United States Patent
`
`[191
`
`[11]
`
`4,193,983
`
`Ullman et al.
`[45] Mar. 18, 1980
`
`
`
`use in immunoassays, as well as irnrnunoassays employ-
`ing such novel compositions. The compositions com-
`prise discrete charged colloidal particles comprised of
`small molecules which particles are capable of retaining
`their discrete character in an aqueous medium and com-
`posed of aggregates of lipophilic and/or amphiphjlic
`organic molecules to which are bound non-covalently a —
`label capable of producing a detectible signal and a
`ligand or an analog of the ligand capable of competing
`with a ligand for a ligand receptor. The discrete colloi-
`dal particle serves as a hub or nucleus for retaining the
`ligand or its analog and the label within a limited locus.
`The compositions are prepared by individually cova-
`lently bonding the ligand and the label. when not natu-
`rally lipophilic, to a lipophilic (includes amphiphilic)
`compound, normally a phospholipid. Depending upon
`the nature of the particle, the amphiphilic conjugated
`ligand and label are combined with the particle or alter-
`natively may be combined with the compounds em-
`ployed for preparing the particle under particle forming
`conditions. Particles are then obtained having the ana-
`log of the ligand and the label bound to the particle.
`The compositions find use in immunoassays where an
`interaction between the label and receptor provides a
`means for modulating a detectible signal. The interac-
`tion can be as a result of quenching or modification of
`fluorescence, where the label is a lluoresoer, steric inhi-
`bition of the approach of a signal modifier to the label,
`such as a label receptor or with an enzyme label, an
`antienzyme or enzyme inhibitor, the inhibition of cleav-
`age of an enzyme labile bond or the cooperative interac-
`tion of two labels, such as two enzymes, where the
`product of one enzyme is a substrate of another enzyme.
`
`[54] LABELED LIPOSOME PARTICLE
`COMPOSITIONS AND IIVIMUNOASSAYS
`THEREWITH
`
`[T5]
`
`Inventors: Edwin F. Ullmau, Atherton; John M.
`Brinkley, Oakland, both of Calif.
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] Appl. No.: 906,514
`
`[22] Filed:
`
`May 16, ms
`
`[51]
`
`im. C13 ................... __ GIJIN 31/no; com 33/15;
`GOIN 23/on; A6lK 37/00
`[521 US. Cl. .................................. .. 424/12; 23/230 B;
`2&0/112 R; 260/I12 B; 424/1; 424/3; 424/7;
`424/s;424/11; 424/13; 424/33; 435/7; 435/133
`[53] Field of Search ....................... .. 424/1, 3, 7, 3, 11,
`424/12, 13, 33; 250/112 R, 112 B; 23/230 B;
`195/103.5 A, 103.5 L. 53
`
`[56]
`
`I
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`Beaumont ........................ .. 424/13 X
`11/ 1974
`Forgione
`424/ll X
`5/1975
`
`6/i975 McConnell
`424/13 X
`4/1976
`Forgione
`424/13 X
`l2/1976 Ullman
`...... 424/8
`3/19'.-'8 Maier
`424/8 X
`
`
`
`3,849,546
`3,882,224
`3,337,698
`3.949.065
`3,996,345
`4,l0-11.029
`
`OTHER PUBLICATIONS
`
`Sato, Japan J. Exp. Med., vol. 46, 1976. pp. 213-221.
`Schwenk, The J. of Immunology, vol. 120, No. 5, May
`1973, pp. 1612-1615.
`
`Primary Exom:'ner—Anna P. Fagelson
`Attorney. Agent. or F:'rm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`The subject invention concerns novel compositions for
`
`17 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech '
`IPR2016-00710
`Genentech Exhibit 2059
`_Genentech Exhibit 2059
`
`IPR2016-00710
`
`

`
`1
`
`4,193,983
`
`2
`
`LABELED LIPOSOME PARTICLE
`COMPOSITIONS AND IMMUNOASSAYS
`TI-IEREWITH
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`Protein binding assays have become of increasing
`importance in the diagnosing of diseased states,
`the
`monitoring of the administration of drugs, and the de-
`termination of trace amounts of organic compounds
`unrelated to human health. Protein binding assays are
`particularly applicable in the determination of specific
`compounds which are present at concentrations of
`10-5 M or less, particularly where they are present in a
`mixture of other compounds having similar properties.
`Protein binding assays depend upon the ability of
`labeling an analyte, where the label provides a detecti-
`ble signal, and where binding of the receptor to the
`labeled analyte permits discrimination between bound
`and unbound label. The presence of the receptor can
`either allow for a mechanical separation of bound and
`unbound labeled analyte or can affect the label in such
`a way as to modulate the detectible signal. The former
`situation is normally referred to as heterogeneous and
`the latter as homogeneous, in that the latter technique
`avoids a separation step.
`there are a
`In developing protein binding assays,
`number of considerations. Considerations related to the
`choice of label include the sensitivity it provides, syn-
`thetic problems, stability, its sensitivity to chang in
`environment and the like. Other considerations are the
`purity required for the analyte and/or analyte receptor
`in preparing the reagents and in the assay. Additional
`considerations are the etlect of varying analyte on the
`synthetic procedures, the properties of the label, and the
`sensitivity and accuracy of the assay. By having an
`assay technique which is generally applicable to a wide
`variety of analytes and is not significantly affected by
`variation in analyte, the preparation of reagents and the
`performance of the assay can be readily adapted to new
`and varying arialytes.
`2. Description of the Prior Art
`Badley. “Fluorescent Probing of Dynamic and Mo-
`lecular Organization of Biological Membranes,” Mod-
`em Fluorescence Spectroscopy, Vol. 2 Ed. E. L.
`Wehry, Plenum Press, N.Y. 1976 and Kanaoka, Angew.
`Chem. Int. Ed. Engl. 16, 137 (197?) discuss fluorescent
`probes in biological systems. Wu et al, Biochemistry, I6,
`3936 (1977), Harris, Chemistry and Physics of Lipids 19,
`243 (1917) Smolarsky et al, J. of Imm. Meth., 15, 255
`(1977) and Waggoner and Stryer, Proc. Nat. Acad. Sci.
`6?, 579 (l9'l'0) describe the use of fluorescent probes
`with biological membranes. Llemura, et al, Biochemis-
`try IS, 1572 (l9'l4), Alving and Richards, Immuno-
`chemistry M». 373 (1977), Geiger and Smolarslty, J.
`Imm. Meth., 17, 7 (I977), Inoue and Nojima, Chem.
`Pharm. Bull. l6, ‘I6 (1963) and Tamamura, et al, Japan J.
`Exp. Metd. 41, 31 (191-'1) describe antibody phospho-
`lipid interactions.
`U.S. Pat. Nos. 3,850,578 and 3,88'l,698, and the refer-
`ences cited therein, teach the use of liposomes in assays
`where complement mediated lysis of the liposome as a
`result of antibody binding to an antigen bound to the
`liposome results in release of stable free radicals con-
`tained in the liposome.
`
`10
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`65
`
`SUMMARY OF THE INVENTION
`
`Novel compositions are provided which find use in
`homogeneous protein binding assays, which composi-
`tions are discrete colloidal particles substituted with at
`least one label and at least one ligand. The particles are
`formed with small
`lipophilic (includes amphiphilic)
`molecules and the label and ligand are joined to the
`particle nomcovalently, but in a substantially fixed av-
`erage spatial relationship. The particles may be vesicles,
`oil droplets or the like, having an ordered surface layer _
`bearing a net electrostatic charge.
`The particles are fonned by combining in an aqueous
`medium the lipophilic particle forming molecules, ei-
`ther individually or as a particle, any auxiliary compo-
`nents, and the lipophilic conjugates, including the li-
`gand conjugates, as the naturally occurring ligand or as
`a conjugate with a lipophilic compound, and the label
`conjugate.
`The particles find use in protein binding assays where
`the presence of receptor bound to ligand adjacent the
`label can be used to modulate the signal provided by the
`label. Illustrative of such situations are assays where the
`presence of receptor inhibits the approach of another
`molecule to the label or the receptor is conjugated with
`a molecule which interacts with the label to modulate
`the signal.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Novel compositions are provided which find use in
`the performance of protein binding assays. The compo-
`sitions ploy a colloidal particle as hub or nucleus for
`controlling a spatial relationship between a label and a
`ligand, where the label and ligand are non-covalently
`bound to the central nucleus. The central nucleus is _a
`stable colloidal particle that consists ofa discrete phase
`different from the aqueous solvent and is comprised of
`organic molecules, which are at least in part lipophilic.
`The particles are comprised of a hydrophilic normally
`electrostatically charged surface layer surrounding a
`hydrophobic layer or core. By employing ligands and
`labels which are naturally lipophilic or made so by
`conjugation with lipophilic compounds, both the label
`and the ligand become non-covalently bound to the
`discrete particle. By providing for appropriate amounts
`of the label and ligand, a substantial proportion of the
`label and ligand will be in relatively close spatial prox-
`imity on the surface of the particle.
`In protein binding assays, the ligand of interest is
`normally labeled and the assay allows for discrimination
`between the amount of labeled ligand which is bound to
`receptor and the amount of labeled ligand which is
`unbound. In heterogeneous assays, the discrimination is
`a result of physical separation. In homogeneous assays,
`the discrimination is a result of modulation of the signal
`provided by the label. By virtue of having the label and
`ligand in relatively close proximity on the surface of the
`particle, the proximity of the label and the receptor
`bound to the ligand adjacent the label can be used in
`accordance with the prior art techniques to modulate
`the signal from the label.
`By employing the subject invention, numerous ad-
`vantages ensue. One advantage is that one has a simple
`hub nucleus to which a wide variety of labels and li-
`gands may be easily bound in a relatively uniform man-
`ner and at a variety of ratios. Secondly, with large li-
`gands, one can readily bond the relatively small lipo-
`
`
`
`

`
`4,193,983
`
`3
`philic compound to-the large ligand. Thirdly, one does
`not directly bond the label to the ligand. This can be
`very important where the ligand is not readily obtain-
`able in pure form. Where the ligand is primarily obtain-
`able as a complex mixture e.g. naturally occurring prod- S
`ucts, when labeling the ligand, one will also label the
`contaminant. The labeled contaminants will therefore
`provide a background which can seriously interfere
`with the sensitivity of the assay. In the subject invention
`since the ligand is indirectly labeled, there will be no 10
`labeled contaminants which interfere with the assay.
`Also, by preparing a single ligand lipophilic conjugate,
`one can employ this compound with a wide variety of
`labels. Similarly, one can prepare labels and employ
`them with a wide variety of ligands. In addition, for 15
`those ligands which require the presence of a liposome,
`the subject method inherently employs the necessary
`environment.
`In order to enhance the understanding of the subject
`invention, a number of the terms which find repetitive
`usage will be defined.
`
`20
`
`4
`colloidal particle. Receptor e.g. antibodies and Fab
`fragments, which recognize or specifically bind to such
`lipophilic compound, including the haptenic or anti-
`genic portion thereof. may be conjugated with label.
`The labeled receptor may then bind to the lipophilic
`compound in the colloidal particle indirectly labeling
`the particle. In this manner, the number of labels bound
`to the particle may be greatly enhanced.
`Lipophile—For the most part are hydrocarbons or
`lipids. Lipids are amphipathic elongated molecules,
`much smaller in weight and size than polymers, but
`sufficiently large to have two distinct regions of greatly
`differing polarity. One is the polar (hydrophilic) region
`at one end of the molecule, which favors the interaction
`with water, and the other is the apolar (hydrophobic)
`region consisting of hydrocarbon chains. The am-
`phipathic nature of the molecule is responsible for the
`molecular association phenomena characteristic of lip-
`ids in both crystals and lipid-water systems. See, Water,
`Volume 4, Aqueous Solutions of Amphiphiles and Mac-
`romolecules. Chapter 4, Lipids, page 213, Plenum Press,
`New York. See also, Tute, Chem. Ind. (London) 1975
`(3). 100-5.
`Amphiphile—see above definition of lipids. Amphi-
`philes may be neutral or charged: when negatively
`charged, usually having as the anionic group, phos-
`phate, carboxylate, sulfonate or sulfate, particularly
`phosphate; when positively charged, usually having as
`the cationic group, ammonium e,g. pyridiniurn, tetra]-
`lcyl ammonium, etc., phosphonium, or sulfonium, par-
`ticularly ammonium.
`Colloidal Particle—tl1e colloidal particle is a small
`discrete particle capable of maintaining its integrity in
`an aqueous environment and comprised of lipophilic,
`usually amphiphilic, molecules. These particles may be
`considered as members of the class of association col-
`loids, which are thermodynamically stable systems in
`which the dispersed phase consists of aggregates of
`molecules (or ions) of relatively small size and mass. See
`Remington's Pharmaceutical Sciences, 15th ed., Mack
`Publishing Co., Eastin, PA, l975, page 300.
`For the most part the colloidal particles will have
`regular shapes such as spheres, cylinders or plates. With
`amphiphilic molecules, the particle is composed of a
`hydrophilic, normally charged outer layer and a hydro-
`phobic interior layer or core. The layer may be mono-
`or polylarnellar and there may be an aqueous phase
`between layers. For lipophilic molecules, there will
`nonnally be a layer of amphiphilic molecules, usually
`charged, on the surface of the particle.
`For the most part, the molecules will have molecular
`weights of at least about l50 and not greater than about
`2,500, usually not greater than 1,500. The molecules
`will have at least twelve aliphatic carbon atoms (in-
`cludes alicyclic), usually at least 18, and more usually at
`least 28 and usually not more than about 175 carbon
`atoms. The amphiphilic compounds will normally have
`an aliphatic chain of at least l0 carbon atoms, usually at
`least 12 carbon atoms and usually not more than about
`36 carbon atoms, and will have from 0 to 3 sites of
`aliphatic unsaturation, usually ethylenic. The particles
`generally have a size of from about 2x10-6 to 103;).-",
`more usually from about 6 X l0"‘5to 133:} and frequently
`from about Ex 10-5 to l0'3p.3. With liposomes, the
`membrane thickness will be about 50 to 100 A.
`In referring to particles prepared from lipophilic
`compounds other than amphiphilic compounds, these
`particles will be referred to as droplets. In referring to
`
`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 a receptor, the modi-
`fication providing means to join a modified ligand to
`another molecule. The ligand analog will differ from
`the ligand by more than replacement of a hydrogen
`with a bond which serves to link the modified ligand to
`another molecule.
`Receptor-—any compound or composition capable of
`recognizing a particular spatial and polar organization
`of a molecule i.e. epitopic site. Illustrative receptors
`include naturally occurring receptors, antibodies, en-
`zymes, Fab fragments. lectins, and the like. For any
`specific ligand. the receptor will be referred to as antih-
`gand. For any specific label, the receptor will be re-
`ferred to as antilabel. The receptor-antiligand- or antila-
`bel and its homologous ligand or label form a specific
`binding pair.
`_
`l..abel—a compound which is either directly or indi-
`rectly involved with the production of a detectible
`signal and is bonded directly to one or more lipophilic
`molecules.
`Illustrative labels include chromogens, e.g. fluoresc-
`ers and chernilurninescers, catalysts, both enzymatic
`and non-enzymatic, molecules having an enzymatically
`labile bond which upon enzymatic cleavage provides a
`compound which can be detected, either directly or
`indirectly, and the like.
`Conjugate—a conjugate intends the covalent bond-
`ing together of two molecules which serve different
`functions in the subject invention. The label-lipophile
`conjugate joins the label to one or more lipophiles. The
`ligand- or ligand analog-lipophile conjugate, hereinafter
`referred to as ligand~|ipophile conjugate, joins the li-
`gand or modified ligand to one or more lipophiles.
`' In some instances the label may be indirectly bound
`to the colloid particle. This can be achieved by employ-
`ing a lipophilic compound, which includes conjugates
`of lipophilic groups with haptens and antigens, in the
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`4,193,983
`
`5
`particles prepared from amphiphilic compounds, these
`particles will be referred to as vesicles or liposomes.
`Colloidal particle reagent-——the colloidal particle rea-
`gent is a colloidal particle which includes the label
`conjugate and the ligand or ligand conjugate (includes
`ligand analog conjugate) as part of the outer molecular
`layer, so that the ligand and label are confined to the
`particle surface in closer proximity than if randomly"
`distributed in solution. The two conjugates or the label
`conjugate and amphiphatic ligand are bound non-cova-
`lently to the particle by virtue of the presence of the
`lipophilic group.
`Kit—a combination of reagents, usually formulated
`with ancillary reagents, such as buffer, salts, stabilizers,
`etc., where the reagents are premeasurcd so as to at least
`substantially optimize the assay sensitivity. Where the
`analyte is antiligand.
`the kit will comprise colloidal
`particle reagent and, as appropriate antilabel, modified
`or unmodified or modified antiligand. Where the ana-
`lyte is ligand, the kit will comprise colloidal particle
`reagent, antiligand and as appropriate antilabel, with
`the receptors being modified or unmodified.
`Modified receptor—-receptor to which is conjugated
`a compound which interacts with the label to modulate
`the detectible signal. Illustrative compounds for recep-
`tor modification include chromogens which act as
`quenchers or energy receptors, enzymes or non-
`enzymatic catalysts.
`
`Assay
`
`6
`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 assay. The temperatures
`for the determination will generally range from about
`1{J°—S0° C., more usually from about 15°—4-0° C.
`The concentration of analyte which may be assayed
`will generally vary from about l0"4 to 10-15 M, more
`usually from about 104’ to 10*” 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 concentrations of the other
`reagents.
`While the concentrations of the various reagents will
`generally be determined by the 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.
`
`The total binding sites of the member of the specific
`binding pair, which is reciprocal to the analyte, will be
`not less than about 0.1 times the minimum concentration
`of interest based on binding sites of the analyte and not
`more than about 1,000 times the maximum concentra-
`tion of interest based on analyte binding sites, usually
`about 0.l—l00 times, more usually about 0.l~l0 times
`the maximum concentration of interest. Where receptor
`is the analyte and a modified receptor is employed as a
`reagent, the amount of modified receptor employed
`based on binding sites will be not less than about 0.01
`times the minimum concentration of interest and usually
`not more than l00 times the maximum concentration of
`
`interest of the receptor. The auxiliary reagents em-
`ployed with the label will be present
`in suflicient
`amount so as not to be rate controlling or interfere with
`the amount of signal as a function of analyte concentra-
`tion. The amount will vary depending upon the nature
`of the label and the auxiliary reagents.
`The order of addition of the various reagents may
`vary widely, depending upon the particular label, the
`nature of the analyte, and the relative concentrations of
`the analyte and reagents.
`Also affecting the order of addition is whether an
`equilibrium mode or rate mode is employed for the
`determination.
`In determining a particular order of addition, there
`will be certain basic considerations. Usually, the rate of
`association of receptor and ligand is much greater than
`the rate of dissociation, although this will be a factor not
`only of the binding constant, but also of the relative
`concentrations of the members of the specific binding
`pair. Another consideration is that one wishes to mini-
`mize the background. so that normally where a rate is
`measured, production of the signal will not be initiated
`prior to all of the components being present. Finally,
`the order of addition must not interfere with the ability
`of the analyte to affect the modulation of the detectible
`signal.
`Where ligand is the analyte, the unknown sample
`may be combined with the ligand receptor and the
`resulting mixture combined with the colloidal particle
`reagent and any auxiliary reagents. Where receptor is
`the analyte, and a modified receptor is not employed as
`a reagent, the receptor may be simply added to the
`colloidal particle reagent, followed by the addition of
`
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`The subject assay is carried out in an aqueous zone at
`a moderate pH, generally close to optimum assay sensi-
`tivity, normally without separation of the assay compo-
`nents or products. The assay zone for the determination
`of analyte is prepared by employing -an appropriate
`aqueous solution, normally buffered, the unknown sam-
`ple, which may have been subject to prior treatment.
`the colloidal particle reagent, any auxiliary materials
`associated with production of the detectible signal, as
`well as when appropriate, modified or unmodified
`receptor(s).
`The presence of ligand or antiligaud as the analyte in
`the unknown will affect the degree to which antiligand
`will bind to the colloidal particle reagent and influence
`the production of the detectible signal. In some in-
`stances, antiligand will be modified by having mole-
`cules bonded to it which interact with the label, and it -
`is this interaction which modulates the detectible signal.
`In other instances, the proximity of the antiligand to the
`label without any modification of the antiligand will
`affect the detectible signal.
`In carrying out the assay an aqueous medium will
`nonnally be employed. Other polar solvents may also
`be employed, usually oxygenated organic solvents of
`from 1-6, more usually from 1-4 carbon atoms. includ-
`ing alcohols, ethers and the like. Usually these cosol-
`vents will be present in less than about 40 weight per-
`cent, more usually in less than about 20 weight percent.
`The pH for the medium will usually be in the range of
`about 4-1 1, more usually in the range of about 5-10, and 60
`preferably in the range of about 6.5-9.5. The pH is
`chosen so as to maintain a significant level of specific
`binding by the receptor while optimizing signal produc-
`ing proficiency. 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-
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`
`7
`auxiliary reagents. Where modified receptor is em-
`ployed,
`the unknown sample and modified receptor
`may be combined and then added to the liquid particle
`reagent, again followed by the addition of auxiliary
`reagents. Alternatively, all of the primary reagents may
`be added simultaneously, concomitantly with or prior
`to the addition of the auxiliary reagents. One or more
`incubation steps may be involved. which will normally
`involve periods of from 0.1 min. to 6 hrs., more usually
`from about 1 min. to 1 hr., usually from about 5 min. to
`30 min. Incubation temperatures will generally range
`from about 4° to 50° C., 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 or visible light, either absorption or
`emission, thermal, volumetric, electrochemically, and
`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.
`.
`The temperature at which the signal is observed will
`generally range from about 10°—50° C., more usually
`from about l5°—40° C.
`Standard assay media can be prepared which have
`known amounts of analyte. The observed signals with
`the standard assay media may then be graphed, so as to
`relate concentration to signal. Once a standard curve
`has been established, a signal may be directly related to
`the conentration of the analyte.
`After all the materials have been combined, the read-
`ings may be made as a rate mode or equilibrium mode.
`One can normally begin the reading immediately or
`within about 2 sec. of having completed the additions of
`the various materials and in a rate mode can take a
`second reading within 0.2 min, usually 0.5 min. or
`longer, normally not exceeding an hour, preferably not
`exceeding 5 min. In an equilibrium mode, one must wait
`until the mixture has stabilized to a fairly constant read-
`ing which can be as little as 0.5 min. and as long as 1 hr.
`or longer.
`
`Materials
`
`The materials involved in the assay are the analyte
`(ligand or antiligand),
`the colloidal particle reagent,
`which includes the label conjugate and ligand analog
`conjugate or ligand conjugate, ligand receptor or modi-
`fied receptor as appropriate, and auxiliary reagents as
`appropriate.
`
`Analyte
`
`The ligand analytes of this invention are character-
`ized by being monoepitopic ' or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poly(amino acids) is. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`For the most part, the polyepitopic ligand analytes
`employed in the subject invention will have a molecular
`weight of at least about 5,000, more usually at least
`about 10,000.
`In the poly(amino acid) category, the
`poly(amino acids) of interest will generally be from
`about 5,000 to 1,000,000 molecular weight, more usu-
`ally from about 20,000 to 600,000 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
`
`4,193,983
`
`related to specific microorganisms, particularly disease
`causing microorganisms, etc.
`The following are classes of proteins related by struc-
`ture:
`protamines
`histones
`albumins
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropimprolactin, insu-
`lin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`ct;-Lipoprotein
`at-Acid glycoprotein
`ct1—Antitrypsin _
`at-Glycoprotein
`Transcortin
`4.6S-Postalbumin
`Tryptophan-poor
`ct: -glycoprotein
`ct1X-Glycoprotein
`Thyroxin-binding globulin
`Inter-ct-trypsin-inhibitor
`Gc—globulin
`(Gr: 1-1)
`(Gc 2-1)
`(Gc 2-2)
`Haptoglobin
`(HP 1-1)
`{Hp 2-1)
`(HP 2-2)
`Ceruloplasmin
`Cholinesterase
`oz-Lipoprotein(s)
`Myoglobin
`C-Reactive Protein
`ct;-Macroglobulin
`az-HS-glyooprotein
`Zn-ct;-glycoprotein
`dz-Neuramino-glyboprotein
`Erythropoietin
`B-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`B2-glycoprotein I
`B3-glycoprotein II
`Immunoglobulin G
`(IgG) or ‘yG-globulin
`Mol. formula:
`‘rare: or 7212
`lmmunoglobulin A (IgA) or 7A-globulin
`Mel. formula:
`lctztcz)" of (a27l2)"
`lmmunoglobulin M
`(lgM) or 'yM-globulin
`Mol. formula:
`{p.zIc2)5 01' (1-t2?l2)5
`
`10
`
`15
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`
`
`

`
`9
`
`4,193,983
`
`10
`
`mntinued
`
`I
`Brad E,
`Huminupilgoental iactogen
`Peptide Hormones from the Neurohgflphgsis
`omoc,-J,
`Vasoptwsin
`
`.
`
`our-', I-‘SH-RF, PIP, MI?
`
`Other polymeric materials of interest are mucopoIy—_
`sacchandes and polysacchandes.
`.
`‘
`Illustrative antigenic polysaccharides cleaved from
`microorganisms are as follows:
`
`,
`or 7E'GlObulm-
`
`Immunoglobulin D(lgD) or 7D-Globulin ('yD)
`Mo]. formula:
`(BZKZ) or (62.3%)
`Im-munoglobl-llm E
`Mol. formula:
`(-Ezxz} or (egltg)
`Fr=°‘<a"d1'“SW§**°i"s
`C?mP1"-'m‘°'“t f“°t°"""
`C 1
`Gig
`Ct},
`C15
`cg
`(33
`,3“;
`tn!)
`C'4
`C5
`I
`C 5
`C’?
`C'g
`.
`.
`.
`C 9
`Important blood clotting factors Include:
`
`BLOOD CLOTTING FACTORS
`
`International designation
`I
`11
`118
`III
`V and VI
`
`Name
`FlbTl“°Sm_
`P"°““'°""b"‘
`- Thrombin
`Tissue tltrombopltin
`Proaccelerin. accelerator
`5l°b““"‘
`"“°°°""=“i“
`-"\mih9m°Ph"l° Elubl-|Jil'l (AHG)
`Christmas factor,
`Flflmfl ‘l'll'°’mb0P'“-95“
`°°'|'|P°“°'“ {FTC}
`5“==fl-Pr°W *’=“=*°r-
`°“'°P’“‘h'°“‘bl“ 1"
`Plfm "=:='(rl';';_°“l>)'3-Stia
`1" '=°=d===
`Hagemann factor
`Fib'i""‘ah“i'zi"3 facmf
`.
`.
`Important protein hormones include:
`
`V"
`VH1
`IX
`
`3‘
`
`X1
`XII
`X!"
`
`.
`
`Peptide and Protein Hormones
`Parathyroid hormone
`(parathromonel
`Thyrncalcitonin
`Insulin
`Glucagon
`Renxin
`Erythroroietin
`Melanotropin .
`,
`(melano-cyte—stIn1tL|atIn,g
`hormone; intennodin)
`Somatotropin
`(growth hormone)
`Corticotropin
`(adrenocorticotropic hormone}
`Thyrotropiu
`Follicle-stimulating hormone
`Lute.-inizing hormone
`(interstitial cell-stimulating
`honrtone)
`Luteomammotropic horrnone
`(lutentropin. prolactin}
`Gonadoiropin
`(chorionic gonadotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`
`5
`
`10
`
`I5
`
`30
`
`25
`
`3o
`
`40
`
`45
`
`so
`
`55
`
`65
`
`5
`
`.
`of M.
`.
`lCl'0OI’gfl.l'!ISD1S
`peclefl
`Streptococcus pyogenes
`Dtploooccus pneurnontae
`Neisseria meningitidis
`Neisseria gonorrhoeae
`Coryrtebocteriurrl diplltlieriae
`Actinobacillus mallei.
`Actinobacillus whiternon‘
`p,,,,,c,5,,", ,u.,,,,,,,,,is
`
`Pastcurella pstis
`Pasteurella pestis
`l'1.lCC 3 3
`[IS
`l|;aste|a£eIl:-|nr:tltocida
`Haemophiltm influenza:
`Haemopitilus pertussis
`Treponema reiteri
`vemmlena
`Erysipelmhriu
`Listeria mooocytogenes
`Cltrontobactesiutn
`Myoobacterium tuberculosis
`
`35
`
`-
`
`d .
`Ol.ll'l
`ll'l
`
`H
`
`.I. F
` I Il'I
`Polysaocharicle
`Folysaochande
`Polysaocharide
`Polysaocharide
`Polysaocharide
`Crude extract
`
`[_iP0p°]y-saocharide
`Polysaccharide
`
`Polysacclaaride
`l'I.l
`8.1
`Eapsdtélfl anagsm
`Folysaccharide
`Crude
`Polysaccharide
`Lipopoiysacchafide
`poiysaachmide
`Polysaocharide
`Lipopolysaocharide
`Saline extract of 90%
`Phmol gmmcmd
`rnyoobacteria and poly-
`sacoharide fraction of
`cells and tuberculin
`Polysaccharide
`Polysaocharide
`Lipopotysacch ‘cl
`potyflmhafidem c
`Polysaccharide
`3
`pohmcharide
`Crude. polysaccharide
`Cfudg gnmgt
`Polysaocharide
`Crude extract
`
`.
`
`Klebsiella aerogenes
`Klebsiella cloacae
`Sal
`Ila
`la
`mom wp ma
`Salmonella typhi-rnurium.
`Salmonella derby
`Salmnnell
`I!
`shisena d:,;';,:::;"
`Shisella ftenuieri
`Shigella sonnei
`Rickgjtfiae
`Candida albicans
`Entamoeba histolytica
`Other materials of irueresl include allergens
`_
`,
`_
`,
`The monoepttoplc 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 includes morphine, codeine, heroin, dex-
`tromethorphan, their derivatives and metabolites; co-
`caine alkaloids, which includes cocaine and bettzoyl
`ecgonine,.their derivatives and metabolites; ergot alka-
`loids, which includes the diethylamide of lysergic acid;
`steroid alkaloids; iminazoyl alkaloids; quinazoline alka-
`loids; isoquinoline alkaloids; quinoline alkaloids; which
`includes quinine and quinidine; diterpene alkaloids,
`their derivatives and metabolites.
`The next group of drugs includes steroids, which
`includes
`the estrogens. gestogens, androgens,
`an-
`drenocortical steroids, bile acids, cardiotonic glycosides
`
`
`
`

`
`11
`and aglyoones, which includes digoxin and digoxigenin,
`saponins and sapogenins, their derivatives and metabo-
`lites. Also included are the steroid mimetic substances,
`such as diethyl stilbestrol.
`The next group of drugs is lactams having from 5 to 5
`6 annular members, which include the barbiturates, e.g.
`phenobarbital and secobarbital, diphenylh