[191
`United States Patent
`[11]
`4,235,869
`[45] Nov. 25, 1980
`Schwarzberg
`
`
`
`[54] ASSAY EMPLOYING A LABELED
`FAB-FRAGMENT LIGANT) COMPLEX
`
`['75]
`
`Inventor:
`
`Moshe Scltwarzllerg, Sunnyvale,
`Calif.
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] App}. No.: 906,388
`
`[22] Filed:
`
`May 16, 1978
`
`[51]
`
`Int. CL? ................... .. com 21/no; GOIN 31/00;
`GOIN 31/14; GOIN 33/16
`[52] U.S. Cl. .................................... .. 424/8; 23/230 B;
`250/302; 424/1; 424/12; 424/13; 435/?
`[531 Field of Search ......................... .. 424/1, 3, 12. 13;
`250/302; 23/230 13; 435/?
`
`[56]
`
`I
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3.935,o74
`3,996,345
`3,998,943
`4,104,029
`
`Ruberlstein ....................... 195/ 103.5
`1/l 976
`...... .. 424/12
`E2/1916
`
`4241-/12
`S2/1976
`S/1918 Maier ................................. .. 424/8 X
`
`OTHER PUBLICATIONS
`
`Ternynck et al., Ann. Immunol. (Inst. Pasteur). vol. 12?
`C 1976 pp. 197-208.
`Weir (Ed.), Handbook of Exptl. Immunology, Black-
`well Sci. Pub. London, 2nd ed., 1973, pp. 14.19-14.25.
`Carrico, et
`211., Anal. Biochem. vol. 72,
`1972 pp.
`271-232, 283-292.
`Forsurn, I. of Immuno. Methods, vol. 2, 19';'2 pp.
`183495.
`
`Primary Exam:'ner—Anna P. Fagelson
`Attorney. Agent. or Fi‘rm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Methods and compositions are provided for improved
`protein binding assays by preparing compositions hav-
`ing indirectly labeled ligands substantially free of label
`conjugated to materials other than the indirectly labeled
`ligand. The method for preparing the compositions
`involves employing amonovalent receptor to which the
`label is conjugated and combining the monovalent re-
`ceptor labeled conjugate to the ligand, either in pure or
`impure form. The mixture is then segregated according
`to molecular weight and the ligand conjugated to the
`labeled receptor isolated. This conjugate may then be
`directly used as a reagent in a protein binding assay,
`where the assay mixture is substantially free of label
`other than labeled receptor bound to ligand.
`The labels will be for the most part of relatively low
`molecular weight, while the receptor is preferably a
`Fab fragment. The ratio of receptor to ligand will be
`chosen so as to provide reasonable molecular weight
`distinctions between unbound ligand. unbound labeled
`receptor, ligand bound to labeled receptor, and other‘
`materials which may be in the mixture. Various tech-
`niques may be used for the separation.
`The assays are performed in accordance with known
`methods employing a second receptor composition,
`which may be labeled or unlabeled.
`
`10 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2056
`
`Genentech Exhibit 2056
`
`IPR2016-00710
`
`

`
`1.-
`
`4,235,869
`
`2
`
`ASSAY‘ EMPLOYING A LABELED
`FAB-FRAGMENT LIGAND comersx
`
`. BACKOROUNO _ OF THE INVENTION
`l. Field of the Invention
`
`The availability of molecules which are able to specif-
`ically bind toia particular-spatial and polar organization
`is the basis for a wide variety of techniques referred to
`as competitive protein binding assays. These techniques
`depend upon having a member of a specific binding pair
`conjugated with the label which is involved with the
`production of a detectible signal.
`Depending upon the nature of the label, various
`methods have evolved for distinguishing between an
`analyte which is bound to the corresponding member of
`the specific binding pair and analyte which is unbound.
`With the various techniques, either the receptor or the
`ligand is labeled. Particularly, where the receptor is
`labeled, the ‘receptor is normally one component of a
`complex mixture of analogous composition and molecu-
`lar weight. Fonegrample, antibodieswhich are isolated
`from serum will be present with globulins and other
`antibodies which are either non-specific or specific for a
`wide variety of ligands other than the ligand of interest.
`When labeling the receptor composition. both the re-
`ceptor of interest as well as the contaminating globulins
`will be labeled. The label
`involved with extraneous
`receptors or other materials will act as a background in
`the assay. interfering with the sensitivity of the assay.
`While affinity chromatography may be employed to
`enhance the purity of the receptorof interest, this tech-
`nique has many deficiencies. One deficiency is that the
`most strongly binding antibodies tend to be retained by
`the affinity chromatography column. Secondly, there
`are normally substantial losses of the antibodies of inter-
`est and substantial reduction in the binding constant of
`the recovered antibody. It is therefore desirable to find
`alternative methods to provide labeled reagents having
`reduced amounts of label bound to extraneous materi-
`als.
`
`2. Brief Description of the Prior Art
`US. Pat. No. 3,998,943 discloses a fluorescent immu-
`noassay involving a ligand conjugated to a lluorescer,
`employing receptor to ligand and receptor to fluo-
`rescer, where the receptor to fluorescer is inhibited
`from binding to fiuorescer when receptor to ligand is
`bound to ligand. U.S. Pat. No. 3,935,074 describes an
`immunoassay where a receptor for a detector label and
`a receptor for ligand are employed with the detector
`label labeled to ligand. Various labels are described.
`U.S. Pat. No. 3,996,345 describes an assay employing a
`chromogenic pair, where one of the chromogens fluor-
`esces emitting light at a wavelength within the absorp-
`tion band of the other chroruogen. Copending patent
`application Ser. No. 315,636, filed July 14, 1977 now
`U.S. Pat. No. 4,160,145. discloses the use of a non-
`enzymatic cata'lyst- as a label
`in competitive protein
`binding assays.
`Co-pending application Ser. No. 893,910, filed Apr. 5,
`1978, describes -a‘ chemiluminescent label in a competi-
`tive protein binding assay. Assays dependent upon the
`presence of enzyme labile bonds are described in Car-
`rico, et al, Anal. Biochem. 72, 271-282 (1976); ibid 72,
`283-292 (1972).
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`S5
`
`65
`
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for protein
`binding assays. The method of preparing reagents in-
`volves conjugating a relatively low molecular weight
`label to a monovalent receptor for a polyepitopic li-
`gand, the ligand and receptor being members of a spe-
`cific binding pair. The composition is then purified by
`separating the mixture according to molecular weight,
`so as to isolate the ligand bound to labeled receptor
`substantially free of other labeled compounds.
`The resulting purified ligand bound to labeled mono-
`valent receptor (labeled ligand) may then be employed
`as a reagent in a protein binding assay, where the pres-
`ence of labeled or unlabeled polyvalent e.g. antibody,
`receptor bound to the labeled ligand affects the detecti-
`ble signal produced by the label. Illustrative labels in-
`clude fluorescers, chemiluminescers, nonenzymatic cat-
`alysts, groups having enzyme labile bonds, and the like.
`Compositions are provided having labeled Fab anti-
`bodies bound to a p-olyepitopic ligand in the substantial
`absence of other labeled materials, as well as the label.
`These labeled ligand compositions are provided in kits
`with ligand receptor in premeasured amounts for use in
`protein binding assays.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`The subject invention is concerned with novel com-
`positions for use in protein binding assays, methods for
`preparing such compositions, combinations of reagents
`for use in protein binding assays, and improved methods
`for performing protein binding assays by employing the
`reagents of this invention.
`The reagents of this invention are prepared by conju-
`gating a relatively low molecular weight label capable
`of providing a detectible signal to a monovalent recep-
`tor, where the receptor is one component of a mixture
`of components having analogous chemical properties.
`The labeled mixture,‘ preferably segregated as to molec-
`ular weight, is then combined with a ligand which will
`bind solely to its reciprocal receptor. The mixture is
`then subjected to segregation by molecular weight,
`segregating and isolating those fractions involving one
`or more of the monovalent labeled receptor bound to
`ligand. The resulting product is then substantially free
`of other materials bound to label, as well as the label,
`and also unbound ligand. Since these compositions are
`substantially free of label which is uninvolved with the
`assay, as well as other interferants, the observed signal
`is solely derived from the label which is bound to ligand
`through the monovalent receptor. Thus,
`the back-
`ground which would result from label unrelated to
`ligand and its reciprocal receptor is substantially dimin-
`ished or absent.
`'
`The labeled receptor-ligand complex may then be
`employed in a protein binding assay, the technique de-
`pending upon the particular label. The complex may be
`provided as a reagent in a kit in combination with recep-
`tor for ligand, where the two reagents are premeasured
`so as to substantially optimize the sensitivity of the
`assay.
`'
`
`Definitions
`
`Analyte—the compound or composition to be mea-
`sured, which may be a polyepitopic ligand, normally
`antigenic, having at least two epitopic sites, a mixture of
`
`

`
`3
`compounds which share at least two common epitopic
`sites, or a receptor.
`Ligand—any compound for which a receptor natu-
`rally exists or can be prepared.
`Receptor—any compound or composition capable of
`recognizing a spatial and polar organization of a molec-
`ular i.e. epitopic site. In the subject invention, there will
`normally be twodifferent receptors employed. The first
`receptor which will be labeled, will be monovalcnt,
`having only one binding site, which binding site is spe-
`cific to a particular spatial and polar organization. For
`the most part, the monovalent receptors will be Fab
`fragments of antibodies, conveniently obtained by pep-
`tidase digestion of an antibody. Since for the most part,
`the rnonovalent receptors will be Fab fragments, these
`receptors will normally be referred to as Fab fragments,
`it being understood that Fab fragments is illustrative of
`a broader class of monovalent receptors. The other
`receptors which will be employed will normally be
`polyvalent receptors. These receptors include antibod-
`ies, enzymes, lectins, and the like. The receptor and its
`reciprocal ligand form a specific or homologous bind-'
`ing pair. In referring to receptors to a ligand, the recep-
`tors will be referred lo as “antiligan .”
`Label—a molecule under about 5,000 molecular
`weight which is capable in combination with electro-
`magnetic radiation or auxiliary chemical reagents of
`producing a detectible signal, which capability is af-
`fected by the presence of labeled or unlabeled receptor
`bound to ligand in spatial proximity to the label. Illus-
`trative labels include fluorescers, chernilurninescers,
`nonenzymatic catalysts, groups having enzyrnatically
`labile bonds, and the like.
`Label-Fab—'a conjugate in which the label is cova-
`lently bound to a Fab antiligand.
`there‘ being on the
`‘
`average at least one label per Fab antiligand.
`Ligand-label complex—-—a complex having at least one
`label-Fab non-covalently bound to ligand and retaining
`at least one free epitopic site.
`
`Ligand-Label Complex
`
`The ligand-label complex has three components: (1)
`ligand; (2) monovalent receptor (abbreviated as Fab);
`and (3) label. The first component to be discussed will
`be the ligand.
`
`Ligand
`
`The ligands which will be employed in the subject
`invention will generally have at least 15,000 molecular
`weight, more usually at least 25,000 molecular weight
`and for the most part at least 50,000 molecular weight.
`There is no real upper limit on molecular weight, al-
`though most compositions which will be of diagnostic
`interest will generally be below 2,000,000 molecular
`weight, more usually below l.000,000 molecular
`weight. The polyepitopic ligand analytes will normally
`be poly(arnino acids) i.e. polypeptides and proteins,
`polysaccharides. nucleic
`acids,
`and combinations
`thereof. Such combinations or assemblages include bac-
`teria. ‘viruses, chromosomes, genes. mitchondria, nuclei.
`-cell membranes, and the like.
`For the most part, hormones of interest will generally
`be from about 20,000 to 100,000 molecuiar weight,
`more usually from about 20,000 to 60,000 molecular
`weight. Enzymes of interest will generally range from
`about 20,000 to 600,000 molecular weight, more usually
`from 20,000 to about 300,000 molecular weight. Immu-
`
`5
`
`10
`
`IS
`
`20
`
`25
`
`30
`
`35
`
`I15
`
`50
`
`55
`
`65
`
`4,235,869
`
`4
`noglobulins will generally range from about 150113) to
`1,000,000 molecular weight.
`The wide variety of proteins may be considered as to
`the family of proteins having similar structural features,
`proteins having particular biological functions, proteins
`related to specific microorganisms, particularly disease
`causing microorganisms, etc.
`The following are classes of proteins related by struc-
`ture:
`protamines
`histories
`albumins
`
`giobulins
`scleroproteins
`phosphoproteins
`rnucoproteins
`chromoproteins
`lipoproteins
`_ nucleoproteins
`-
`glycoproteins
`unclassified proteins, e.g. sornatotropin. prolactin,
`insulin, pepsin
`A number of proteins found in the human ptasma are
`important clinically and include:
`Prealbumin
`'
`Albumin
`ct]-Lipoprotein
`ct;-Acid glycoprotein
`a1-Antitrypsin
`ct;-Glycoprotein
`Transcortin
`4.68-Postalbumin
`Tryptophan-poor at-glyooprotein
`aux-Glycoprotein
`Thyroxin-binding globulin
`Inter-at-trypsin-inhibitor
`Gc-globulin
`(Gr: 1-1)
`(Ge 2-1)
`(Ge 2-2)
`I-Iaptoglobin
`(HP 1-1)
`(Hp 2-1)
`(Hp 2-2)
`Ceruloplasmin
`Cholinesterase
`ct:-Lipoprotein(s)
`ct;-Macroglobulin
`ct;-HS-glycoprotein
`Zn-oz-glycoprotein
`ct;-Neuramino-glycoprotein
`Erythropoietin
`,8-lipoprotein
`Transferrin
`
`'
`
`Hemopexin
`Fibrinogen
`Plasminogen
`,0;-glycoprotein I
`.62-glycoprotein ll
`Imrnunoglobulin G (IgG) or 'yG-globulin
`Mol. formula:
`Til-:2 Or ')'2?\2
`Immunoglobulin A (IgA) or 7A-globulin
`Moi. formula:
`(a2K2)" or (a27\2)*’
`Immunoglobulin M (lgM) or 'yM-globulin
`Mol. formula:
`
`(iL2K2)5 or (iL17L2)5
`
`

`
`4,235,869
`
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF), CRF, LRF, TRF, Somato-
`tropin-RF, GRF, FSH-RF, PIF, MIF
`Other polymeric materials of interest are mucopoly-
`saccharid and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`Hemosensitin Found in
`Species of Microorganisms
`Polysacchatide
`Streptococcus pyogenes
`Polysaccllaride
`Dtlplccocctrs pneu monlae
`Polysaccharidc
`Nelsserfa meningltldls
`_
`Nelswrla gonorrlmeae
`Polysaccharide
`Polysaccharide
`Corynebacterfum dijtrltrlaektae
`Crude extract
`Actlnobaclllus mallel;
`Actlncboclllus wltltemcrl
`Frartctsella mlarertsls
`
`Lipopolysaccharide
`Polysaccharidt:
`
`5
`Immultoglobulin D(IgD) or 'yD-Globulin (1/D)
`Mol. formula: _
`(fizltz) or (82713)
`Irnmunoglobulin E. (IgE) or )IE—Globuiin ('yE)
`Moi. formula:
`{e1Ic2) or (QM)
`Free K and 7 light chains
`Complement factors:
`C’l
`C’1q
`C’1r
`C15
`C2
`C3
`31A
`cc2D
`C4
`C5
`C6
`C’?
`C8
`09
`Important blood clotting factors include:
`
`I
`
`'
`
`-
`
`BLOOD CLOTTING FACTORS
`International designation
`Name
`I
`Fibrinogen
`ll
`Prolhrombin
`[Ia
`Thrombin
`[II
`Tissue thromboplastin
`V and VI
`Proaccelerin. accelerator
`globulin
`Proconverti-n
`Antihemophiiic globulin (AHG)
`Christmas factor,
`-
`plasma thromboplastin
`component (FTC)
`Stuart-Prower factor,
`autoprotltrombin III
`Plasma thromboplastin
`antecedent (PTA)
`Hagernann factor
`XII
`
`XIII I-“uh:-in-stabilizing factor
`
`X
`
`XI
`
`VI!
`VIII
`IX
`
`Important protein hormones include:
`
`5
`
`10
`
`t5
`
`20
`
`25
`
`30
`
`4,3
`
`45
`
`50
`
`Peptide and Protein Hormones
`Parathyroid hormone (parathromone)
`Thyrocalcitonin
`Insulin
`Glucagon
`.
`Relaxin
`Erythropoietin
`Melanotropin (melanocyte-stimulating hormone; in-
`terrnedin)
`Somatotropin (growth hormone)
`Corticotropin (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone (interstitial cell-stimulating hor-
`mone)
`Luteomammotropic hormone (luteotropin, prolactin) 60
`Gonadotropin (chorionic gonadotropin)
`Tissue Hormones
`
`55
`
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`
`55
`
`Pasrettrella pestle
`Pasta-urella pestle
`Pasteumlla multaclda
`Brttoella abormr
`Haemophllus influenza:
`Haemapltllus pertussls
`Treponemo relrerl
`Vetllanella
`Erysfipelotlirlx
`Listeria monocjxtogencs
`Cltmmolmcterium
`Mycobacterlnm ttaberculasir
`
`35
`
`Klebclella aerogenes
`Klebslellra claacae
`Salmonella typlicmt
`
`Polysaccharide
`Capsular antigen
`Crude extract
`Poiysaccharidc
`Crtrde
`Poiyseccharidc
`Ljpopclysacehatide
`Polysaccharide
`Poly-saccharide
`Lipopolysaccharide
`Saline extract of 99%
`phenol extracted
`mycobactcria and poly-
`saccharide fraction of
`cells and tuberculin
`Polysaccharide
`Polysaccharide
`Lipopolysacchnride.
`Polysaccharide
`Poly-saccharide
`
`Salmonella typlu‘-mm-lam.’
`Salmonella derby
`Salmonella pnllomrn
`Slrfgella tlysentcrlae
`Sltlgella flexnert‘
`Slnfgelle scum.-:1‘
`Crude. polysaccharide
`Crude extract
`Rlcltettslae
`Candida albloans
`Polysaccharide
`Crude extract
`Entamoelm lnsrolym-a
`
`Polysaccharide
`
`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:
`
`Coryne-bacteria
`
`Corynebacterium diptlterlae
`Pneumococci
`
`Dtplococcus pnestmonlae
`
`Streptococci
`
`Streptococcus pyogenes
`Streptococcus salivarus
`
`Staphylococci
`
`Staphylococcus aureus
`Staphylococcus albus
`Neisseriae
`
`Neisserla men :‘ng1'tla't‘s
`Neisseria gonorrheae
`
`

`
`7
`.__...j._._.........j......_
`
`4,235,869
`
`Nacardfa brasiffensis
`
`8
`
`The Spirochetes
`_
`_
`Trap”-Wm": Pamdflm
`Treponema perrenue
`T"-”°”_""”“ ‘°""‘“e‘_"’"
`B9’7'91“? mcflrre-“£35
`Le_yro.rp1'ra fcrerohemorrhagfae
`Le :05 f a canicola
`_ 1° "
`_
`Spmllum mm us
`Srreprobacillus monfftfbrmis
`
`5
`
`10
`
`Mycoplasmas
`15 Mycopfasma przeumombe
`
`Other pathogens
`
`_
`_
`Ltsrena mcmocytogenes
`Etysgaeforhrix rhusioparhfae
`Streprobacilfm morziii ormis
`Domrzmfa gmnulomatis
`Banofleua bag‘-m-Jfirmr-S
`_
`,
`,
`_
`_
`Rlckettslae (bactena-like parasites)
`R:'ckerrs1':: pmwazekff
`Rickertsfa moaseri
`R:‘ckeIrs:'a rt'ckeztsf:'
`Rkkem”-G mm”;
`Ricketrsfa 0'£t$I?fll'£-S
`?L;""’:e“'”_a flzmfms
`_
`63533 a an
`Rickeflsta tsuzmgam usht
`Rickerrsia burners?
`Rfckemia qm'm'ana
`.
`.
`.
`.
`.
`Chlamydm (unc_lass1fiable parasltes bactenal/viral)
`Chlam dia 21
`t
`in uncertain
`3'
`gen 5 (mm g
`)
`
`
`
`_
`Fungi
`
`2
`
`0
`
`25
`
`30
`
`35
`
`40
`
`Cijrprocaccus mzofitarmam"
`Bfasramyzces demaridis
`45 Histapfasma ::apsu.:'amm
`cmrdmdes immffis
`‘Pam‘?““:’°‘.f95 bmmnm
`Crmd':d'a abfum.-13
`A_,Wgm."sf"m,:ga,“
`Mucnr can-mb{',I5:r {Absidfa corymbtfera)
`50 Rfxizopus oryzae
`Rhfzopus arrfirfzus
`}
`Phycomycetes
`‘_
`-'W=0P*f5 "58”1'“"5
`?"'°""'J'"”‘d""""‘{""‘
`F:;;:::: ffimrgfiia
`55 Fmmm, d__,mM_,_;d£,
`Cfadmporium cam‘om'I'
`Pbiafqahom verrucasa
`-4-W!!!"-'-’=45 m'dW-‘H5
`M““""'¢"’“ ""J_’*-‘°‘°""‘
`60 M"d"'9H“ 3"“
`A flescherfa boydfi
`Pfrfafaaphara jeansefmei
`Micrmparum gypseum
`Trfchqahyton :nenragmp.F:yIe5
`Kemrinomyces ajeffof
`65 Micmtpomm cam’!
`Trfchophyton rubmnf I
` .j..j.
`
`%f=_1=_*I£t°_$i_8£l!.°_..
`Exherfchia cab"
`Aerabacrer aemgwenes
`Kijhfeffgfmeuronfae
`Sn mane a Iyp am
`sa:mm:ra droferaesuir
`Salmonella ryp.‘u‘mI.rr¢'um
`3-figs-’-‘vi dfiwiter-“R9
`shgwn" whmftzif
`Shgeffa ammamrda
`S.‘:.-‘grfla fleimeri
`Sfiisefffl bard-‘f
`Siaigefla Snmm.-I
`
`'
`The coliform bacteria
`The salmonellae
`
`)
`D
`
`‘The Shigellae
`
`Other enteric baciiii
`Pyofgm vy-Igarfif
`Proteus mimbffzlr
`Proteus morgam‘
`Pseudomonas aeruglnm
`Arcarrgms faeculis
`
`Wbrfv -'-'-WW9
`
`Proteus sptcips
`
`J»
`
`_
`Hemophtlusnflorcletella group
`Hemaphifus‘ fnfluenzae,
`H_ dmmyf
`43- h9m9P}"7“5
`H- “EEPPNWS
`H‘ P"m"”fl”e"zf”
`Borderefla perrums
`
`Paswufellae
`
`Pasteurefla pemls‘
`Pasreureffa tulareusfs
`
`Brucellae
`
`Bmcefla mefirerrsfs
`Bmcefla charms
`Bmcefla Sufi
`.
`.
`.
`.
`Aeroblc Spore-forming Bacllll
`Bacillus antkracfs
`Bacillus subrflis
`
`BRCHIHS megaterfum
`Bacillus cereus
`
`_
`Anaerobic Spore-formmg Bacilli
`Clastridium bum finum
`,
`_
`.
`I
`CIOsm.d:.um reranf
`gig“? Pe’3’rf’13e“5
`051?! mm HOV}?!
`Cfosfrfdfum septicum
`Closrridium htlrrofyficum
`Closrridium rertmm
`C!ostr::d1:um bxfermemans
`Clmtrrdtum sporagenes
`
`.
`Mycobactena
`Mycabacterfum rubemufosis h0m1'm’s
`Mycabacterfum bovis
`Mycobacrerfum avium
`Mycobacterium ieprae
`Mycobacterium paratuberculosis
`
`Actinomycetes (fungus-like bacteria) '
`Actfrramyces :'srae!':'1'
`Acrmomyces bow}
`Acrinomyces naeslundii
`Nocardfa asteroides
`
`'
`
`

`
`9
`
`Viruses
`
`Adenoviruses
`
`Herpes viruses
`
`Herpes simplex
`Varicella (Chicken pox)
`Herpes Zoster (Shingles)
`Virus B
`Cytornegalovirus
`
`Pox Viruses
`
`Variola (smallpox)
`Vaccinia
`Poxvims bovfs
`Paravaccinia
`Molluscum contagiorttm
`Pioornaviruses
`
`Poliovirus
`Coxsackievirus
`Echoviruses
`Rhinoviruses
`
`Myxoviruses
`
`Influenza (A, B, and C)
`Parainlluenza (1-4)
`Mumps Virus
`Newcastle Disease Virus
`Measles Virus
`Rinderpest Virus
`Canine Distemper Virus
`Respiratory Syncytial Virus
`Rubella Virus
`
`Arboviruses
`
`Eastern Equine Encephalitis Virus
`Western Equine Encephalitis Virus
`Sindbis Virus
`
`Chikugunya Virus
`Sernliki Forest Virus
`Mayors Virus
`St. Louis Encephalitis Virus
`California Encephalitis Virus
`Colorado Tick Fever Virus
`Yellow Fever Virus
`Dengue Virus
`
`Reoviruses
`
`Reovirus Types 1-3
`
`Hepatitis
`
`Hepatitis A Virus
`Hepatitis B Virus
`
`Tumor Viruses
`
`Rauscher Leukemia Virus
`Gross Virus
`Maloney Leukemia Virus
`
`Allergens
`
`Receptor
`
`10
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`The next component is the monovalent receptor.
`While there will be some naturally occurring n1onova-
`lent receptors, for the most part, the monovalent recep-
`tors will be Fab fragments of antibodies. Fab fragments
`are well known and a description may be found in Ad-
`vanced Immunochemistry, Eugene Day. The Williams
`
`65
`
`4,235,869
`
`10
`& Wilkins Company, Baltimore, 1972, pp. 33ff. Fab
`fragments can be produced by digestion with a pepti-
`dase, such as papain, trypsin, or pepsin. Other treat-
`ments may be involved such as reduction, substitution,
`e.g. aminoethylation, carboxyalkylation with, for exam-
`ple,
`iodoacetamide, and the like. The fragment will
`normally have about 40,000 molecular weight, be
`monovalent and retain a high degree of the specificity
`and binding constant of the intact antibody.
`Label
`
`The label is characterized by being a small molecule,
`generally below about 2,000 molecular weight, more
`usually below about 1,000 molecular weight, greater
`than about 100 molecular weight and preferably from
`about 125 to about 800 molecular weight.
`The label is further characterized by having either a
`chemical or physical (electronic) transformation af-
`fected by the spatial proximity of the receptor or a label
`on the receptor. The effect of the spatial proximity of
`the receptor may be one of steric bulk impeding the
`approach of a large molecule to the label or a change of
`environment in the area of the label. Alternatively, the
`receptor may be labeled with a companion label, which
`interacts with the label of the complex when in close
`proximity, so as to modify the signal from the label of
`the complex.
`While the label can be involved in a single event,
`desirably, the label will be capable of producing a plu-
`rality of events, so that a multiplication of the effect
`may be achieved.
`The labels will be for the most part simple organic
`molecules which either absorb electromagnetic radia-
`tion or produce a product which will absorb electro-
`magnetic radiation.
`The first class of compounds of interest are fluoresc-
`ers. These compounds will for the ‘most part absorb
`light above 300 nm, preferably above 350 nm and more
`preferably above 400 nm. These compounds will prefer-
`ably have extinction coefficients of at least 103, prefera-
`bly at least 104 above the indicated wavelengths.
`Various chromophores which may be employed as
`lluorescers include the xanthene dyes, which include
`the fluoresceins derived from 3,6-dihydroxy-9-phenylx-
`anthhydrol and rosamines and rhodatnines, derived
`from 3,6-diamino-9-phenylxanthhydrol. The rhoda«
`mines and fluoresceins have a 9-0-carboxyphenyl group
`and are derivatives of 9-o-carboxyphenylxanthhydrol.
`These compounds are commercially available with
`substituents on the phenyl group which can be used as
`the site for bonding or as the bonding functionality. For
`example, amino and isothiocyanate substituted lluores-'
`cein compounds are available.
`Other dyes which may be used as fluorescers include
`3-phenyl-7-isocyanatocoumarin, acridines, such as 9-
`isothiocyanatoacridine and acridine orange N-(p-(2-
`benzoxazolyl)phenyl)n1aleimide; benzoxadiazoles, such
`as 4-chloro-7-nitrobenzo-2—oxa-1,3-diazole and 7-(p-
`rnethoxybenzylarnino)-4-nltrobenz.o-2-oxa-1,3-diazole;
`stilbenes, such as 4-dimethylainino-4'-isothiocyanatos-
`tilbene
`and
`4-dimethylamino-4’-maleimidostilbene
`N,N'-dioctadecyl oxacarbocyanine p-toluenesulfonate;
`pyrenes, such as 8-hydroxy-l,3,6-pyrenetrisulfonic acid,
`and 1-pyrenebutyric acid; merocyanines e.g. merocya-
`nine 540;
`rose bengal; 2,4-diphenyl-3(2H)-furanone;
`cyanines: anthraquinones; porphyrins; triarylrnethanes;
`as well as other readily available dyes which are capable
`
`

`
`11
`of fluorescing. These dyes, either have active function-
`alities for conjugation or such functionalities may be
`readily introduced.
`It should further be noted that the absorption and
`emission characteristics of the dye may vary from being
`free in solution and being bound to a protein or ligand.
`Therefore, when referringto the various wavelength
`ranges and characteristics of the dyes, it is intended to
`indicate the dyes as employed and not the dye which is
`unoonjugated and characteristizecl in an arbitrary sol-
`vent
`
`The next type of label is a chemiluminescent com-
`pound, which by reacting with a compound in solution
`is capable of producing light. Therefore, a chemilumi-
`nescent source will normally have at least two compo-
`nents.
`_
`
`For purposes of convenience, the chemiluminescent
`source will be divided into two categories: those which
`do not involve the intermediacy of enzyme catalysis;
`and those which do involve enzyme catalysis.
`Considering chemiluminescent sources which do not
`involve enzyme catalysis, only those sources can be
`employed which chemiluminesce under conditions
`which neither inhibit the binding of the receptor to the
`ligand, nor degrade the receptor and ligand at an unac-
`ceptable rate during the period of measurement. While
`ordinarily chemiluminescent sources which are depen-
`dent upon nonaqueous solvents and strong basic condi-
`tions, greater than pHIl, will not be useful, techniques
`can be employed involving rapid injection or llow tech-
`niques where the modulated emission is substantially
`completed before the protein is denatured and signifi-
`cant dissociation occurs. After injection of base, one
`would observe a burst of light which could be mea-
`sured.
`
`A diverse number of families of compounds have
`been found to provide chemiluminescence under a vari-
`ety of conditions. One family of compounds is 2,3-dihy-
`dro-1,4-phthalazinediones. The most popular com-
`pound is luminol, which is the 5-amino compound.
`Other members of the family include the 5-amino-6,7,8_-
`trimethoxy and the dimethylamino[ca]benz analog.
`These compounds can be made to luminesce with alka-
`line hydrogen peroxide or calcium hypochlorite and
`base. Another family of compounds is the 2,4,5—tri-
`phenylimidazoles, with lophine as the common name
`for the parent product. Chemiluminescent analogs in-
`clude para-dimethylamino and -methoxy substituents.
`The next group of chemiluminescent compounds are
`indolen—3-yl hydroperoxides, precursors thereto and
`derivatives thereof.
`
`The next group of compounds are the bis-9,9’-bia-
`cridinium salts. of which lucigenin, N,N’-dimethyl-9,93
`biacridiniurn dinitrate is illustrative. These compounds
`chernilurninesce upon combination with alkaline hydro-
`gen peroxide.
`The next group of compounds are aeridinium salts
`which are substituted in the 9 position. Particular sub-
`stituents are carboxylic esters, particularly the aryl es-
`ters, acyl substituents, particularly benzoyl, and cyano.
`Alkaline hydrogen peroxide is employed to induce che-
`miluminescence.
`_
`Another group of compounds are various acyl per-
`oxy esters and hydroperoxides, which may be formed in
`situ in combination with compounds such as 9,10-
`diphenylanthracene.
`Another source of chemiluminescence is hydroperox-
`ides e.g.
`tetralin hydroperoxide in combination with
`
`10
`
`I5
`
`10
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,235,869
`
`12
`metal complexes, particularly porphyrins and phlhalo-
`cyanines, where the metals are iron and zinc.
`Preferred systems are those which provide a satisfac-
`tory quantum efficiency of
`emission
`from the
`chemiluminescer at a pH at or below ll, preferably at
`or below !0.
`The next group of compounds are based on-
`chemiluminescers which chemiluminesce under enzy-
`matic catalysis. Primarily, there are two groups of enzy-
`matically catalyzed cherniluminescers. The first group
`are those compounds which chemiluminesce in combi-
`nation with alkaline hydrogen peroxide. By employing
`a peroxidase e.g. horseradish peroxidase, in combina-
`tion with hydrogen peroxide and the chemiluminescer,
`chemiluminescence can be achieved. Illustrative sys-
`tems include 2,3-dihydro-l,4-phthalazinediones.
`The second enzymatic source of chemiluminescence
`is based on luciferins and their analogs and luciferases.
`The next group of compounds concerns non-
`enzymatic catalysis. These catalysts are involved with
`electron transfer agents. One agent acts by the transfer-
`ring of two electrons (R1), the second electron transfer
`agent acts by the transferring of one electron (R1) and
`an intermediate or catalyst agent serves as the label and
`is able to receive and transfer one and two elections. In
`some instances, the intermediate or catalyst may react
`as either 11* or R1, where the particular catalyst cannot
`react with a particular electron transfer reagent or fam-
`ily of reagents.
`The label will for the most part be either metal com-
`plexes or aromatic compounds capable of assuming a
`neutral or changed quinone structure with a heteroatom
`of atomic number 7 to 8 i.e. oxygen and nitrogen. For
`the most part, the label will be aromatic having from
`one to five, usually one to four, fused or non-fused rings,
`where one or more heteroatoms may be involved as
`annular atoms. The labels will be able to assume either
`an o-quinone or p-quinone structure, either outsideofor
`as part of a cyclic structure. Therefore, common to the
`label which does not involve a metal will be the follow-
`ing formulli.
`
`x
`\\
`
`Y
`/1
`
`where X and Y can be the same or different and X is
`oxygen or nitrogen and Y is oxygen, nitrogen or carbon,
`and wherein X and Y, as well as the. annular atoms may
`be further substituted.
`The first group of compounds are the quinones, either
`oi-tho or para, where the heteroatoms are not involved
`in a heterocyclic structure. These compounds will for
`the most part have the following formulii.
`
`X1
`II
`
`1'
`Y‘
`
`“,3
`
`‘wt
`
`w,
`
`w1
`
`xi
`ll
`
`wl
`
`I Y./
`
`w‘
`
`W,
`
`w3
`
`wherein
`
`X‘ and Y1 are oxygen or imino, more usually oxygen;
`
`

`
`4,235,869
`
`13
`W14 may be hydrogen, halogen, particularly of
`atomic number 9 to 35, more particularly of atomic
`number 17 to 35, alkyt of from I to 6 carbon atoms,
`more usually of from 1 to 4 carbon atoms or W
`groups on adjacent carbon atoms, particularly W1
`and W3 or W3 and W4, may be taken together to
`form an aromatic ring, particularly a benzene ring,
`either substituted or unsubstituted, normally hav-
`ing not more than about 2 substituents which are
`alkyl or heteroatorn or atomic numbers 7 to 8.
`which include hydroxy, alkoxy, amino having
`from 0 to 2 alkyl groups, e.g. alkylamino, and dial-
`kylamino, wherein the alkyl groups are of from 1 to
`6, more usually of from 1 to 3 carbon atoms.
`Generally, the-quinones will have from 1 to 3 rings,
`usually fused rings, and will be of from about 6 to 20
`carbon atoms, more usually of from about 6 to I6 car-
`bon atoms. The total number of heteroatoms will gener-
`ally be in the range of from about 2 to 8, more usually
`from 2 to 6.
`Illustrative quinones include alizarirl, l,2-naphthoqui-
`none, cliloranil, 2,6-dichlorophenolindophenol and 2,6-
`dibromophenolindophenol.
`The next group of compounds are those which are
`basically internal o-quinonediimines. These compounds
`for the most part will have the following forrnulii.
`
`D’
`
`D4
`
`1‘
`E‘
`
`W
`
` f
`
`N
`
`IA
`
`!
`
`o‘
`
`D2
`
`wherein:
`the broken lines indicate the presence or absence of a
`double bond,
`the double bonds normally being
`present in the oxidized form, usually not more than
`one of the double bonds being present in the re-
`duced form;
`A and A1 may be the same or different and may be an
`unshared pair of electrons, hydrogen, alkyl of from
`1 to 6 carbon atoms, hydroxyallcyl of from S to 6
`carbon atoms, particularly sugars e.g. ribityl, A and
`A‘ normally being other than hydrogen when the
`broken line forms a double bond, and usually one of
`A and Al is other than hydrogen or an unshared
`pair of electrons;
`D14 may be th