United States Patent
`
`[19]
`
`[11]
`
`4,281,061
`
`[45] Jul. 28, 1981
`Zuk et al.
`
`
`
`having a signal producing system, which provides a
`detectable signal, which system includes a macromolec-
`ular member. The determination of the analyte, which is
`a member of "a specific binding pair consisting of a li-
`gand and its homologous receptor,
`is performed by
`creating an extensive matrix in the assay medium by
`having in the assay medium in addition to the analyte,
`ligand labeled with one of the members of the signal
`producing system, antiligand either present as the ana-
`lyte or added, a polyvalent receptor for antiligand, the
`macromolecular member of the signal producing sys-
`tem, and any additional members of the signal produc-
`ing system. The labeled ligand, antiligand, and polyva-
`Ient receptor for the antiligand create a matrix which
`modulates, e.g. inhibits, the approach of the macromo-
`lecular member of the signal producing system to the
`labeled ligand. The extent and degree of formation of
`the matrix is dependent upon the concentration of the
`analyte in the medium. By comparing the signal from an
`assay medium having an unknown amount of analyte,
`with a signal obtained from an assay medium having a
`known amount of analyte, the a.mount_of analyte in the
`unknown sample may be determined qualitatively or
`quantitatively.
`
`Kits are provided having predetermined amounts of the
`various reagents to allow for enhanced sensitivity of the
`method.
`
`1'.-" Claims, No Drawings
`
`[54] DOUBLE ANTIBODY FOR ENHANCED
`SENSITIVITY IN IMMUNOASSAY
`
`[75]
`
`Inventors: Robert F. Zak, San Francisco; Ian
`Gibbons, Menlo Park; Gerald L.
`Rowley, Cupertino; Edwin F. Ullmsul,
`Atherton, all of Calif.
`
`_Syva Company, Palo Alto, Calif.
`[73] Assignee:
`[211 Appl.No.: 61,542
`
`[22] Filed:
`
`Jul. 27. I979
`
`[51]
`
`Int. GL3 ..................... .. CIZN 9/96; GDIN 33/54;
`GOIN 33/43; GOIN 31/14
`[52] US. Cl. ......................................... .. 435/7; 435/5;
`435/138; 424/3; 424/ 12; 23/230 B
`[53] Field of Search .................... 424/3, 12; 23/230 B;
`435/5, 7, 184, 183, 810
`
`{S6}
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`424/12 x
`435/'.-'
`424/8
`. 23/230 B
`435/?
`
`
`
`3.9353?-t
`3,966,893
`4,161,515
`4,139,466
`4,193,983
`
`1/1916 Rubenstein
`6/1976
`Sjdquist et a1.
`7/1979 Ullman
`2/1980 Ainis et al.
`3/I980 Ullman et al.
`
`Primary Examiner—-Thomas G. Wisetnan
`Attorney, Agent, or Firm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Method and compositions are provided for performing
`homogeous imrnunoassays. The method involves
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2047
`Genentech Exhibit 2047
`
`IPR2016-00710
`
`

`
`1
`
`.._4,2s1,_0._6_1
`
`DOUBLE ANTIBODY FOR ENHANCED ,
`SENSITIVITY IN IMMUNOASSAY
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`There is a continuing and increasing need for accu-
`rate, sensitive techniques for measuring trace amounts
`of organic materials in a wide variety of samples. This
`need includes the measurement of drugs, naturally oc-
`curring physiologically active compounds or nutrients
`in physiological fluids, the presence of trace amounts of
`contaminants or toxic materials in foods, water or other
`fluids, and the like, as well as monitoring materials for
`trace contamination introduced during chemical pro-
`cessing.
`Among the various techniques which have found
`increasing exploration are techniques involving recep-
`tors which recognize or bind to a specific polar and
`spatial organization of one or more molecules. For the
`most part, the receptors are antibodies and the tech-
`niques are referred to as immunoassays. These tech-
`niques conventionally employ a labeled ligand where
`the binding to the receptor allows for distinguishing
`between a bound labeled ligand and an unbound labeled
`ligand. Certain techniques, generally referred to as het-
`erogeneous, rely on segregating the bound from the
`’unbound labeled ligand. Other techniques, generally
`referred to as homogeneous, rely on the bound labeled
`ligand providing a signal level different from unbound
`labeled ligand.
`In many of the homogeneous techniques, the label
`must interact with another substance in order to differ-
`entiate the signal. For example, in one technique, the
`label is an enzyme and when receptor is bound to the
`ligand the enzymatic activity is inhibited. This requires
`that the enzyme ligand combination be such that when
`receptor is bound to the enzyme ligand conjugate, ei-
`ther substrate is inhibited from entering the active site
`or the enzyme is allosterically modified, so that its turn-
`over rate is substantially reduced. In another technique,
`a fluorescent label is employed in conjunction with a
`receptor for the fluorescer. The binding of the receptor
`to the lluorescer substantially diminishes the fluores-
`cence when the fluorescer is irradiated with light which
`normally excites the fluorescer. When the fluorescer is
`conjugated to ligand, and antiligand is bound to the
`conjugate, the antifluorescer is inhibited from binding
`to the fluorescer.
`While the techniques have found great use or show
`great promise, there is still an interest in "enhancing the
`sensitivity of techniques which do not require separa-
`tion. As lower and lower concentrations of aualytes are
`encountered, improvements in available techniques are
`required to allow for accurate determination of the
`presence of extremely small amounts of the analyte.
`Therefore, there has been an ongoing elfort to find new
`and improved ways
`to measure extremely small
`amounts of organic molecules in a wide variety of envi-
`ronments.
`
`2. Description of the Prior Art
`U.S. Pat. No. 3,817,837 describes a homogeneous
`enzyme immunoassay. U.S. Pat. No. 3,996,345 describes
`a homogeneous immunoassay employing two chromo-
`phores related by being a lluorescer and a quencher.
`Co-pending application Ser. No. 893,650, filed Apr. 5,
`1978 describes a technique employing a plurality of
`enzymes, where the substrate of one enzyme is the
`
`5
`
`10
`
`15
`
`20
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`25
`
`30
`
`35
`
`45
`
`50
`
`SS
`
`60
`
`65
`
`2
`product of the other enzyme. U.S. Pat. No. 3,935,074
`describes an immunoassay involving steric hindrance
`between two antibodies. Co-pending application Ser.
`No. 815,487, describes an enzyme immunoassay, em-
`ploying antienzyme as an inhibitor.
`SUMMARY OF THE INVENTION
`
`Method and compositions are provided for enhancing
`the sensitivity of imrnunoassays requiring the proximity
`of two reagents for modulating a signal related to the
`amount of analyte in the assay medium. The subject
`method employs a second receptor, which is polyva-
`lent, and binds to a receptor for ligand to affect the
`degree of interaction between the two reagents, particu-
`larly inhibiting interaction. The role of the second re-
`ceptor is manifold depending upon the nature of the two
`reagents involved in signal production. The second
`receptor is employed in forming or extending matrices
`of ligand and ligand receptor which results in modula-
`tion of entry or exit from the matrix. Depending upon
`the role of the second receptor, different protocols will
`be employed.
`- A kit is provided having predetermined amounts of
`the various reagents, as well as ancillary reagents for
`optimizing the sensitivity of the immunoassay.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Method and composition are provided for determin-
`ing small amounts of organic compounds in a wide
`variety of media by employing an organic receptor
`which recognize-s'a specific spatial and polar organiza-
`tion of a molecule, either the organic compound or its
`receptor being the analyte of interest. In the subject
`method, a signal producing system is employed having
`at least two members which provides a detectible signal
`which may be modulated in accordance with the con-
`centration of the analyte in the assay medium. The mod-
`ulation of the signal is as a result of two members of the
`signal producing ‘system being brought into proximity,
`which results in either an enhancement or reduction in
`the signal level. Two receptors are involved in the as-
`say: the first receptor, which is added when the organic
`compound or ligand is the analyte, or is inherently pres-
`ent when the first receptor is the analyte; and a second
`receptor, which is polyvalent and specifically binds to
`the first receptor. The presence of the second receptor
`enhances the differentiation in signal level as a result of
`the degree of proximity of the two reagents of the signal
`producing system.
`In the broadest sense in performing the assay, the
`analyte, which is a member of a specific binding
`pair—ligand and its homologous receptor—is intro-
`duced into an assay medium in combination with: (1)
`antiligand, when ligand is the analyte; (2) ligand conju-
`gated to a label, where the label is a member of a signal
`producing system; (3) a second member of the signal
`producing system which interacts with the first member
`to modulate the signal depending on the proximity of
`the second member of the signal producing system to
`the first member of the signal producing system; (4) a
`pclyvalent receptor for the antiligand; (5) and any ancil-
`lary reagents necessary for the signal producing system.
`Depending upon the particular signal producing sys-
`tem, various protocols will be employed.
`By modulation is intended to create, destroy, modify,
`affect or change the signal, so as to allow a detectable
`
`

`
`4,281,061
`
`3
`difference by virtue of the interaction of the label and
`macromolecular reagent.
`'
`-
`Definitions
`
`5
`
`Analyte-—the compound or composition to be mea-
`sured, which may be a ligand, which is mono- or
`polyepitopic (antigenic determinants) or haptenic, a
`single or plurality of compounds which share at least
`one common epitopic site, or a receptor capable of
`binding to a specific polar or spatial organization.
`Specific binding pair—two different molecules.
`where one of the molecules has
`area on the surface or
`in a cavity which specifically binds to a particular spa-
`tial and polar organization of the other molecule. The
`members of the specific binding pair are referred to as
`ligand and receptor (anl:iligand}.
`Ligand-any organic compound for which a recep-
`tor naturally exists or can be prepared.
`Receptor—any compound or composition capable of
`recognizing a particular spatial and polar organization 20 '
`of a molecule i.e. epitopic site. Illustrative receptors
`include naturally‘ occurring receptors. e.g.
`thyroxine
`binding globulin, antibodies, enzymes. Fab fragments.
`lecttns, and the like.
`In the subject invention, a polyvalent receptor, nor-
`mally an antibody, for the ligand receptor anti(antili-
`gand) will be employed. The anti(antiligand) is pre-
`pared by employing the antiligand as an imimmogen in
`in vertebrate different from the source ofthe antiligand.
`Producing &rste.m—the signal producing sys-
`tem will have at least two components. at least one
`component being conjugated to ligand and another
`component which will be a macromolecular ragent.
`The goal producing system produces a measurable
`signal which is detectible by external means, usually the
`measurement of electromagnetic radiation. For the
`most part.
`the signal producing system will involve
`enzymes,
`antibodies,
`and
`chromophores, where
`chromophores include dyes which absorb light in the
`ultraviolet or visible region, fluoresccrs, phosphors and
`chemiluminescers. While for the most part, the signal is
`conveniently the absorption or
`of electromag-
`netic radiation. usually in the ultraviolet or fisiblc
`range, electrochemical changes, thermal changes, neph-
`elometric, and the like may also find application. The
`subject signal producing system requires that the mac-
`romolecular reagent interact with or react with, di-
`rectly or indirectly, the member of the signal producing
`system (label) bound to the ligand, resulting in the pro-
`duction, enhancement or diminution of the signal ob-
`served for the label.
`Macromolecular reagent—-the macromolecular rea-
`gent is a member of the signal producing system, which
`reactsorinteracts with, directlyorindirectly, them-
`her ofthe signal producing system bound to the ligand,
`so as to modulate the signal produced by the label. The
`macromolecular reagent will be at ieast I X10‘, usually
`at least 5 X 10‘ molecular weight, and preferably greater
`than 1x105 molecular weight. and may be 10 million
`molecular weight or more. The macromolecular rea-
`gent may be a single compound, a group ofcompounds,
`covalently or noncovalently linked together, or one or
`a plurality, usually a plurality, of molecules bound to a
`hub nucleus. The hub nucleus may be water soluble or
`insoluble, and is a polyfunctional material, nonnally
`polymeric, having a plurality of functional groups, such .
`as hydroxy, amino, mercapto, ethylenic, etc., as sites for
`linking. The hub nucleus will generally have a molecu-
`
`4
`to or greater than 10.000, usually
`lar weight equal
`50,0(Il molecular _weight. Illustrative hub nucleii in-
`clude polysaccharides, polypeptides, including proteins,
`nucleic acids, ion exchange resins, vinyl polymers, such
`as polyacrylainides and vinyl alcohols, polyethers,
`polyesters, and the like. The significant factor of the
`macromolecular reagent is that its approach to the label
`bonded to the ligand will be affected, when a large
`immunological matrix is formed by polyvalent recep-
`tors about the labeled ligand associated with antiligand.
`Ligand-receptor matrix-—the matrix is a matrix
`formed from a plurality of ligands, ligand receptors, and
`antireceptors, where at least the antireccptors are poly-
`valent. and normally all the mbers of the matrix are
`polyvalent. What is intended is that a plurality of li-
`gands.whichareboundtoligaI1dreceptors,arecon—
`nected by a plurality of bridges formed by ant.irecep-
`tors, which results in relatively large reticulated micro-
`structures, capable of modulating, usually reducing, the
`rate of difiiision of a molecule in the environment of the
`matrix up to and including steric exclusion.
`Labeled Ligand-'—~the conjugate of the ligand member
`of the specific binding pair covalently bonded to a mem-
`ber of the goal producing system, either joined by a
`bond, li.ul:i.ng group or hub nucleus. The labeled ligand
`mayhaveoneormoreligandsoroneormcrelabelsor
`a plurality of both. Where conjugation of the label and
`ligand are required, the ligand will normally be modi-
`fied to provide for a site for linking. The modified li-
`gandisreferredtoasligandanalog. aadtheligand
`analog may difler from the ligand by replacement of a
`hydrogen or more usually by the introduction of or
`modification of a functional group.
`Polyfligand analog}—a plurality of baptenic ligand
`analogs bonded to a water soluble bub nucleus of at
`least about 10,!!!) molecular weight, usually 30,(IJO to
`dtllflll, e.g. proteins, polysaccharides. nucleic acids,
`synthetic polymers. etc.
`Method
`
`The subject asay is carried out in an aqueous zone at
`a moderate pl-I, generally close to optimum assay
`tivity, without separation of the assay components or
`products. The away zone for the determination of ana-
`lyle is prqiared by employing an appropriate aqueous
`medium, normally buffered, the unknown sample which
`may have been subject to prior treatment, the labeled
`ligand. ligand receptor (antiligand), the macromolecu-
`lar reagent member of the signal producing system, and
`antireceptor as well as any additional materials required
`for the signal producing system for producing a detecti-
`ble signal. In the event that antiligand is the analyte,
`antiligand need not he added.
`The presence of ligand or its homologous receptor
`(antiligand) in the unknown will affect the extent to
`which the macromolecular reagent interacts with the
`labeled ligand.
`In carrying out the assay, an aqueous medium will
`normally be employed. Other polar solvents may also
`be included. usually oxygenated organic solvents 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 per-
`cent.
`.
`
`The pH for the medium will usually be in the range of
`about 4 to 11, more usually in the range of about five to
`ten, and preferably in the range of about 6.5-9.5. The
`
`

`
`4,281,061
`
`5
`pH is chosen so as to maintain a significant level of
`specific binding by the receptor while optimizing signal
`producing efficiency. In some instances, a compromise
`will be made between these two considerations. Various
`buffers may be used to achieve the desired pH and
`maintain the pH during the determination. Illustrative
`buffers include borate, phosphate, carbonate, tris, barbi-
`tal, and the like. The particular buffer employed is not
`critical to this invention, but in individual assays one
`buffer may be preferred over another.
`Moderate temperatures are normally employed for
`carrying out the assay and usually constant tempera-
`tures during the period of the measurement, particularly
`for rate determinations. The temperatures for the deter-
`mination will generally range from about 10° to 50° C.,
`more usually from about 15° to 40° C.
`The concentration of analyte which may be assayed
`will generally vary from about 10-4 to 1045M, more
`usually from about
`l0‘—5 to 10'-13M. Considerations
`such as whether the assay is qualitative, semiquantita—
`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.
`While the concentrations of the various reagents in
`the assay medium will generally be determined by the
`concentration range of interest of the analyte, the final
`Eooncentration of each of the reagents will normally be
`determined empirically to optimize the sensitivity of the
`assay over the range of interest. The total binding sites
`of the members of the specific binding pair which are
`reciprocal to the analyte will be not less than about 0.1
`times the minimum concentration of interest based on
`binding sites of analyte and usually not more than about
`1,000 times the maximum concentration of interest
`based on analyte binding sites, usually about 0.1 to 100
`times, more usually about 0.3 to 10 times the maximum
`concentration of interest. By concentration is intended
`the available concentration, that is, the concentration at
`saturation and not necessarily the actual concentration
`where all members of the specific binding pair may not
`be equally available for binding.
`Depending upon the particular signal producing sys-
`tem, as well as the manner in which the specific binding
`pair members are employed, the amount of the various
`members of the signal producing system can be varied
`relatively widely. That is, relatively large excesses of
`the macromolecular reagent may be employed, where
`either the rate of formation of the matrix is high as
`compared to the interaction between the label and the
`macromolecular reagent or the matrix is allowed to
`form initially, followed by introduction of the macro-
`molecular reagent. As suggested above, the order of
`addition of the various reagents may vary widely, de-
`pending on the particular label and signal producing
`system, the nature of the analyte, the relative concentra-
`tions of analyte and reagents, the mode of determina-
`tion, and the sensitivity desired.
`When ligand is the analyte. the addition of ligand will
`normally diminish complex formation involving Iabeled
`ligand, so that the interaction between the labeled li-
`gand and the macromolecular reagent will be enhanced
`with increasing amounts of ligand being present in the
`assay medium. By contrast, when ligand receptor is the
`analyte, there can be no complex formation in the ab-
`sence of ligand.
`For ligand analyte, conveniently the ligand may be
`combined with antiligand, so that available binding sites
`
`6
`of the antiligand become filled by the available ligand.
`To the extent that ligand is present, the amount of antili-
`gand available for binding labeled ligand will be re-
`duced. Alternative ways would be to combine both the
`ligand and labeled ligand simultaneously with the an1ili-
`gand, but one would normally not combine the labeled
`ligand with the antiligand prior to addition of the ligand
`analyte. This is due to the fact that with many receptors,
`the association of the specific binding pair members is
`almost irreversible during the time period of an assay.
`While for the most part, ligand analytes will be anti-
`gens, which have a plurality of determinant (epitopic)
`sites, haptenic (mono-epitopic) compounds can also be
`employed by having a plurality of haptens conjugated
`to a large label (often greater than 10,000, usually
`greater than 30,000 molecular weight) or a plurality of
`haptens and labels bonded to a water soluble hub nu-
`cleus.
`One or more incubation steps may be involved in
`preparing the assay medium. For example, it will usu-
`ally be desirable to incubate an antigen analyte with the
`antiligand. In addition, it may be desirable to have a
`second incubation after addition of the antireceptor.
`Whether to employ an incubation period and the length
`of the incubation period will depend to asubstantial
`degree on the mode of determination—-rate or equilibri-
`um——and the rate of binding of the receptors to their
`homologous members. Usually, incubation steps will
`vary from about 0.5 min to 6 hrs, more usually‘l‘rom
`about 5 min to 1 hr. Incubation temperatures will- gener-
`ally range from about 4° to 50° C., more usually from
`about 15" to 37" C.
`-
`-
`After the reagents are combined the signal will then
`be determined. The method of determination may be
`the observation of electromagnetic radiation, particu-
`larly ultraviolet and visible light, either absorption or
`emission, calorimetric, electrochemical, nephelometric,
`or the like. Desirably, the signal will be read as electro-
`magnetic radiation in the ultraviolet or visible region,
`particularly from about 250 to 750 nm, usually from
`about 350 to 650 nm.
`The temperature at which the signal is observed will
`generally range from about 10° to 50° C., more usually
`from about 15° to 40° C.
`Standard assay media can be prepared which have
`known amounts of the analyte. The observed signal for
`the standard assay media may then be plotted, so as to
`relate concentration to signal level. Once a standard
`curve has been established, a signal level may be di-
`rectly related to the concentration of the analyte.
`The time for measuring the signal will vary "depend-
`ing on whether a rate or equilibrium mode is used, the
`sensitivity required, the nature of the signal producing
`system and the like. For rate modes, the times between
`readings will generally range from about 5 sec to 6 hrs,
`usually about 10 sec to 1 hr. For the equilibrium mode,
`after a steady state is achieved, a single reading may be
`sufficient or two readings over any convenient time
`interval may suffice.
`For the most part, the labels bound to the ligand will
`catalysts, particularly enzymes or oxidation-
`be
`reduction catalysts, chromogens, which absorb or emit
`light in the ultraviolet or visible region, such as dyes,
`fluorescers, cherniluminescers, donor-acceptor
`fluo-
`rescer combinations, phosphorescers, or enzyme bind-
`ing substances e.g. chromogenic substrates, cofactors
`and inhibitors, and the like.
`
`10
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`15
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`4,281,061
`
`7
`
`.
`Materials
`The components employed in the assay will be the
`labeled ligand, antiligand when ligand is the analyte,
`macromolecular reagent, and receptor for the ligand 5
`receptor, anti(antiligand),' as well as any additional
`members of the signal producing system.
`Analyte
`
`The ligand analytes of this invention are character- It}
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poly(amino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations of assemblages include bacteria, 15
`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 20
`about 10,000. In the poly(amino acid) category,
`the
`poly(an1ino 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 25
`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 30
`related to specific microorganisms, particularly disease
`causing microorganisms, etc.
`The following are classes of proteins related by struc-
`U-“'35
`llijlfotamines
`1510335
`315‘-‘mills
`siobulms
`5C1e1’0l31'0l€i115
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`proteoglycans
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`C¢1'Lil30P¥‘07—ei11
`ct;-Acid glycoprotein
`0:1-Antitrypsin
`a_l_Glyc0pr-otein
`T1-anscoflin
`4-.6S-Postalbumin
`Tryptophan-poor
`akglycoprotein
`0'-IX'G'lyc°Pr°tei“
`Thyroxin-binding globulin
`Inter-ct-trypsin-inhibitor
`-
`Gc-globulin
`(G0 1-1)
`(Go 2-I)
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`,
`
`8
`
`(Hp I-1)
`(Hp 2_1)
`(Hp 2-4 2)
`Ceruloplasrnin
`Cholinesterase
`a2.Lipop1-ote-in(s)
`Myoglobin
`C-Reactive Protein
`a.2~Macroglobulin
`Q12-HS.glycop1-Qtein
`Zn-ct:-glycoprotein
`a2..Neu1'a,[nino-glycopfotein
`E1-ythropoiatin
`,3.1ipgpm;ejn
`Transferrin-
`Hemopexin
`Fibrinogen
`Plasminogcn
`,32-g1ycopm¢,ein 1
`)3;-glycoprotein ll
`Immunoglobulin G
`(IgG) or *yG-globulin
`Mol. formula:
`3123:; or 72);;
`Immunoglobulin A (lgA) or 7A-globulin
`Mo], fm-muja;
`(a.2K2)’* or (a2?t2)"
`Immunoglobulin M
`(IgM) or -yM-globulin
`Mo]. formula:
`
`.
`(r~t1K2)5 or (ll-27\2)5
`Immunoglobulin D(IgD) or 'yD-Globulin ('yD)
`Mo], formuia;
`.
`(figlcz) or 5212)
`Immunoglobulin E (IgE) or -yE-Globulin ('yE)
`M01. formula:
`'
`(ezrcz) or (£212)
`Free at and it light chains
`Complement factors:
`C1
`C'1q
`C’ 11-
`C'1s
`C2
`C3
`31A QZD
`C'4.
`05
`C6
`C’?
`C3
`cg
`Important blood clotting factors include:
`
`
`
`V and Vi‘
`
`BLOOD. CLOTTING FACTORS
`I
`International designation
`Name
`I
`ll-jibniinogeg
`11
`mt ronl in
`El:-lJ1x:l’ll1Ii'o1nboplaslin
`Proaccelerin, accelerator
`globulin
`_
`.-
`P‘°?°“""‘”? .
`Annhemoplnlic globulin
`(AB6)
`Christmas factor.
`plasma ll'|l'Ol('l;l;0glaSl.ll1
`COITI Oflflfll
`Stualgt-Prower faglcr,
`autoprothrornbin III
`
`V"
`VIII
`
`Ix
`
`X
`
`

`
`4,281,061
`
`9
`-continued
`BLOOD CLONING FACTORS
`International designation
`Name
`x1
`Plasma thrombopiastin
`teoedent (PTA)
`XII
`iinagmnann factor
`
`x111
`Fibrin-stabilizing factor
`
`
`
`10
`-continued
`Species of Microorganisms
`
`Found In
`
`in M M _
`e
`2
`cerogeries
`Klebsfella clmcae
`55-'m°"€l-'9 WPWW
`
`Salmonella rypln‘-murium:
`- Salmonella derby
`Salmonella pt.-liar-um
`Slrigella clysenrefiee
`Sliigelloflaxneri
`Sinigelia samtei
`Rickerrsfae '
`C"""T'd" """"."""
`Entamoelm limolyrim
`
`and ?°1y“c,
`:51-w_d=
`f
`"9‘7“.°" °
`“"5 ud‘
`g=r:>er=;;1]=1ndde
`o yea
`it
`Polysaocharide
`c
`n e.
`Lli!t:°,t;°1¥5&¢-
`Poirswcharide
`Polysaoeharide
`
`Polysaccharirie
`Crude. Poly-
`saccltnriade
`Crude extraet
`P°""‘°°l‘““"°
`Crude extract
`
`The microorganisms which are assayed may be in-
`tact, lysed, ground or otherwise fragmented. and the
`resulting composition or portion, e.g. by extraction,
`assayed. Microorganisms of interest include:
`
`Important protein hormones include:
`Peptide and Protein Hormones
`_
`_
`Parathyrold hormone (parathromone)
`'I‘hyrocalcitonin
`Insulin
`gllilclfgon
`C 33111
`E1-ythxgpoietin
`Melanotropin (melanocyte-stimulating hormone; in-
`.
`termedm)
`Somatotropin (growth hormone)
`Corticotropin (adrenoeorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone (interstitial cell-stimulating hor- 25
`rnone)
`Luteomammotropic hormone (luteotropin, prolactin)
`Gonadotroptn (chonomc gonadotropin)
`Tissue Hormones
`
`5
`
`In
`
`15
`
`20
`
`30
`
`emmh
`Cmnmmwn dwmw
`PWLLW
`Diplococ-'.‘I.I': pneumonine
`_
`Strggococei
`Secretm
`Smepromocus pyugene:
`Gastrin
`Srmprocacrus saliwrms
`A_ngioten5iJ1 I and II
`
`:_J.L_.m“P;'1°°°°°1
`Bradykinin
`ta ylacaceus aureus
`Smpwwmw mm
`35
`Human placental lactogen
`Peptide I-lormones from the Neurohypophysis % id
`_
`eisseria meningif is
`Oxytocm
`Nekrenfu gonamkeae
`Va§o,pr.,éssin
`EmmIra~=teaiatc_iae_
`Releasing factors (RF) CRF, LRF, TRF, Somatotro- 40
`Eicfivrivlia to-‘I’
`pin-RF, GRF, FSH-RF, PIF, MIF
`"'-"°~"f’“'-"“’-"’°3”“’~.‘
`.
`.
`.
`Klebwella pneumamae
`Other polymeric materials of interest are _mucopol_y-
`_W,,,,,,,eH, wk“
`saccharides and polysaccharides.
`Salmonella cliolemesmk )
`Illustrative antigenic polysaccharides derived from
`f.VPlH'f'=W=‘=t*'=
`microorganisms are as follows:
`sag”: fl’::’_f;';;"
`Shlgella omliiuorarda
`S'"}’="° flew-jfi
`55139775 5079'"
`_
`_
`Stare!-'0 Sanftef
` ll!..._
`Proteus vulgurir
`Pmrenr mirabilis
`Pmreur morgani
`Pseudomorm aewginm
`Almligenes faecaliir
`Vfbrfo clmleme
`Hemoghilus-flordetella gmug
`Hemopliilus influenzae.
`
`_
`T" °?“‘°"“
`bnetena
`The Salmonella:
`
`The Shisellae
`
`Proteus species
`
`H. demyi
`H_ ;wmop;”-hm,
`H_ “gm”-_._.u_,
`H Nmfnflwme
`
`45
`
`so
`
`55
`
`}
`
`60
`
`55
`
`3,,,.,gm,r;,, Padang
`pmeureuae
`.
`"“"""""""“""'
`.
`g:""""“ pm"
`entails rnlareuso
`§’—'5°°"i
`_
`3"'““'”‘7 memefll"
`5’''‘“H“ ‘’l’‘’”‘'-‘
`Bmoelln suis
` flL
`Bacillus onllmrcis
`Bacillus subrilis
`
`Species of Microorgartisms
`Srreprocacruspyogenes
`Diplococcns pneumonia:
`Neisseria menirigiddir
`Neinreria gmiamlmre
`Grrynebarterium diielttlreriae
`Aciinobacillus mailer‘;
`Arrinabacillus wlcirenzori
`Fmncisella rularensir
`
`‘P"'”e""'9'l"'" Pal’?
`Pastcurclla pesns
`Pasteurella multocizln
`En.-cello aborrus
`flaemopliilus Lqflaenaae
`Haernophilus pertussis
`Tneponema neirerl
`Veiiloneffa
`Bglsiaelorhrix
`Listeria manocyrogener
`Cltromabacterium
`
`Hycobacterium tuberculosis
`
`Hemosensitin Found in
`Polysaécharide
`Polysaccharide
`Polysaecharide
`Polysaceharide
`Polysaccharide
`Crude extract
`Lipopolysac-
`eharide
`Polysaccltaride
`‘
`Polysaoohande
`Capsular antigen
`Crude extract
`Polysacoharide
`Crude
`Polysaccluu-ide
`Lipopolysa.c-
`chm“
`Polysaccharide
`P-olysaccharide
`Lipopolysao
`chm-[dc
`Saline extract of
`90% phenol
`extracted
`
`

`
`4,281-,0'_6'l
`
`I2
`
`Ttichfilnn mbninr
`Hhngollrm andauniln‘
`Virasu;
`'
`
`§;
`V‘
`
`Variuclh (Chicken you)
`Herpes Znstcr (Singles)
`
`ClIiIg—yIV'IIB
`Til-"'cIwV'III
`lhynnVirIs
`9. Innis IE-huqInI'nisV‘-ms
`
`|.'.h|II'lhTit:ll%uuV‘n-is
`Ycflnw‘FcucI'\"'|IB
`Dcg2V'lI:
`lntvuilaes
`
`louuins'l}1iI:sl-C!
`
`El
`
`kp-I:'n'sAVirus
`&n'.'I'sBV’n'Is
`“l‘UIanr\P'irIs:s
`IInschcr‘i.i1iV"nIs
`GIIEVI3
`Ennnylaulhliaifnrnus
`
`10
`
`I5
`
`35
`
`45
`
`Tlumtmoqitnpicliglndnlnlytlswmgmaaflybe
`frunabouH(I|Io2,£Illmnlecularweight,nnnensnally
`fmu:l25tnl.tII)molecularweigln.Theamlytesof
`pol}u1-
`mlgandthefikahdndedammgdrugsdhtutstam
`
`SS
`
`lu'ds,whichinchliesmorpl|iue,q:o:ldne.hemi11,de1—
`
`mine alkaloid; which includu cunning and bu.-nzoyl
`
`Ioidswiachincluaesrhediuhylunideorlysugicacsd;
`su_:mida!kaloids;itniuuoylaIhluids-,quinaz:olint:a!ka-
`
`
`
`indudes'qmm;ne'‘
`
`
`
`
`
`and qmnuime:;'' '
`
`d,1tu'penc' alkaloid:
`
`65
`
`’I‘henextgroupofdmgsincIudesste11oid§.,which
`includes the -wtrogcns, gestogens, andmgens, an-
`drenoourtica] slscmids,-bile acids. canliottmic glycnsides
`andaglyeones,w}:ichinclntlesdigoxinanddigoxigeI1in.,
`snpuninsandsapogeniIIs.theirderivalivesamdmctaho-
`
`

`
`13
`lites. Also included are the steriod mimetic substances,
`such as diethylstilbestrol.
`The next group of drugs is lactams having from 5 to
`6 annular members, which include the barbiturates, e. g.
`phenobarbital and secobarbital, diphenylhydantonin,
`primidone. ethosuxirnide, and their metabolites.
`The next group of drugs is aminoalkylbenzenes, with
`alkyl of from 2 to 3 carbon atoms, which includes the
`amphetamines, catecholamines, which includes ephed-
`rine, L-dopa, epinephrine, narceine, papaverine, their
`metabolites.
`
`The next group of drugs is aminoalkylbenzenes, with
`allcyl of from 2 to 3 carbon atoms, which includes
`ephedrine, L-dopa, epinephrine. narceine, papverine,
`their metabolites and derivatives.
`The next group of drugs is benzheterocyclics which
`include oxazepam, chlorpromazine,
`tegretol,
`imipra-
`mine, their derivatives and metabolites, the heterocyclic
`rings being azepines, diazepines and phenothiazines.
`The next group of drugs is purines, which includes
`theophylline, caffeine, their metabolites and derivatives.
`The next group of drugs includes those derived from
`marijuana, which includes cannabinol and tetrahydro-
`cannabinol.
`
`The next group of drugs includes the vitamins such as
`A, B complex, e.g. B12, C, D, E and K, folic acid, thia-
`mine.
`
`10
`
`15
`
`20
`
`25
`
`The next group of drugs is prostaglandins, which
`differ by the degree and -sites of hydroxylation and
`unsaturation.
`
`30
`
`The next group of drugs is antibiotics, which include
`penicillin, chlorornycetin, actinomycetin