[191
`United States Patent
`[1 1}
`4,287,300
`Gibbons et al.
`[45 Sep. 1, 1981
`
`
`
`[54] CHARGE EFFECTS IN ENZYME
`IMMUNOASSAYS
`
`[TF5]
`
`Inventors:
`
`Ian Gibbons, Menlo Park; Gerald L.
`Rowley, Cupertino; Edwin F. Ullman,
`Atherton, all of Calif.
`
`I’.-'3] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`:21] Appl. No.: 51,099
`
`{Z2} Filed:
`
`J til. 26, 1979
`
`_
`
`say, a New Immimochemicoi Tec.imi‘qae”, Bfoehem. Bio-
`phys. Res. Comm, vol. 4?, No. 4 (1972) pp. 846-351.
`Nargessi et 211., ‘‘Use of Antibodies Against the Label in
`Non-Separation Non~Iostopic Immunoassay:
`Indirect
`Quenching Fluoi-immunoassay of Proteins"J’. Immzmof.
`Merh, vol. 26, No. 4, (1979) pp. 307-313.
`Gibbons et al., "Homogeneous Enzyme Immunoassay
`for Proteins Employing B—Galactosidase", Anal. Bio»
`chem, vol. 102 No.
`1 (1930) pp. l6':'—l70.
`“Enzyme Immunoassays Revisited", The Lancet, vol.
`1], (1979), pp. 730-781.
`Schuurs et ai., “Enzyme—Imm_unoassay", Chin. Chim.
`Aelo, vol. Si, No. 1, (197?) pp. 1-40.
`
`Primary Exorm'ner—Thomas G. Wisernan
`Attorney, Agent, or Fi'rm—Bert1'am I. Rowland
`
`[57]
`
`ABSTRACT
`
`A method for determining a member of a specific bind-
`ing pair-ligand and receptor (antiligand). Reagents em-
`ployed include a first modified member which provides
`an electrical field due to the presence of a plurality of
`ionic charges and a second modified member labeled
`with a component of a signal producing system, which
`system may have one or more components. The average
`proximity in the assay medium of the first and second
`modified members is related to the amount of anaiyte.
`where the observed signal from the signal producing
`system is related to the effect of the electrical field on
`the signal producing system.
`Also, compositions are provided, as well as reagents, in
`predetermined ratios for optimizing the signal response
`to variations in analyte concentration.
`
`15 Claims-, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2038
`
`Genentech Exhibit 2038
`
`IPR2016-00710
`
`£51]
`
`[58]
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`' 6/ 19% Rubenstein et al.
`................... .. 435/‘I
`12/1976 Uilrnan et al.
`. ..... ..
`435;‘?
`B/1978 Maier et al.
`.......
`435/ 188
`3/1980 Ullman et al.
`435/‘!
`5/ 1980
`Rubenstein et al.
`................... .. 435/?
`
`
`
`OTHER PUBLICATIONS
`
`3,81'i',83'.I'
`3.996.345
`4,104,029
`4,193,983
`4,203,802
`
`Walienfels et al., The Enzymes, vol. 3, (Boyer ed.), Aca-
`demic Press, NY. (1972) pp. 617-663.
`Rowley, et al. “Mechanism by which Antibodies Inhibit
`Hapten-Maiate Dehydrogenase Conjugates",
`J’. Biol
`Chem. vol. 250 No. 10, (1975), pp. 3759-3766.
`Engasser, et al., “Diffusion and Kinetics with Immobi-
`lized Enzymes", Applied Biochemistry and Bioengineer-
`ing, vol. I, (Wingard et al. ed), Academic Press, N.Y.
`(1976) pp. 12?-145, 194-205.
`Rubenstein, et al.. “Homogeneous‘ Enzyme Immunoas-
`
`[52] U.S. Cl.
`
`Int. Cl.’
`
`CIZQ 1/79; GOIN 33/S4;
`Cl2N 9/96
`435/5; 435/7;
`435/188; 435/310; 424/12; 23/230 B
`Field of Search ..................... .. 435/4, 7, 177, 188,
`435/805. 810, 5; 424/8, 12, 85; 260/112, 121;
`252/408 R; 73/230 B; 422/8
`
`

`
`CHARGE-.rir‘1mcrs1i~z Enzyme '
`IMMUNOASSAYS.
`
`BACKGROIillNlD OF THE INVENTION
`1. Field of the Invention '
`'
`'
`Protein binding assays or immunoassays have been
`the subject 'of-thorough investigation and commercial-
`ization. The ability to specifically determine a drug or
`other compound of interest, particularly physiological
`interest, which is presentin extremely low concentra-
`tion, usually Iess thanimicrograms per milliliter, ‘has
`opened up new opportunities in clinical laboratories.
`The ability to monitor”-therapeutic drug administration
`or drug addiction, «to rapidly and elliciently determine
`diseased states, and to monitor the condition of a patient
`during times of stress,'has provided significant opportu-
`nities for irn'provement='ol'-health ‘care.
`The assays in the'.clinical-laboratory frequently re-
`quire not only high sensitivity but accuracy over a rela-
`tively narrow-range. —'I'herefore,- new techniques are
`being developed which ~recognize these requirements
`by providingfor greater sensitivity, reduced responseto
`non-specific effects,“-and easier and simplier protocols.
`In addition, many antigenscan be obtained only in im-
`pure fonn or in pure form at elevated costs. Therefore,
`assays must accomodate-the-possibility that the antigen
`will be impure. Parallelto. this situation is that most
`antibodies which are .obtained “by antigenic injection
`will have less than about 30 weight %, or frequently less
`than about 20 \_vei'gl1_£«% of _t__l_1e total protein as the anti-
`body of interest"--When:-preparing reagents which in-
`volve reactions with- the -antibody composition, _ the
`presence of the. large amount of contaminant must be
`taken into account.
`l.._.--._
`.
`-
`Other considerations. in developing an assay include
`the necessity for 'and._ru.I_rnber of incubations, the period
`required for tl1e,incubation._tl1e period required for the
`measurement, .the:-'sensitivity- of the measurement
`to
`extraneous factors, the stability of_ the reagents,
`the
`formulation of the reagents,-and thelilte.
`2. Description of t._h_e,_Prior Art
`.
`U.S. Pat. No. 3,296,345 describes the employment of
`a chromophore, pa_i_r_—fluorescer
`and quencher-—-,
`where the members "oi; the_. pair are bonded to different
`members of a specific _bi_ndi_ng pair, so that the amount
`of fluorescer and quencher which come within an.inter-
`acting distance is _d_epen_dent upon the amount of analyte
`in the medium.__ U.S. Pat._ No. 3,935,074 describes an
`immunoassay depem,'lent_.upon._the inability of two anti-
`bodies to simultaneo_usly_.bi_nd.to-a reagent having at
`least two deteqninant.sites.,_Co-pending application Ser.
`No. 893,650, filed Apr_..5, -1,9‘.-'B,_U.‘_5. Pat. No. 4,233,402,
`teaches the ooncept;;;.iu immurioassays of bringing to-
`gether two enzymes, whose, substrates or products are
`in some ways related to the production of a detectible
`signal, where thejuxtaposition of the enzymes is related
`to the amount of analyte in the medium. Co-pending
`application Ser. No. 8.l5,632, filed July 14, I977, U.S.
`Pat. No. 4,208,479. teaches:—the employment of a macro-
`molecular modifier .of' _.a_ label bound to an antibody,
`where the modifier is inhibited from approaching the
`label when the labeled-antibody is bound to antigen.
`Co-pending application Ser. No. 815.487, filed July .14,
`1977, U.S. Pat. No. -4,233,401, discloses an enzyme im-
`munoassay where ligand is labeled -with enzyme and an _.
`enzyme inhibitor. .is
`inhibited from approaching the
`enzyme, when antibody is bound to the ligand. Co-
`
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`4,287,300
`
`2
`pending application Ser. No. 964,099, filed Nov. 24,
`1978. discloses the use of macromolecular particles to
`provide discrimination between a label bound to the
`particle and a label free in the solution, where the
`amount of label bound to the-particle is related to the
`amount of analyte in the medium, and the observed
`detectible. signal is dependent upon the distribution of
`the label between the particle and the medium. U.S. Pat.
`No. 3,317,337 describes a homogeneous enzyme immu-
`noassay.
`-
`
`SUMMARY OF THE INVENTION
`
`A protein binding assay is provided involving mem-
`bers of a specific binding pair, the members being ligand
`and receptor (antiligand). As a first reagent
`in the
`method, one of the members is substituted with a plural-
`ity of ionizable functionalities which are capable of
`providing a charge, either positive or negative, under
`the assay conditions. A second member, either the same
`or homologous member, is labeled with a component of
`a signal producing system, which signal producing sys-
`tem is able to produce a detectible signal under the assay
`conditions.
`The signal produced by the signal producing system
`is affected by the juxtaposition of the electrical field
`resulting from the charged first member. By appropri-
`ate choice of the members in relation to the analyte, the
`average proximity of the first and second members in
`the assay medium can be related to the amount of ana-
`lyte in the assay medium. Thus, the observed signal can
`be related to the amount of analyte in the assay medium.
`By employing appropriate standards having known
`amounts of analyte, one can establish a relationship
`between concentration of the analyte and the level of .
`observed signal.
`In addition, reagents are provided for the assay, as
`well as combinations of rengentswhich provide for
`substantial optimization of the sensitivity of the assay or
`level_ of response of the assay to variations in the con-
`centration of the analyte.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`The subject invention is concerned with a protein
`binding assay, involving a specific binding pair, ligand
`and receptor. The basis of the invention is to relate the
`proximity of two reagents to the amount of analyte in
`the assay medium. The first reagent, is a first member of
`the specific binding pair which is modified with a plu-
`rality of ionic charges, so as to create a charged field in
`an aqueous environment at a predetermined pH. A
`second member is modified with a label, whichis one
`component of a single or multicomponent signal pro-
`ducing system; the level of the observed signal is depen-
`dent upon the juxtaposition of the signal producing
`system to the field created by the charged member. The
`field affects the signal producing system by enhancing
`or diminishing the localized concentration of certain
`ions in the assay medium, which ionic concentration
`affects the level of the observed signal. In addition to
`the analyte and the first and second members, other
`materials may also be added depending upon the nature
`of the signal producing system.
`The binding of homologous members of the specific
`binding pair results in the fonnation of a complex,
`where two or more, usually three or more members are
`involved. In view of the polyvalent nature of antibodies
`
`

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`4,287,300
`
`4
`concentration range of interest of the analyte, the final
`concentration of each of the reagents will normally be
`determined empirically to optimize the sensitivity of the
`assay over the range of interest. That is, a variation in
`‘concentration of the analyte which is of significance
`should provide an accurately measurable signal differ-
`ence.
`
`3
`and antigens, the complex can be extended to create a
`network of antibody and antigen bringing a plurality of
`signal labels and ionic charges into proximity, where
`interactions can occur.
`
`the following
`In discussing the subject invention,
`order will be involved. First. the assay method will be
`considered. This will be followed by definitions delin-
`ing various terms in relation to the materials employed.
`Following the definitions,
`the materials will be dis-
`cussed as to the analyte, signal producing system, and
`charged member. This will be followed by the experi-
`mental and demonstration of the utility of the subject
`method.
`
`ASSAY METHOD
`
`In accordance with the subject method, the analyte, a
`reagent having a plurality of ionizable groups which are
`substantially ionized under the conditions of the assay, a
`labeled reagent, where the label is a member of a signal
`producing system and any necessary additional compo-
`nents are combined in a buffered aqueous medium. The
`observed signal may then be read and compared to an
`assay medium having a known amount of analyte.
`The analyte is a member of a specific binding pair,
`consisting of ligand and its homologous receptor, and
`either the ligand or the receptor may be the analyte.
`The assay medium is normally aqueous, which is nor-
`mally buffered in suflicient amount to a moderate pH,
`generally close to providing optimum assay sensitivity.
`The assay is perfonned without separation of the assay
`components or products.
`The aqueous medium may be solely water, or may
`include from zero to 4-0 volume percent of a cosolvent,
`nonnally a polar solvent, usually an oxygenated organic
`solvent or from one to six, more usually of from one to
`four carbon atoms, including alcohols, ethers, and the
`like.
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`The total binding sites of the members of the specific
`binding pair which are reciprocal to the analyte will
`vary widely depending upon the nature of the reagents,
`that is, whether the reagents are the same as or different
`from the analyle. For example, both reagents may be
`receptors or both reagents may be ligand or one of the
`reagents may be ligand and the other reagent receptor.
`At least one reagent will be the receprocal bonding
`agent of the analyte.
`-
`The ratio of labeled reagent to analyte based on bind-
`ing sites in the assay medium will generally be from
`about 0.5 to about 100 binding sites of labeled reagent per
`binding site of analyte, usually from about 1 to 50, and
`more usually from about 1 to 20 over the analyte con-
`centration range. These members refer to available sites
`at saturation since all sites will not be equally available.
`It should be appreciated that
`these numbers are
`merely intended to be illustrative of the ratios of most
`likely interest. The ratio will vary depending upon the
`manner of measurement, equilibrium or rate, the bind-
`ing constant of the labeled reagent, the concentration of
`the analyte, the sensitivity of the signal producing sys-
`tem to charge effects, the nature of the ligand, and the
`sensitivity with which the signal may be detected.
`The mole ratio of the charged member to the labeled
`member may also be varied widely, depending upon the
`nature of the label, the sensitivity of the label to charge
`effects, and the nature of the ligand. Usually, the mole
`ratio of the charged member to the labeled member will
`be from about'0.5—l00:l, more usually from about 1 to
`2&1. This can vary quite dramatically, depending upon
`the particular protocol, the order of addition, the nature
`of the label, the relative binding affinities and the like.
`The order of addition may vary widely, although
`frequently all of the components of a multicomponent
`signal producing system will not be added simulta-
`neously. Usually, the membcr labeled with a component
`of the signal producing system will be added to the
`assay medium prior to at least one of the other compo-
`nents of the signal producing system. This will be par-
`ticularly true, where an enzyme is a label and the other
`components are substrates and cofactors.
`The two reagent members, may be added simulta-
`neously or consecutively to the analyte. Conveniently,
`the signal producing system label will frequently be
`added prior to the charged member. The two reagents
`may be provided as a single composition or as separate
`compositions, depending upon the nature of the proto-
`col.
`.
`
`Two particular protocols may be indicated as illustra-
`tive. The first protocol involves the addition of the
`signal producing system labeled member to the analyte
`and incubating for a sufficient time for the system to at
`least approach equilibrium. To the mixture may then be
`added the charged member and at the same time or
`immediately thereafter, any additional components of
`the signal producing system.
`An alternative protocol would be to add substantially
`simultaneously,
`the signal producing system labeled
`member and the charged member and either incubate or
`not, as required. Desirably, one may add a signal inhibi-
`
`The pH for the medium will usually be in the range of
`about 4 to 11, more usually in the range of about 5 to 10,
`and preferably in the range of about 6.5 to 9.5. The pH
`is chosen so as to maintain a significant level of specific
`binding by the receptor, while optimizing the sensitivity
`or response of the signal producing system to variations
`in analyte concentration.
`Various buffers may be used to achieve the desired
`pH and maintain the pH during the determination. Illus-
`trative buffers include borate, phosphate, carbonate,
`tris, barbital and the like. The particular. buffer em-
`ployed is not critical to this invention, but in individual
`assays, one or another buffer may be preferred.
`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. Incubation temperatures will
`normally range from about 5° to 45° C., more usually
`from about 15° to 40° C. Temperatures during measure-
`ments 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-‘ to 10-15 M, more
`usually from about 104’ to 10-” M. 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 concentrations of the other
`reagents.
`While the concentrations of the various reagents in
`the assay medium will generally be determined by the '
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`5":
`tor; that is, a material which interacts, with the signal
`producing system label, so as to inhibit production of
`the signal, when the signal producing system labeled
`member is not bound to its homologous member.
`Theanalyte may act to bring the charged member
`and the signal label member together or enhance the
`separation of the charged and signal label members. For
`example, when the analyte is an antigen or poly(1igand
`analog) and the two reagent members are antibodies,
`within a limited concentragion range, the analyte will
`serve to bring on the average _'t'_he__t_wo reagent members
`in closer proximity than when the members are diffus-
`ing freely in solution. On the other hand, when the
`analyte is an antigen and the signal label member is an
`antigen, then the analyte and signal label member will
`compete for a charged antibody.
`One or more incubation steps may be involved in
`preparing the assay medium. For example, it may be
`desirable to incubate an antigen analyte with labeled
`receptor. In addition, it may be desirable to have a
`second incubation step, depending upon the nature of
`the other reagents employed. Whether to employ an
`incubation period and the length of the incubation per-
`iod will depend to a substantial degree on the mode of
`determination—rate or equi1ibrium—and the rate of
`binding of the receptor to the ligand. Usually, the time
`for incubation steps will vary from about 0.5 min to 6
`hrs, more usually from about 5 min to l hr. Incubation
`temperatures will generall§r"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 will.nor-
`mally be the observation of electromagnetic radiation,
`particularly, ultraviolet and visible light, more particu-
`larly visible light, either absorption or emission. Desir-
`ably, where fluorescence is involved. the light emitted
`should have a wavelength in excess of 400 am, more
`desirably in excess of 45_0.nm, a_nd'p_referably in excess
`of 500 nm. Where absorption is involved, the absorption
`will normally be_ in the range of about 250 to 900 1'lI'I1,
`more usually from about 325 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 obser'ved signal for
`the standard assay media may then be plotted so as to
`relate concentration to. signal. Once a standard curve
`has been established, a signal may be directly related to
`the concentrat_ion.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
`5)/51~°-111 and th,6.li1_$__€-_
`mode, the times between
`readings will generally'vary=-from about 5 sec to 6 hrs.
`usually about 10 sec to l--hr.-"For the equilibrium mode,
`after a steady state -is ‘achieved, a single reading may be
`sufficient or two r_ead_i:r'|g's __over any convenient time
`interval may suffice.
`I.
`
`--DEFINITIONS
`
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`Analyte—the com'p'_ou'nd_' oi: composition to be mea-
`sured, which may be a ligand','a single orplurality of
`compounds which‘ share at least one common epitopic
`or determinant site,"-‘or a receptor.
`Specific binding pair—two different molecules.
`where one of the molecules has an area on the surface or
`In a cavity which specifically binds to.‘ a par_ticular spa-
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`65
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`4,287,300
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`6
`tial and polar organization of the other molecule. The
`members of the specific binding pair are-referred to as
`ligand and receptor (antiligand).
`Ligand—any organic compound for which a recep-
`tor naturally exists or can be prepared.
`Receptor (antiligand)-—any compound or composi-
`tion capable of recognizing a particular spatial and polar
`organization of a molecule i.e. determinant or epitopic
`site. Illustrative receptors include. naturally occurring
`receptors, e.g. thyroxine binding globulin, antibodies,
`Fab fragments, enzymes, lectins and the -lilte.
`Ligand Analog-—a modified ligand-which can com-
`pete with the analogous ligand for a receptor, the modi-
`ftcation providing means to join the ligand analog to
`another molecule. Depending upon the available func-
`tionalities on the ligand, the ligand analog may differ
`from the ligand by more than replacement of a hydro-
`gen with a bond which links the ligand analog to a hub
`or label.
`'
`
`Polyfligand-analog)—a plurality of ligand analogs
`joined together covalently, frequently to a hub nucleus.
`The hub nucleus is a polyfunctional material, normally
`polymeric, usually having a plurality of functional
`groups e.g. hydroxy, amino, mercapto, ethylenic, etc. as
`sites for linking. The hub nucleus may be water soluble
`or insoluble, usually water soluble, and will normally be
`at least about 10,000 molecular weight. usually at least
`about 35,000 molecular weight and may be 10 million or
`more molecular weight, usually under 600,000, more
`usually under 300,000 molecular weight. Illustrative
`hub nuclei include polysaccharides, polypeptides,
`in-
`cluding proteins, nucleic acids, ion exchange resins and
`the like. Water insoluble hub nuclei can include glasses,
`addition and condensation polymers, both cross-linked
`and non—cross-linked, naturally occurring particles, or
`the like, either having a plurality of functionalities or
`capable of functioualization.
`_
`Signal Producing System-—the signal producing sys-
`tem may have one or more components, there being one-
`component conjugated to'a specific binding pair mem-
`ber. The signal producing system produces a measur-
`able signal which is detectible by external means, nor-
`mally the measurement of electromagnetic radiation. In
`the subject invention, the level of observed signal pro-
`duced by the signal producing system will be suscepti-
`ble to the proximity of the charged member to the la-
`beled member. For the most part, the signal producing
`system will involve enzymes and chromophores, where
`chromophores include dyes which absorb light in the
`ultraviolet or visible region, phosphors, lluorescers and
`chemiluminescers. The enzymes will normally involve
`either the formation or destruction of a substance which
`absorbs light in the ultraviolet or visibleregion or the
`direct or indirect production of emitted light by fluores-
`cence or chemiluminescence.
`Label—any molecule conjugated to another mole-
`cule, particularly, the former being a member of the
`signal producing system. In the subject invention, the
`labels will be the signal producing system component
`bound to a member of the specific binding pair and will
`be referred to as the signal label.‘
`Labeled Ligand—the conjugate of the ligand member
`(ligand analog) of the specific binding pair with a mem-
`ber of the signal producing system, either "covalently or
`non-covalently bound, when covalently bound, either
`bound by a bond, linking group or hub nucleus. The
`labeled ligand may have one or more ligands or one or
`
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`4,28'i',300
`
`7
`more labels or a plurality of both, the latter being re-
`ferred to as polyfligand analog)-polylabel.
`Labeled Receptor—the conjugate of receptor with a
`member of the signal producing system, where the two
`are bound either covalently or noncovalently, usually
`covalently by a linking group, where there may be one
`or more receptors bound to the label or one or more‘
`labels bound to the receptor.
`Charged Mernber—a soluble member of the specific
`binding pair, usually the receptor, more usually anti-
`body, being polyionic by being either covalently or
`non-covalently substituted with a plurality of function-
`alities of the same charge, either negative or positive, so
`as to create a relatively high localized density of a par-
`ticular charge, which may be a single member or a
`plurality of members linked together.
`MATERIALS
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`The components employed in the subject assay will
`be the analyte, which is a member of the specific bind-
`ing pair, the signal labeled member, any additional com-
`ponents of the signal producing system, the charged
`member, and as appropriate receptor or polyfligand
`analog).
`Analyte
`The ligand analytes of this invention are character-
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poly(atnino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations of assemblages include bacteria,
`viruses, chromosomes, genes, mitochondria, nuclei, cell
`membranes, and the like.
`For the most part, the polyepitopic ligand analytes
`employed in the subject invention will have a molecular
`weight of at least about 5,000, more usually at least
`about 10,080. In the poly(amino acid) category, the
`poly(amino acids) of interest will generally be from
`about 5,000 to 5,000,000 molecular weight, more usu-
`ally from about 20,000 to 1,000,000 molecular weight;
`among the homtones of interest, the molecular weights
`will usually range from about 5.000 to 60,000 molecular
`weight.
`'
`The wide variety ofproteins may be considered as to
`the family of proteins having similar structural features, 45
`proteins having particular biological functions, proteins
`related to specific microorganisms, particularly disease
`ca%hegl'o‘llii::v{ir(irg:rli:iSt:l1a:si:cof roteins related b st
`mm
`5
`P
`5’ “"‘
`prmamjnes
`histones
`
`so
`
`'
`
`a1-Lipoprotein
`at]-Acid glycoprotein
`ct:-Antitrypsin
`at-Glycoprotein
`Transcortin
`4.6S-Postalbumin
`Tryptophan-poor
`a1—glycoprotein
`cnx-Glycoprotein
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin
`(Go 1-!)
`(Go 2-1)
`(Gc 2-2)
`Haptoglobin
`(Hp 1-1)
`(Hp 2-I)
`(Hp 2-2)
`Ceruloplasmin
`Cholinesterase
`ct;-Lipoprotein(s)
`Myoglobin
`'
`C-Reactive Protein
`ct;-Macroglobulin
`ct;-HS-glycoprotein
`Zn-ag-glycoprotein
`oz-Neuramino-glycoprotein
`Erythropoietin
`:3-lipoprotein
`' Transferrin
`I-lemopexin
`Fibrinogen
`Plasminogen
`B;-glycoprotein I
`,0;-glycoprotein ll
`Itmnunoglobulin G
`(IgG) or 7G-globulin
`Mo]. formula:
`'?2K2 Or 7212
`Immunoglobulin A (IgA)
`O1, ,yA_g]°bufin
`_
`ME: £°?,n:1l_a{'a M),
`Immfmzo lobuliil M
`a M)gm_ M_ lobulin
`M013formal:
`3
`(I-':2K2)5 01' '0-t13'l2)5
`Immunoglobulin D(lgD)
`or -yD-Globulm (-yD)
`
`
`albllmifls
`globulins
`scleroproteins
`nhosphopmceins
`mucoproteins
`cl'l1'OII10pl’0tBlI'lS
`ljpoprogeim
`nucleoproteins
`glycoprateins
`proteoglycans
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`I
`- h
`-
`_
`A number of proteins found in the human plasma are 65 P mpm-mm pl-mam ormones mcluda
`important clinically and include:
`eggiihafiiigrfirggglgrmones
`Prealbumin
`.
`(parathromone)
`Album!“
`Thyrocalcitonin
`
`55
`
`tit)
`
`vu
`VIII
`
`‘X
`
`X
`
`XI
`XII
`xlt
`
`'
`
`Proconyertin
`Antihemophilic globulin
`(M16)
`°,’:,";:"::;:::,:*-.,,,,
`:,,,,,P,,,,,,,, (“-5,
`Stuart-Prower factor.
`autoprothrombin lIl_
`:;::‘eD":'d;:"("l:.'{.‘;l’)la"'“
`Hagernnnn Factor
`Fibrin-stabilizing factor
`
`'
`
`

`
`4,287,300
`
`10
`-continued
`Species of Microorganisms
`Remosensitin Found in
`Safmonefla derby
`Satmmetfa "pr:ttomm
`Skfgetta dysemertae
`Shtlgetta flexneri
`Sktgetta sot-met"
`
`Poiysaccharide
`
`Crude. Poly-
`saccharine
`Crude extract
`Pclysaccharide
`Crude extract
`
`Rickettsiae
`Cn‘rtd't'a’a aibfmcs
`Entamoeba »‘tt'.rt‘ot'ytt'ra
`
`_
`
`Insulin
`Glucagon
`Reiaxin
`Erythropoietin
`Meianotropin
`(melanocytestimulating hormone; intermedin)
`Somatotropin
`(growth hormone)
`Corticotropin
`(adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone
`(interstitial cell-stimulating hormone}
`Luteomammotropic hormone
`(luteotropin, prolactin)
`Gonadotropin
`(chorionic gonadotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`'
`Vasopressin
`Releasing factors (RP)
`CRF, LRF, TRF, Somatotropin-RF,
`GRF, PSI-I-RF, PIF. MIF
`Other polymeric materials of interest are mucopoiy-
`saccharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`5
`
`10
`
`15
`
`30
`
`25
`
`30
`
`35.
`
`The microorganisms which are assayed may be in-
`tact, lysed, ground or otherwise fragmented, and the
`resulting composition or portion, eg by extraction,
`assayed. Microorganisms of interest include:
`Corynebacteria
`Cmynebacterfum dtptheriae
`Pneumococci
`. Dtplacoccus pnettmontae
`Streptococci
`Streptococcus pyogenes
`Streptococcus sativarus
`Staphylococci
`_ Staphylococcus tmrerts
`Staphylococcus alb its
`Neisseriae
`Neisserta meningtttdxls
`Netsserta gonorrheae
`Enterobacteriaciae
`
`Escherichia an-it
`aierahaeteraemgenes
`Kfebsiettc pneumonia:
`Salmonella typhasa
`Sefmanetta ctroieraerttts
`Salmonella rygahimurium
`Shtjgeifa dysenreriae
`Shaigeffa sctimttzti
`Siu'get't'a ambfnotarda
`.S'.’n'gEHO flexnen‘
`Shxgeffa boydit
`Shtgetta sat-met‘
`
`
`}
`-
`)
`
`The coiiforrn
`bacteria
`
`The Saimorieilae
`
`The Shigeiiae
`
`Other enteric bacilli
`
`P.-crews vutgaris
`'
`Proteus‘ mfrahitfs
`Proteus species
`
`P.-arm: morgam‘
`
`Pseudamonas aemginara
`Alcattgenes faecalts
`Vibrfo cholerae
`Hemophilus-Bordetella group
`Hemopffilus tnfluenzae.
`H. dztcreyt
`H. hemophilus
`H. aegypticus
`H. paratrzfluenzae
`Barderelta pertussis
`Pasteurellae
`Pastettretla pests":
`Pasreztretla tttlareusts
`Brucellae
`Bmceffa melftensfs
`Bmcefia abcrttts
`Bmcetta saris
`Aerobic Spore-forming Bacilli
`Bactfltts anthrccts
`
`-
`
`
`
`Species of Microorganism
`Streptococcus pyogener
`Dfpfamocus pneumo.-rice
`Nefmy-ta meningttfdir
`Neisserta ganorniteae
`Carynebicrvedum dtluhtherthe
`Acrtnobactttus matter‘:
`Acrfnohaciiius Wa'tr'I‘errton‘
`Fmnciseffa mfarensis
`
`Pcgteureffa pertts
`Pasmrretta pestir
`Pt.-steuretta muttacida
`Bntcetta abortus
`
`Hnemophifu: influenzae
`Haemopfrifus pertussis
`Treponema miteri
`Veillonella
`
`Erysipelothrix
`Listeria monotjrtogertes
`Chrornohacterium
`Mycabacterium tnbemutosis
`
`Ktebsfefta uerogener
`Kirbsiefta ctoacae
`Satmoneffa typiiosa
`
`Safmonefla .ryptn'-murt'um.'
`
`Hernoseltsitirl Found in
`Folysaccharide
`Polysaccharide
`Polysuccharide
`Polysaccharide
`Polysaccharide
`Crude extract
`
`Lipopolysam
`charide
`Potysacchsride
`
`Polysaccharide
`Capsular antigen
`Crude extract
`
`Polysaocharide
`Crude
`Poiysaocharide
`LipopoIysac-
`charide
`Polysaccharide
`Poiyseccharide
`LipopoIysac-
`charide
`Saline extract of
`90% phenol
`extracted
`myoobacterie
`and polysacw
`charide
`fraction of
`certs and
`turberculirl
`Polysaccharide
`Poly-saccheride
`Lipopolysac-
`charide,
`Pclysaccharide
`Polysaccharide
`
`40
`
`45
`
`so
`
`55
`
`- 60
`
`65
`
`

`
`11
`
`_
`
`Bacillus 3ubu'!i's
`BGCIWUS megaterium
`Bacillus cereus
`Anaerobic Spore-forming Bacilli
`C!o:rrt'a'ium botulfnum
`Closrridium re.-‘um’
`Clostridium perfringens
`Closrridfum novyi
`Clostridium septicum
`Closrridfum Irfsrolytfcum
`Closirfdium rertfum
`Closrrfdfum bifermemans
`Clostridium spomgenes
`Mycobacteria
`Mycobacrerfum tuberculosis hominis
`Mycobacierium bovis
`Mycobacrerium avfum
`Mycobacrerium Ieprae
`Mycabacrerium pararuberculosfs
`Actinomycetes (fungusdike bacteria)
`Actfuomyces israei'i:'
`Acrinomyces bow’:
`Actinomyces naeslundif
`Nocardfa asrerofdes
`Nocaniia bmsfiiensis
`
`The Spirochetes
`Trepanema palladium.‘ Spirillum minus
`Trepouema perren HE.‘ Srreptobacillus monfhfformis
`Trepouema camteum
`'
`Borrelfa recumemis
`Leptaspira :'c:erohemorr}:agi'ue
`Leptaspira cunfcola
`Mycoplasrnas
`Mycopfasma pneumomhe
`Other pathogens
`Listeria monacyrogenes
`Erysijpelothria: rhusiopenfiae
`Srreprabucillus monfltjfbrmfs
`Douvonia gmnulomufis
`Barronella bacfllfformtic
`Rickettsiae (bacteria-like parasites)
`Rfckettsfa pro wazekff
`Rickerrsie mooseri
`Rfckettsia r1'cketrsi1'
`Rickertsiu conori
`Rfckeusia australis
`Rfckertsfa sibirfcus
`Rickettsia alum‘
`Ri'cketrst’a tsursugamushi
`R:'cker£sfa bumem‘
`Rfckertsia quintuna
`Chlamydia (unelassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`Fungi
`Clj’p£0€0CCl£$ neoformaus
`Blastomyces dermarfdis
`Hxlfloplasma capsuiorum
`Cbceidfaides .-‘mm.-'rr's
`Paracoecfdfofdes brasiiiensis
`Candida albicans
`Aspergillus fumigcuus
`Mucor carym bifer (Absfdia corymbifera)
`
`
`_
`
`Rhizopu