykinin
`Human placental Iactogen -
`
`pal-_;_;o"eHa
`5 Erysr'peloIlrrr'x
`Listeria monocyrag.-mes
`Clrmrnobocrerium
`Myrobccrerluna rubercrrlosls
`
`to
`
`Klebflella aerogenes
`Klebsiella claacrre
`Salmonella orplmsa
`
`Ljpopolysaochan-de
`Polysaccharide
`Polysaccharide
`ljpupalysaccharide
`Saline extract of 90%
`phenol extracted
`rnycobacteria and poly-
`saceharide fraction of
`cells and mg-,e;-eurin
`Polysaccharide
`Polysaccharide
`Lipopoiysaccharide,
`Polysaccharide
`Polysacclzaride
`
`I5 Salmonella ryplH'~m:.rrl:.rm.'
`e::;::::::::::w.
`Slrlgellc dyscnterrhe
`Slilgella flexueri
`Crude, polysaccharide
`Slniseiia samrel
`20 Rrbkertrlae
`Crude extract
`Cnmfirla albicanr
`Polysaccharide
`
`Ea-rtamwba lrisrafyrfca
`Crude extract
`
`Polysaccharide
`
`_
`_
`_
`_
`The mlcroorgaisms winch are assayed may be intact,
`25 !.Y-39¢ 81'0W_|§ 01' 0Th31'W'155 f1‘3B"1?-“led. and the result-
`mg composition or portion, e.g. by extraction, assayed.
`Mlcroorgamsms of interest mclude:
`Cnrymbamefia
`Corynebacterium diptheriae
`Pneumococci
`
`30
`
`Diplococcus pneumoniae
`
`35
`
`40
`
`Streptococci
`Streptococcus pyogenes
`Streptococcus salivarus
`'
`Sta h l
`P 3’ omccl
`Staphylococcus aureus
`Staphylococcus alblls
`
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF, Somatotropin-RF,
`GRF, FSH-RF, PIF, MIF
`Otlherdpolymericlmatergalegf interest are muoopo1y- so
`53°C an 3? and
`y5_acc an 55-
`_
`_
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`45
`
`Ncisseriae
`Neisseria meningitidis
`Neisseria gonorrheae
`
`E
`b
`_
`1
`E:fiE:':::_:'::£ge"es
`Klebrlella p-rreumonlae
`Salmonella .-ypizm
`Salmonella clioleraemis
`Salmonella Jyplrimurlum
`Sldgella dysenreriac
`
`The commm bacteria
`The Sairnonellae
`
`}
`}
`
`re’
`
`6
`
`55
`
`60
`
`Slf
`ll
`bi
`Smgefla schmllzfi
`srlfigrrg}3Zi..L’Ii’“
`5;,‘-gdgg boydfi
`3;”-gay“ some,-
`other emeric bacim
`""""o_—
`Pmm“ wlganfs.
`Plmeus ”“"""""‘_‘
`‘
`humus m°'3‘””
`Psemlornanar aenrgrnase
`Alcallgenesfaecalis
`V‘b":"’ ""°l9m"
`'°“
`55 Hem“ h'l“S’B°‘d'“““”‘
`Hemapliilus frrfluenzae.
`
`H
`The Sh’
`age ac
`
`,
`P““°“5 spam”
`
`H. drrcreyr‘
`H. lremaplrllns
`H, gggypfl'gy_{
`H, parqfgflgenzgg
`
`
`
`Species of Microorganisms
`Swrrawww Pmms
`Diplomrcus prreumaniae
`Neisrerfa nrenlrrgirrrlir
`Nemerfa gonorrlroeae
`Corynebacrerfum drphtlierfae
`Ac.r:'nobece'l'lus mallet.’
`Acrlmbaclllus wfi-r'rcmon'
`Fmncisella mlrmzi-rs.-'5
`
`p‘,_,_.Em._._,Ha Pam.
`Pasreurella peso‘:
`Pasmrrella mulrocirla
`Bra-rella aborrus
`Haemaplrflars influerrzae
`Haemoplxllus rni;-rrussalr
`Tneponema reiren
`
`Hemoeensitin Found in
`Polvsaccharide
`Polysaccheride
`Polysaccharide
`Polysaccharicle
`Polysaccharicle
`Crude extract
`
`.
`
`Lipopolysaccharide
`Polysaccharjdfl
`
`Polysaccharide
`Capsular antigen
`Crude extract
`Polysaccharide
`Crude
`_
`Polysacchande
`
`
`
`

`
`4,208,479
`
`11
`continued
`
`
`
`Bardeteffa perius.-‘s
`
`Pasteurellae
`
`Pasteurella pestis
`Pasteurella tulareusis
`
`Brucellae
`
`Brueella melitensis
`Brucella abortus
`Bruoella suis
`
`Aerobic Spore-forming Bacilli
`Bacillus anthracis
`Bacillus subtilis
`Bacillus megaterium
`Bacillus cereus
`
`Anaerobic Spore-forming Bacilli
`Clostridium botulinum
`Clostridium tetani
`Clostridium perfringens
`Clostridium novyi
`Clostridium septicum
`Clostridium histolyticum
`Clostridium tertium
`Clostridium bifermentans
`Clostridium sporogenes
`
`Mycobacteria
`
`Mycobacterium tuberculosis horninis
`Mycubacterium bovis
`Mycobacterium avium
`Myoobacterium leprae
`Myoobacterium paraluberculosis
`
`Actinomycetes (fungus-like bacteria)
`Actinomyces israelii
`Actinomyces bovis
`Actinomyces naeslundii
`Nacardia asteroides
`Nocardia brasiliensis
`
`
`The Spimchetes
`Tnepamvma pafffdum
`Traepemema pertemm
`Tneponema carateum
`Bans-Iia remrrenris
`Leprospim icterabemorrftagiae
`Lepfaqpim canfcala
`
`Spirfllum minus
`Srrep:obac:‘.!'!us monffxfarmis
`
`Mycopiasmas
`Myooplasrna pueumoniae
`
`Other pathogens
`
`Listeria monocytogenes
`Erysipelothrix rhusiopathiae
`Streptobacillus moniliformis
`Donvania granulomatis
`Bartonella bacilliformis
`
`Rickettsiae (bacteria-like parasites)
`
`Rickettsia prowazekii
`Rickettsia moose:-i
`
`15
`
`25
`
`35
`
`45
`
`SD
`
`55
`
`65
`
`12
`
`Rickettsia rickettsii
`Rickettsia conori
`Rickettsia australis
`Rickettsia sibiricus
`Rickettsia alum’
`Rickeusia tsutsugamushi
`Rickettsia burnetii
`Ricketlsia quintana
`
`Chlamydia (unclassifiable parasites bacterial/viral)
`
`Chlamydia agents (naming uncertain)
`
`}
`
`Fungi
`Corpmcmwus neqfin-mans
`Blasromyces demattdis
`Histoplasma capwlatum
`Coccfdioldes Immflis
`Pamcoccfdfaides brarfliensls
`Candida afbicans
`Aspe1gm‘u.¢fi;mfgaru:
`Mum! mormbifer (Absidta mrymbafem)
`Ritfzopus sauce
`Rltfzapus annkizus
`Rldzapus mfgrimns
`Sputum:-:J‘mm scimikff
`Farueoaea pedrosaf
`Fmsmmm cangpacm
`Fbrgrecuea dermatixidis
`Cladngoarium onrriomi‘
`Piltafophom Hermann
`Aspergfllus niduians
`Madureflu myoeromf
`Madumfla grime
`Aflescfleria boydif
`Phialaspkom kamwfmei
`Microspamm gjweum
`Trichapfiyron mentagwphyre:
`Kemtfmmynes ajeflai
`Mkmpomm cam’:
`T‘-fichqnhyran mbmm
`
`Micmqporunz audoufui
`
`Phycomyeetes
`
`Viruses
`
`Adenovimses
`
`Herpes viruses
`
`Herpes simplex
`Varioella (Chicken pox)
`Herpes Zoster (Shingles)
`Virus B
`Cytomegalovirus
`
`Pox Viruses
`
`Variola (smallpox)
`Vaccinia
`Poxvirus bovis
`Paravaccinia
`Molluscum contagiosum
`Picornaviruses
`
`Paliovirus
`Coxsackievirus
`Echoviruses
`Rhinoviruses
`
`Myxoviruses
`
`Influenza (A, B, and C)
`Parainfleuenza (1-4)
`Mumps Virus
`Newcastle Disease Virus
`Measles Virus
`
`
`
`

`
`13
`
`Rinderpest Virus
`Canine Distemper Virus
`Respiratory Syncytial Virus
`Rubella Virus
`
`Arboviruses
`
`Eastern Equine Encephalitis Virus
`Western Equine Encephalitis Virus
`Sindbis Virus
`Chikungunya Virus
`Semliki Forest Virus
`Mayora Virus
`St. Louis Encephalitis Virus
`California Encephalitis Virus
`Colorado Tick Fever Virus
`Yellow Fever Virus
`Dengue Virus
`
`Reoviruses
`
`Reovirus Types 1-3
`
`Hepatitis
`
`Hepatitis A Virus
`Hepatitis B Virus
`
`Tumor Viruses
`Rauscher Leukemia Virus
`Gross Virus
`Maloney Leukemia Virus
`The monoepitopic ligand analytes will generally be
`from about 100 to 2,000 molecular weight, more usually
`from 125 to 1,000 molecular weight. The analytes of
`interest include drugs, metabolites, pesticides, pollut-
`ants, and the like. Included among drugs of interest are
`the alkaloids. Among the alkaloids are morphine alka-
`loids, which inclu