llited Mates Patent
`
`[19]
`
`Sehwarzherg
`
`[54] ASSAY EMPLOYING A LABELED
`FAB-FRAGMENT LIGAND COMPLEX
`
`[75]
`
`Inventor: Moshe Schwarzberg, Sunnyvale,
`Calif.
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] Appl. No.: 906,388
`
`[22] Filed:
`
`May 16, 1973
`
`[51]
`
`lint. CL? ................... .. G01N 21/00; GOIN 31/00;
`G0lN 31/14; GOIN 33/16
`[52] US. Cl. .................................... .. 424/8; 23/230 B;
`250/302; 424/ 1; 424/ 12; 424/13; 435/7
`[58] Field of Search ......................... .. 424/1, 8, 12, 13;
`250/302; 23/230 B; 435/7
`
`[56]
`
`,
`
`References Cited
`-
`U.S. PATENT DOCUMENTS '
`
`3,935,074
`3,996,345
`3,998,943
`4,104,029
`
`195/103.5
`..
`1/1976 Rubenstein.
`...... .. 424/12
`12/1976 Ullman
`
`12/1976 Ullman
`424/12
`8/1978 Maier ................................... 424/8 X
`
`OTHER PUBLICATIONS
`
`Ternynck et al., Ann. Immunol. (Inst. Pasteur), vol. 127
`C 1976 pp. 197-208.
`Weir (Ed.), Handbook of Exptl. Immunology, Black-
`well Sci. Pub. London, 2nd ed., 1973, pp.'l4.l9—14.25.
`Carrico, et al., Anal. Biochem. vol. 72, 1972 pp.
`271-282, 283-292.
`Forsum, J. of Immuno. Methods, vol. 2, 1972 pp.
`183-195.
`
`[11]
`
`[45]
`
`4,235,869
`
`Nov. 25, 1980
`
`Primary Examiner—Anna P. Fagelson
`Attorney, Agent, or Fz'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 a monovalent 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
`
`SANOFI v. GENENTECH
`SANOFI v. GENENTECH
`IPR20 15-0 1624
`IPR2015-01624
`EXHIBIT 2056
`EXHIBIT 2056
`
`

`
`4,235,869
`
`2
`
`Assn‘EMPLOYING A LABELED
`FAB-FRAGMENT ILIGAND COMPLEX
`
`_BAcKoRoUNo OF THE INVENTION
`1. 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 vgeight. For.exan;1ple, 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
`U.S. Pat. No. 3,998,943 discloses a fluorescent immu-
`noassay involving a ligand conjugated to a fluorescer,
`employing receptor to ligand and receptor to fluo-
`rescer, where the receptor to fluorescer is inhibited
`from binding to fluorescer 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 chromogen. Copending patent
`application Ser. No. 815,636, filed July 14, 1977 now
`U.S. Pat. No. 4,160,145, discloses the use of a non-
`enzymatic catalyst 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
`
`55
`
`60
`
`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 polyepitopic 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 polyepit.opic 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 5
`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 monovalent,
`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 monovalent 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 to as “antiligand.”
`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 tluorescers, chemiluininescers,
`nonenzymatic catalysts, groups having enzymatically
`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.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`‘
`
`4,235,869
`
`4
`noglobulins will generally range from about 150,000 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 _
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`_ nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinicallyand include:
`’ Prealbumin
`’
`Albumin
`at-Lipoprotein
`on-Acid glycoprotein
`on-Antitrypsin
`on-Glycoprotein
`Transcortin
`4.6S-Postalbumin
`Tryptophan-poor a1-glycoprotein
`oL1X-Glycoprotein
`.
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin
`(Gc l-l)
`(Ge 2-1)
`(Gc 2-2)
`I-laptoglobin
`(Hp 1-1) 5
`(HP 2-1)
`(HP 2-2)
`Ceruloplasmin
`Cholinesterase
`
`'
`
`:12-Lipopro_tein(s)
`ag-Macroglobulin
`az-HS-glycoprotein
`Zn-a2-glycoprotein
`a2-Neuramino-glycoprotein
`Erythropoietin
`‘,8-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`B2-glycoprotein I
`,8;-glycoprotein II
`Immunoglobulin G (IgG) or 'yG-globulin
`Mol. formula:
`72x2 or 'y2?~2
`Immunoglobulin A (IgA) or 'yA-globulin
`Mol. formula:
`(a2K2)" or (a2>»2)"
`Immunoglobulin M (IgM) or 'yM—globulin
`Mol. formula:
`‘
`(p.2K2)5 or (;t27\2)5
`
`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.
`‘
`
`45
`
`Ligand
`
`The ligands which will be employed in the subject
`invention will generally have at least l5,000 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 1,000,000 molecular
`weight. The polyepitopic ligand analytes will normally
`be poly(amino 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 molecular 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-
`
`50
`
`55
`
`60
`
`65
`
`

`
`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-
`saccharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`Hemosensitin Found in
`Species of Microorganisms
`Streptococcus pyogenes
`Polysaccharide
`Diplacoccus pneumoniae
`Polysaccharide
`Neisseria meningitidis
`Polysaccharide
`Polysaccharide
`_
`Neisseria gonorrhaeae
`Polysaccharide
`Corynebacterium diphtheriae
`Crude extract
`Actinobacillus mallet‘;
`Actinobacillus whitemori
`Francisclla tularensis
`
`Lipopolysaccharide
`Polysaccharide
`
`5
`Immunoglobulin D(IgD) or 'yD-Globulin (yD)
`M01. formula: _
`(52I<2) Or (52>x2)
`Immunoglobulin E (IgE) or 'yE—Globulin (3/E)
`Mol. formula:
`(e2K2) Of (€2?»2)
`Free K and 7' light chains
`Complement factors:
`C’l
`C'1q
`C’1r
`C’1s
`C’2
`C’3
`B 1A
`a2D
`C'4
`C’5
`C’6
`C7
`C’8
`C’9
`Important blood clotting factors include:
`
`.
`
`‘
`
`BLOOD CLOTTING FACTORS
`International designation
`Name
`I
`Fibrinogen
`II
`Prothrombin
`IIa
`Thrombin
`III
`Tissue thromboplastin
`V and VI
`Proaccelerin, accelerator
`globulin
`Proconvertin
`Antihemophilic globulin (AHG)
`Christmas factor,
`»
`plasma thromboplastin
`component (PTC)
`Stuart-Prower factor,
`autoprothrombin III
`Plasma thromboplastin
`antecedent (PTA)
`Hagemann factor
`XII
`
`XIII Fibrin-stabilizing factor
`
`X
`
`XI
`
`VII
`VIII
`IX
`
`Important protein hormones include:
`
`Peptide and Protein Hormones
`Parathyroid hormone (parathromone)
`Thyrocalcitonin
`Insulin
`
`5
`
`10
`
`l5
`
`20
`
`25
`
`30
`
`40
`
`45
`
`50
`
`Glucagon
`.
`Relaxin
`Erythropoietin
`Melanotropin (melanocyte-stimulating hormone; in«
`termedin)
`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
`
`65
`
`Pasteurella pestis
`Pasteurella pestis
`Pasteurella multocida
`Brucella abortus
`Haemophilus influenzae
`Haemaplzilus pertussis
`Trepanema reiteri
`Veillonella
`Erysipelothrix
`Listeria monocytogenes
`Chromobacterium
`Mycobacterium tuberculosis
`
`35
`
`Klebsiella aerogenes
`Klebsiella cloacae
`Salmonella typhosa
`
`Polysaccharide
`Capsular antigen
`Crude extract
`Polysaccharide
`Crude
`Polysaccharide
`Lipopolysaccharide
`Polysacchartde
`Polysaccharide
`Lipopolysaccharide
`Saline extract of 90%
`pihenol extracted
`mycobacteria and poly-
`saccharide fraction of
`cells and tuberculin
`Polysaccharide
`Polysaccharide
`Lip-opolysaccharide,
`Polysacchnride
`Polysaccharide
`..a
`
`Salmonella typhi-murium;
`Salmonella derby
`Salmonella pullorum
`Shigella dysenteriae
`Shigella flexneri
`Shigella sonnei
`Crude, polysaccharide
`Ricketrsiae
`Crude extract
`Candida albicans
`Polysaccharide
`Crude extract
`Entamoeba histolylica
`
`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:
`
`Corynebacteria
`
`Corynebacterium diptheriae
`Pneumococci
`
`Diplacoccus pneumoniae
`
`Streptococci
`
`Streptococcus pyogenes
`Streptococcus salivarus
`
`Staphylococci
`
`Staphylococcus aureus
`Staphylococcus albus
`Neisseriae
`
`Neisseria meningitidis
`Neisseria gonorrheae
`
`

`
`Nocardia brasiliensis
`
`4,235,869
`
`Entcrobacteriaciae
`Escherichia coli
`Aerabacter aerogenes
`Klebsiella pneumoniae
`Salmonella typhasa
`Salmonella choleroesuis
`Salmonella typhimurium
`Shigella dysenteriae
`Shigella schmitzii
`Shigella orabirzotarda
`Sltigella flexneri
`Shigella boydii
`Shigella Sonnet’
`
`Other enteric bacilli
`Proteus vulgaris
`Proteus mirobilis
`
`
`
`¥,_/L.....,._.J§,_/\_.,J
`
`'
`
`The coliform bacteria
`
`The Salmonéllae
`
`5
`
`The Spirochetes
`
`_
`Treponema Pallldum
`Treponema pertenue
`Treponema carateum
`Borrelio recurrentis
`
`The Shigcuae
`
`Proteus species
`
`10
`
`Leptospira icterohemorrlzagioe
`Leptospira camcola
`Spirillum minus
`Streptabacillus monilzformis
`M co lasmas
`y
`p
`15 Mycoplasma pneumoniae
`
`Other pathogens
`
`_
`_
`Ll-579"“ m0”0CJlt0g€’”e-5'
`Eryszpelothrix rhusiopatlziae
`Streptobacillus moniliformis
`Donvanio granulomatis
`Bartonella baa-lb-form]-S
`,
`.
`,
`_
`_
`Rlckettsxae (bacterla-11ke parasltes)
`Rickettsia prowazekii
`Rickettsia mooseri
`Rickettsia rickettsii
`RI-ckem,-a comm-
`Rickettsia australis
`Rickettsia sibiricus
`Rickettsia akari
`
`Rickettsio tsutsugamushi
`Rickettsia burnetii
`Rickettsia quifltall“
`
`Chlamydia (unclassifiable parasites bacterial/viral)
`
`Chlamydla agents (nammg uncertam)
`
`
`
`Flmgi
`
`2
`
`0
`
`25
`
`30
`
`35
`
`40
`
`Cryptococcus neoformans
`Blastomyces dermotidis
`45 Histoplasma capsulotum
`Coccidioides immilis
`Paracoccidiodes brasiliensis
`Candida albims
`Aspergillusfumigatus
`Mucor corjymbtfer (Absidia corymbifera)
`50 Rhizapus oryzae
`Rhizopus arrhizus
`>
`Phycomycetes
`H
`R/uzopus mgncuns
`Sporotrzchum schertku
`Fammea pedmwl
`Fonsecoea compacto
`55 Fonsecaea dermatitidis
`Cladosporium corrionii
`Phialophora verrucosa
`Aspergi//us Hidulalis
`M”d"’*‘-”’" ’"Jf‘e’°""
`60 M"d“'e”" gme“
`Allescheria boydii
`Phialosphora jeansclmei
`Microsporum gypseum
`Trichophyton mentagrophytes
`Keratinomyce: ajellai
`65 Micrasporum Cam's
`T’f"“’l”'3”"" ’“”’"”f _
`
`Mlcrosporum andoumt
`
`Proteus morgani
`Pseudomonas aeruginosa
`Alcaligenesfaecalis
`
`Vibrio cholerae
`
`_
`Hemophllus-Bordetella group
`Hemo hilus in uenzae,
`H gucreyi fl
`H. hemophilus
`H aegylfticus
`H- 1"”‘"”fl“e”Z”9
`Bordetella pertussis
`
`pastem-e]1ae
`
`Posteurella pestis
`Posteurella tulareusis
`
`B1'11Ce113-9
`
`Brucella melitensis
`Brucella abortus
`Brucella suis
`
`Aerobic Spore_fol_ming Bacim
`Bacillus anthracis
`Bacillus subtilis
`
`‘
`
`Bacillus megaterium
`Bacillus cereus
`
`.
`_
`_
`_
`Anaerobnc Spore-formmg Bacnlh
`
`Clostrzdzum botulmum
`Clostridium tetarzi
`_
`_
`_
`_
`C103"ldmm Pelfilngens
`Clostridzum novyi
`Clostridium septicum
`Clostridium histolyticum
`Clostridl-um tern-um
`Clostridium bifermentans
`_
`,
`C105"ldmm Sporogenes
`
`.
`Mycobacterla
`Mycobacterium tuberculosis hominis
`Mycobacterium bovis
`Mycobacterium avium
`Mycobacterium leprae
`Mycobacterium paratuberculosis
`
`Actinomycetes (fungus-like bacteria) -
`Actinomyces isroelii
`Actinomyces bovis
`Actinamyces naeslundl-i
`Nocardia asteroides
`
`

`
`4,235,869
`
`9
`
`Viruses
`
`Adenoviruses
`
`Herpes viruses
`
`Herpes simplex
`Varicella (Chicken pox)
`Herpes Zoster (Shingles)
`Virus B
`Cytomegalovirus
`
`Pox Viruses
`
`Variola (smallpox)
`Vaccinia
`Poxvirus bovis
`Paravaccinia
`Molluscum contagiosum
`Picomaviruses
`
`Poliovirus
`Coxsackievirus
`Echoviruses
`Rhinoviruses
`
`Myxoviruses
`
`Influenza (A, B, and C)
`Parainfluenza (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
`Semliki Forest Virus
`Mayora Virus
`St. Louis Encephalitis Virus
`California Encephalitis Virus
`Colorado Tick Fever Virus
`Yellow Fever Virus
`Dengue Virus
`
`Reoviruses
`
`Reovirus Types 1-3
`
`Hepatitis
`
`Hepatitis A Virus
`Hepatitis B Virus
`
`Tumor Viruses
`
`Rauscher Leukemia Virus
`Gross Virus
`Maloney Leukemia Virus
`
`Allergens
`
`Receptor
`
`is the monovalent receptor.
`The next component
`While there will be some naturally occurring monova—
`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
`
`M
`& Wilkins Company, Baltimore, 1972, pp. 88ff. Fab
`fragments can be produced by digestion with a pepti-
`dase, such as papain,
`trypsin, our 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
`fluorescers include the xanthene dyes, which include
`the fluoresceins derived from 3,6-dil1ydroxy-9-phenylx-
`anthhydrol and rosamines and rhodamines, derived
`from 3,6-diamino-9-phenylxanthhydrol. The rhoda-
`mines and fluoresceins have a 9-o-carboxyphenyl group
`and are derivatives of 9-0-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 fluores-'
`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-
`benzoxazoly1)phenyl)maleimide; benzoxadiazoles, such
`as 4—ch1oro-7-nitrobenzo-2-oxa-l,3-diazole and 7-(p-
`methoxybenzylamino)-4-nitroben zo-2-oxa- 1,3—diazo1e;
`stilbenes, such as 4-dimethylamino-4’-isothiocyanatos—
`tilbene
`and
`4-dimethylamino-4’-maleimidostilbene
`N,N'-dioctadecyl oxacarbocyanine p-toluenesulfonate;
`pyrenes, such as 8-hydroxy-1,3,6-pyrenetrisulfonic acid,
`and 1-pyrenebutyric acid; meroc:yanines e.g. merocya-
`nine 540;
`rose bengal; 2,4-diphenyl-3(2H)-furanone;
`cyanines; anthraquinones; porphyrins; triarylmethanes;
`as well as other readily available dyes which are capable
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`

`
`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
`unconjugated and characteristized 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 pH1 1, will not be useful, techniques,
`can be employed involving rapid injection or flow 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-9,95
`biacridinium dinitrate is illustrative. These compounds
`chemiluminesce upon combination with alkaline hydro-
`gen peroxide.
`The next group of compounds are acridinium 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
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,235,869
`
`12
`metal complexes, particularly porphyrins and phthalo-
`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 11, preferably at
`or below 10.
`The next group of compounds are based on»
`chemiluminescers which chemiluminesce under enzy-
`matic catalysis. Primarily, there are two groups of enzy-
`matically catalyzed chemiluminescers. 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 a"g‘ents. One agent acts by the transfer-
`ring of two electrons (R2), 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 electrons. In
`some instances, the intermediate or catalyst may react
`as either R1 or R2, 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 outside of or
`as part of a cyclic structure. Therefore, common to the
`label which does not involve a metal will be the follow-
`ing formulii.
`
`x
`\\
`
`Y
`//
`
`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
`ortho or para, where the. heteroatoms are not involved
`in a heterocyclic structure. These compounds will for
`the most part have the following formulii.
`
`W‘
`
`‘W2
`
`X1
`
`H
`Y1
`
`W3
`
`A
`
`'w4.
`
`W1
`
`W2
`
`X1
`
`Y]¢
`
`W4
`
`w3
`
`wherein
`X1 and Y1 are oxygen or imino, more usually oxygen;
`
`

`
`4,235,869
`
`‘
`
`113
`W” may be hydrogen, halogen, particularly of
`atomic number ,9 to 35, more particularly of atomic
`number 17 to 35, alkyl of from 1 to 6 carbon atoms,
`more usually of from 1 to 4 carbon atoms or W
`groups on adjacent carbon atoms, particularly W1
`and W2 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 heteroatom 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, thetquinones 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 16 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 alizarin, 1,2-naphthoqui-
`none, chloranil, 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 formulii.
`
`D3
`
`D4
`
`1‘
`N
`
`’@I ‘E
`
`I:
`l
`
`IA
`
`D1
`
`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, hydroxyalkyl of from 5 to 6
`carbon atoms, particularlyisugars e.g. ribityl, A and
`Al normally being other than hydrogen when the
`broken line forms a double bond, and usually one of
`A and A1 is other than hydrogen or an unshared
`pair of electrons;
`D14 may be the same or different and are hydrogen,
`halo, particularly chloro, oxy e.g. al