United States Patent
`
`[191
`
`Rowley et al.
`
`[54] ENZYME-AMINOGLYCOSIDE
`CONJUGATES
`
`[75]
`
`Inventors: Gerald L. Rowley, San Jose; Danton
`Leung, Campbel]; Prithipai Singh.
`Santa Clara, all of Calif.
`
`[73] Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[21] Appl. No.: 125,713
`
`[22] Filed:
`
`Feb. 23, 1980
`
`Related U.S. Applieafion Data
`
`[62]
`
`Division of Ser. No. S':'6,'l'l2. Feb. 10. 1978. Pat. No.
`4,220,722.
`
`Int. Cl.3
`[51]
`.................... .. CIZN 9/96
`
`[52] U.S. CL .................................. .. 435/188; 435/ 177;
`260/112 R
`[58] Field of Search ............................. .. 435/188, 177;
`260/112 R
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`435/138
`321977 Uhnan eta].
`4.039.335
`435/188
`4.057.114 1/ms Ruhenstein et a.
`
`4,100,268 7/ 19138 Sober: ................................. .. 414/'12
`
`[11]
`
`[45]
`
`4,328,311
`
`May 4, 1982
`
`4,144,131
`
`3/1979 Richardson ....................... .. 435/114
`
`Primary E.xaminer—Lionel M. Shapiro
`Attorney, Agent, or Ffrm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Methods and compositions are provided for conjugat-
`ing a wide variety of compounds, particularly polyfunc-
`tional compounds, having a tnercapto group. either
`naturally present or synthetically introduced, to a poly-
`amino compound, particularly a polypeptide (including
`proteins). The method employs a haloalkyl-carbonyl
`compound, which is conjugated to one or more of the
`amino groups under mild acyiating conditions. This is
`followed by combining the acylated polyamino com-
`pound with a rnercapto containing compound, whereby
`the halogen is displaced‘ by the sulfur of the mercapto
`group to form a stable thioether linkage. The resulting
`conjugates, depending on the compounds involved, can
`find uses in immunoassays, as hapten-antigen conjugates
`for the production of antibodies, and as ligand analog
`enzyme conjugates for use as reagents in controlling the
`distribution of substitution of a rnercapto compound to
`a polyamino compound.
`
`17 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2046
`
`Genentech Exhibit 2046
`
`IPR2016-00710
`
`

`
`I
`
`4,328,311
`
`ENZYME-AMINOGLYCOSIDE CONJUGATES
`
`This is a divisional of application Ser. No. 8'?6,722,
`filed Feb. 10, l978, now U.S. Pat. No. 4,220,722 issued
`Sept. 2, 1930.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`There is a continuously expanding interest in being
`able to conjugate, frequently selectively, a compound to
`another compound which is polyfunctional. Where
`both compounds are polyfunctional,
`the problem of
`conjugation is exacerbated, if one does not wish all of
`the functional groups to participate in the reaction.
`Also, the need to functionalize a polyfunctional com-
`pound for conjugating to a polyamino compound will
`frequently require the introduction of protective groups
`for alcohols and amines, so that the reactive functional-
`ity does not polymerize the compound to be conju-
`gated.
`One area of particular interest is the conjugation of a
`wide variety of haptens and antigens to polypeptides
`(including proteins), particularly where conjugation is
`to occur at available amino groups. In preparing anti-
`bodies for use in competitive protein binding assays,
`where the analyte of interest is haptenic, it is generally
`necessary to conjugate the hapten to an antigen, nor-
`mally a protein. Where the analyte has a plurality of
`functionalities which can react with the active function-
`ality to be used for conjugating to the polypeptide, it
`becomes necessary to introduce removable protective
`groups to prevent polymerization of the analyte. After
`conjugation, it is usually difficult to efficiently remove
`the protective groups.
`Where the conjugate is to be used for the preparation
`of antibodies, the resulting antibodies not only recog-
`nize the analyte of interest, but the analyte having the
`protective groups. This may result in substantially re-
`ducing the specificity of the antibody composition for
`the analyte of interest.
`One class of competitive protein binding assays in-
`volves the use of enzymes as a label. It is necessary to
`conjugate the analyte of interest to the enzyme. It is
`desirable that certain reactive site positions on the en-
`zyme be preferentially conjugated as compared to other
`reactive site positions. A method which would provide
`the ability to discriminate to even a partial degree is
`desirable.
`In addition, to have an enzyme which has been modi-
`fied. whereby the same sites will be conjugated to ana-
`lytes, regardless of the particular analyte, can provide a
`number of advantages. For example, in one of the assays
`which employs an enzyme as a label, it is desirable that
`the enzyme retain a substantial proportion of its initial
`activity after conjugation, but when antibody or other
`receptor is bound to the analytes conjugated to the
`enzyme, the enzymatic activity is substantially reduced.
`The fewer the analytes necessary to conjugate to the
`antibody to obtain the desired degree of reduction in
`enzymatic activity upon the binding of antibody or
`other receptor to the conjugated analyte,
`the more
`sensitive will be the assay response.
`In addition, where a universal reagent can be em-
`ployed for conjugation, greatly increased experience
`can be obtained in the handling of the compounds, the
`reacting of the compounds, as well as the subsequent
`handling and treatment after conjugation. This can
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`2
`provide for great efficiencies in synthesizing and subse-
`quent formulation.
`2. Description of the Prior Art
`Kato, et al, Eur. J. Biochem. 62, 285 (1976), discloses
`the use of maleic anhydride with a polyamino com-
`pound to provide one or more maleirnide groups, fol-
`lowed by the addition of a compound with a rnercapto
`group to add to the double bond of the maleimide. See
`also, Lee and Kenny, Clinical Chem. 21, 967 (1975).
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for combin-
`ing a polyfurictional compound having a plurality of
`reactive primary and/or secondary amino functionali-
`ties with a second compound having a rnercapto func-
`tionality, usually polyfunctioual, having functionalities
`reactive to acyl groups e.g. hydroxylic and amino. The
`polyamino compound is initially reacted with a linking
`compound having an active halogen or pseudohalogen
`and a non-oxo carbonyl functionality for reacting with
`at least one of the amino functionalities. The Inercapto
`compound is then added to the halo or pseudohalo
`containing polyarnino compound for substitution of the
`halo groups to provide a thioether linked conjugate of
`the mercapto compound with the polyamino com-
`pound.
`The method finds particular use in the preparation of
`polypeptide and protein conjugates for preparing anti-
`gens, enzyme conjugates for immunoassays, fluorescent
`labeling of polypeptides and proteins and the like. By
`employing the subject method, one can obtain a consis-
`tent pattern of substitution, the conjugation can be car-
`ried out under extremely mild conditions and some
`control of the positions of substitution can be achieved.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`The method of the subject invention involves the
`labeling as a first step of a polyfamino acid) compound,
`normally a polypeptide or protein, with a compound
`having an a.-halo or a-pseudohaloalkylcarbonyl func-
`tionality and a non-oxo carbonyl functionality (includ-
`ing the nitrogen analog,
`imido. and sulfur analog,
`thiono), which may be the same or different from the
`carbonyl of the ct-halo or rt-pseudohaloallcylcarbcnyl
`functionality. The reaction is carried out in a normally
`aqueous medium under mild pH conditions, generally at
`9.5 or below, so as to form an amide (including the
`nitrogen and sulfur analogs, imidine and thioamide re-
`spectively). The product may then be putified under
`conventional conditions and the halo or pseudohalo
`substituted by a mercaptan under mild conditions in an
`aqueous solution at moderate pH, normally basic pH.
`The product may then be worked up and isolated.
`The compounds prepared in accordance with this
`invention have many uses, for example hapteus or anti-
`gens may be conjugated to labels, such as fluorescers
`and enzymes, and the resulting compounds employed in
`immunoassays for the determination of such haptens
`and antigens. In addition, haptens may be conjugated to
`antigens to be used for the production of antibodies,
`which may also serve as reagents in immunoassays.
`The subject method provides a means for preparing
`derivatives of polyamino compounds, which have a
`limited number of active sites. The distribution of these
`active sites may be retained substantially constant, so
`that when conjugating haptens and antigens to the poly-
`amino compounds, substitution will be relatively uni-
`
`

`
`4,328,311
`
`3
`form, regardless of the particular compound which is
`conjugated.
`In addition, it will normally be found that due to the
`position of the halo substituent on the polyamino com-
`pound, the halo compounds may have varying activi-
`ties. One can then distinguish between the varying ac-
`tivities, by employing two different rnercaptan reagents,
`the first reagent being added in a sufficient amount to
`react with all or substantially all of the more reactive
`halogen. In this manner, the mercaptan compound of 10
`interest may be directed either to the more or less reac-
`tive sites. Also, the subject method provides for syn-
`thetic convenience, for so far as the polyamino com-
`pound, the same compound may be repetitively pre-
`pared, regardless of the compound to which it is to be
`conjugated.
`
`5
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`4
`rated and may be straight or branched chain, preferably
`straight chain;
`Z—-OV or OC0-alkyl, wherein alkyl is of from I to 6,
`usually 1
`to 4 carbon atoms and V is hydrogen, p-
`nitrophenyl, N-oxy succinimide, or when Y is NH and
`m is zero, or Y1 is NH and m is 1, alkyl of from 1 to 6
`carbon atoms.
`k, In and p—zero or 1, wherein p is zero when D and
`(CY?) form an isothiocyanate group
`The preferred halo compounds of this invention will
`have the following formula:
`
`Y3
`1'2
`II
`II
`BrCH2CNHD1CZ'
`
`wherein:
`
`Y3 and Y3—0, NH, preferably 0
`D1—alky1ene of from 1 to 8, usually I to 4 carbon
`atoms
`Z'—the same as Z, usually OH or N-oxy succinimide
`Illustrative compounds include
`N-bromoacetyl glycine, N-bromoacetyl valine, N-
`brornoacetyl 4-aminobutyric acid, N-bromoacetyl 3-
`aminopropionic acid, p-chloroacetylbenzoic acid, p-
`bromoacetylphenylacetic
`acid, N-bromoacetyl
`4-
`aminocrotonic acid,
`their p-nitrophenyl esters,
`their
`N-succinimidyl esters, p-chloroacetylphenyl isothiocy-
`auate, and methyl N-bromoacetyl glycinimidate.
`The next group of compounds to be considered, are
`the polyamino functionalized compounds, which are
`primarily polypeptides and proteins, but may also in-
`clude polyglucosamines and nucleic acids. These com-
`pounds may be included in combinations or assemblages
`which include bacteria, viruses, chromosomes, genes.
`mitochondria, nuclei, cell membranes and the like.
`For the most part, the compounds will have a molec-
`ular weight of at least about 5,000, more usually at least
`about 10,000. In the poly(amino acid) category (in-
`cludes polypeptides and proteins),
`the po1y(amino
`acids) of interest will generally be from about 5,000 to
`5,000,000 molecular weight, more usually from about
`20,000 to 1,000,000 molecular wght. In this category,
`hormones of interest will generally range from about
`5,000 to 60,000 molecular weight. Enzymes of interest
`will generally range from about 10,000 to 300,000 mo-
`lecular weight. Immunoglobulins and portions thereof
`e.g. Fab fragments and Bence-Jones proteins, will gen-
`erally range from about 23,000 to 1,000,000, with the
`irnmunoglobulins generally ranging from 150,000 to
`1,000,000.
`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
`histones
`albumins
`globulins
`scleroproteins
`phosphoproteins
`mucoproteins
`chromoproteins
`Iipoproteins
`nucleoproteins
`
`MATERIALS
`
`The materials which are employed in the subject
`invention are the active halogen or pseudohalogen com-
`pound,
`the polyamino polyfunctional compound to
`which the halo or pseudohalo compound is conjugated,
`and the mercaptan which is employed for substitution
`on the halogen or pseudohalogen.
`The first compounds to be considered will be the halo
`or pseudohalo compounds. These compounds will nor-
`mally be of from 2 to 20, more usually of from 2 to 16
`carbon atoms, and preferably of from about 2 to 12
`carbon atoms. Other than the halo or pseudohalo group.
`the compound will normally have at least two heteroat-
`oms, and may have as many as 20 heteroatoms, more
`usually having from about 2 to 12 heteroatoms, and
`preferably from about 2 to 8 heteroatoms. The heteroat-
`oms will normally be oxygen, nitrogen and sulfur or any
`appropriate counterion for a charged species. Oxygen
`will normally be present as in nitro, oxo or ether (an
`ester includes 01:0 and ether oxygens); nitrogen will be
`present as in nitro, amido, or bonded solely to carbon,
`e.g. tertiary amine; and sulfur will be present as thiouo
`or thioether. The compounds will of necessity include
`aliphatic groups, but may also include alicyclic, aro-
`matic, and heterocyclic groups.
`_For the most part, the compounds used for conjuga-
`tion to the amino functionalized compounds will have
`the following formula:
`
`'45
`
`if’
`ii
`XCH2C((A)k{D)m(C)n:)m(Z)p
`
`wherein:
`X--Cl,Br,CH3SO3 (mesylate), preferably Br;
`Y and ‘{‘—0, NH, 5, preferably 0;
`A-—NH, 0, preferably NH;
`D—chain of from 1 to 9, usually I to 6 atoms in the
`chain, having a total number of atoms other than hydro-
`gen or from I to 12, usually 1 to 10, preferably 1 to 6,
`which may be C, O, N and 8, usually C, 0 and N,
`wherein: 0 is present as oxo or ether, particularly non-
`oxo carbonyl; N is present as amido or bonded solely to
`carbon and may be present as terminal nitrogen doubly
`bonded to (CY1) where Y1 is S to form isothiocyanate;
`and S is present as thiono or thioether; preferably hy-
`drocarbon to form a hydrocarbylene group which may
`be aliphatic,
`alicyclic,
`aromatic or
`combinations
`thereof, preferably aliphatic, which may be aliphatically
`saturated or unsaturated having from 0 to i site of unsat-
`uration i.e. ethylenic and acetyienic, preferably satu-
`
`50
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`55
`
`65
`
`

`
`5
`
`4,328,311
`
`C3
`BIA
`a2D
`C4
`C5
`C6
`C”?
`C8
`C9
`
`glycoproteins
`unclassified proteins, e.g. sotnatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`cc;-Lipoprotein
`ct]-Acid glycoprotein
`on-Antitrypsin
`cc;-Glyeoprotein
`Transcortin
`4.68-Postalbumin
`
`5
`
`10
`
`Tryptophan-poor
`ct 1-glycoprotein
`a1X-Glycoprotein
`Thyroxin-binding globulin
`Inter—a—trypsin-inhibitor
`Gc-globulin
`(Gc 1-1)
`(Go 2-1)
`Gc 2-2)
`Haptoglobin
`(Hp 1-1)
`(Ht? 2-1)
`(Hp 2-2)
`Ceruloplasmin
`Cholinesterase
`ct:-Lipoprotein(s)
`oz-Macroglobulin
`0.;-HS-glycoprotein
`Zn-ct;-glycoprotein
`a;--Nenramirio-glycoprotein
`Erythropoietin
`,8-lipoprotein
`Transferrin
`Hernopexin
`Fibrinogen
`Plasminogen
`132.31}-coprotein I
`§2_g1ycopmte,'_n II
`Immunoglobulin G
`{IgG) or -yG-globulin
`Mg1_ fm-1-mfla,
`“K2 0, 1.2;”
`Immunoglobufin A (1gA)
`or -yA_g1obu1§n
`Mo1_ formula,
`(a2,,,2)n 0,. (a2;r2)n
`1mmm,og1o1-,u]j,, M
`(lgM) or -yM—g1obulin
`MOL formula:
`(pzkfls or (pan):
`Immunoglobulin D(IgD)
`or *yD-Globulin ('yD)
`Mo1_ fol-mu}a;
`(339) or (522.2)
`Immunoglobulin E (IgE}
`or «),E.G10bu;gn (713)
`Mol. formula:
`(um) or (5212)
`Free K and -11 light chains
`Complement factors:
`C’)
`(315;
`C1;—
`C15
`C2
`
`25
`
`3!]
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Important blood clotting factors include:
`
`BLOOD CLOTHNG FACTORS
` International designation Name
`
`I
`Fibriruogen
`II
`Prothrombin
`Ila.
`Thrornbin
`1]!
`Tissue thromhopiastin
`V and VI
`Proacceierin. accelerator
`globulin
`Proconveflia
`Antilretnophilic globulin (AI-IG)
`Christmas factor,
`plasma thrornboplnstin
`component (FTC)
`Stuart-Prower factor,
`antoprothrombin HI
`Plasma lhromboplastin
`antecedent (PTA)
`Hagemaxm factor
`XII
`
`XIII Fibrin-stabilizing factor
`
`X
`
`XI
`
`VII
`VIII
`DC
`
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`{P31'3th1'Pm0_1'1€)
`Thyrpcalcstonm
`I115‘-11111
`Glncagon
`R9133”
`Eorthropoietin
`_
`M31’-‘~fl0“'°Pm
`(melanocyte-stunulating hormone; intermedin)
`Somatotropin
`(gfowth hormone)
`Cortwotropm
`(adrenocortiootropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Lutemtzing hormone
`(interstitial cell-stimulating hormone]
`Luteomarnrnotropic hormone
`(Iuteotropin, prolactin)
`Gonadotropin
`(chorionic gonadotropin)
`’
`Tissue Hormones
`Secrefln
`Gasfirm
`Angtotensin I and II
`Bradykinin
`Human placental iactogen
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Rcleasing f3°t°1'5 (RF)
`SOTnat0tr0pifl'RF,
`GRF, FSH-RF, PIF, MIF
`The rnicroorganisnis 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:
`
`

`
`7
`
`8
`
`4,328,311
`
`Corynebacteria
`Corynebocrerium diptherioe
`Pneumococci
`Dipiococcus prieumoniae
`Streptococci
`Streptococcus pyogencs
`Streptococcus salimnu
`Staphylococci
`Staphylococcus aureus
`Staphylococcus albus
`Neisseria
`Neisseria meningiridis pl Neisserio goriofrheae
`
`
`.
`
`5
`
`10
`
`Acrirzomyccs isrczeiii
`Aciinomyces bovis
`Acrinomyces noesizma'ii
`Nocordia asteroides
`Nocardia brosiliensis
`The Spirochetes
`Treporiemo paiiidum
`Treporicma perrerme
`Treponemo camtenm
`Borrelio recurrerriis
`Lepiospiro icrerohemorrhogioe
`Leptospiro conicoio
`Spirillum minus
`Srrepiobociilus moniiifilrmis
`-
`Mycoplasma pneumomae
`0th?r paflhogens
`Listeria monocyrogenes
`_g,3,5,;pe;0;;,,;J¢ ,}m_9fopm«;”'ae
`_
`_
`Srrepiobaciiius moniiifiyrmis
`Dorivamo grarmiomotis
`BGFIGHEHG bcciiizfonnis
`Rickettsiae (bacteria~Iike arasites)
`-
`«
`n
`p
`Rickertsia prowazeku
`R . k
`.
`.
`IC etisia 7.50059?!
`Rickerisio 1'ickeiis1‘i
`Rxckerisuz canon
`Rickerisio ozisrroiis
`Rickeirsia sibiriws
`Ricicetrsia okari
`Rickemia isuisitgomushi
`Rickemio bomeiii
`R,-ckem,-a gm-,1mm
`Chlamydia (unclassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`
`Fungi
`
`Cryptomccus ncojbrmans
`Biauomyces dermaiidir
`gigizizjgii £‘::-‘:_‘fl’;:::'““
`mmcmfdmes ,,,‘,_,m,_,m,
`Candida aibimns
`Aspersiiiusfiznfisatw _
`ifigczrfifrapwoiier (Absrdia carymbtfera)
`Rhizoim afifizus
`J» Phycumycctcs
`Rhizopus nigrimm
`5.00r0m'r-‘Mm Icfieflkii
`§}""“"""’“ P'd”’““"
`32:3: ::T:::$¢-3
`Ciadasparium carrionii
`P»'=='*=-*0?-‘=m Wrmmm
`A‘3"’3m“’ ”'H"f“"‘.
`Moduneffa myrerarm
`Madmm, 8,,-W,
`Aiiescheria boyziii
`Pfiiiawlomiednseimef
`MiF”‘P°”” 3”"""“""
`Emopkym mflmimphym
`emiinomyces ajeiim
`Microsporum mm‘:
`Trfbfiopfiywn mbrrtm
`M"""’-"""“"‘ ‘"’d°“*""
`
`20
`
`25
`
`39
`
`35
`
`4.0
`
`.45
`
`50
`
`55
`
`60
`
`Viruses
`Adenoviruses
`65 Herpes viruses
`Herpes simplex
`Varicella (Chicken pox)
`Herpes Zoster (Shingles)
`
`}
`
`The coliform bacteria
`
`The Salmoncliac
`
`The Shiscllae
`
`ENC id’
`I
`/lembacfer aerogenes
`Kiebsieiia pneumonia:
`Saimoneiia iyplmm
`gaimo.-regs: ci:o:.~_me:zp's
`13.-ziia lf}'9£!i.l'2!.lfl€
`sg_”'°"” " W ".""”"'”
`5_;,,-gem, ,c;,,,,,-,,,-,-
`Shigeiia arabinorarda
`Silllzeifflflexflefi
`Sixi eiic baydii
`Sh?
`.
`Igella Son11e1
`other mneric bacilli
`9,0,2“, mgari,
`Proteus mirabiiir
`‘
`Pmfer-'5 morzwlf
`f;:g°;f;:f;;;§f"m
`’3
`Vibrio ckoiemc
`
`}
`
`Proteus species
`
`-
`
`I-Iemophilus-Bordetella group
`Hem°Ph‘h‘3 f"'fl“e’”m‘-"
`H- d“°"9J"
`H. hemophilus
`H. aegppticus
`H. poraiufluenzoe
`Bordetelio periumis
`Pasteufellae
`Pasteureiia pesiis
`Pasteureiio mloreusis
`Bruce-llae
`Broceiia meiirensis
`Bruceya aborms
`Brmzeua ‘M3
`Aeroblc Spore-forming Bacilli
`Bocifius on thmcis
`Bociiius subiilik
`Bociihis megoterium
`Bocfiius cereus
`Anaerobic Spore-forming Bacilli
`Ciosiridium boiulin om
`.
`.
`.
`Closrridmm teiom
`Ciosiridium perfringens
`Closrridium novyi
`Ciosiriditem sepiicum
`Ciosiridium hisioiyiicum
`_
`_
`_
`Ciosmdmm rertmm
`Ciosrridium bifiermenmns
`Ciosiridium sporogenes
`Mycobacteria
`Mycobocreriom tuberculosis hominis
`Mycobocterium bovis
`Mymbocterium ovium
`Mycobacierium ieproe
`Mycobacterium paramberculosis
`Actinornycetes (fungus-like bacteria)
`
`

`
`9
`
`4,328,311
`
`5
`
`15
`
`21}
`
`25
`
`10
`1.10 Acting on diphenols and related substances as
`donors
`1.10.3 With 02 as acceptor
`1. polyphenol oxidase
`3. ascorbate oxidase
`1.11 Acting on H307, as acceptor
`1 .1 1.1
`6. catalase
`7. peroxidase
`10 3. Hydrolases
`3.1 Acting on ester bonds
`3.1.1 Carboxylic ester hydrolases
`7. cholinesterase
`3.1.3 Phosphoric monoester hydrolases
`1. alkaline phosphatase
`3.1.4 Phosphoric diester hydrolases
`3. phospholipase C
`3.2 Acting on glycosyl compounds
`3.2.1 Glycoside hydrolases
`1. ct-amylase
`4. oellulase
`1?. lysozyme
`23. 33-galactosidase
`2'.-'. amyloglucosidase
`31. B-glucuronidase
`3.4 Acting on peptide bonds
`3.4.2 Peptidyl-amino acid hydrolase
`1. carboxypeptidase A
`3.4.4 Peptidyl-peptide hydrolase
`5. mchyrnotrypsin
`10. papain
`3.5 Acting on OK‘ bonds other than peptide bonds
`3.5.1 In linear amides
`5. urease
`3.6 Acting on acid anhydride bonds
`3.6.1 In phosphoryl-containing auhydrides
`1. inorganic pyrophosphatase
`4. Lyases
`4.1 Carbon-carbon lyases
`4.1.2 Aldehyde lyases
`7. aldolase
`4.2 Carbon-oxygen lyases
`4.2.1 Hydrolases
`1. carbonic anhydrase
`4.3 Carbon-nitrogen lyases
`4.3.1 Ammonia lyases
`3. histidase
`
`Virus B
`Cytomegalovirus
`Pox Viruses
`Variola (smallpox)
`Vaccinia
`Poxvfms bavis
`Paravaccinia
`Molluscum contagiosum
`Picornaviruses
`Poliovirus
`Coxsackievirus
`Echoviruses
`Rhinoviruses
`Myxoviruses
`Influenza (A, B, and C)
`Parainlluenza (1-4)
`Mumps Virus
`Newcastle Disease Vims
`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
`Enzymes of interest are classified in accordance with 45
`the l.U.B. classification as follows:
`1. Oxidoreductases
`1.1 Acting on the CH-OH group of donors
`1.1.1 With NAD or NADP as acceptor
`1. alcohol dehydrogenase
`6. glycerol dehydrogenase
`26. glyoxylate reductase
`27. I.-lactate dehydrogenase
`37. malate dehydrogenase
`49. glucose 6-phosphate dehydrogenase
`I1’. mannitol 1-phosphate dehydrogenase
`1.1.2 With cytochrome as an acceptor
`3. L-lactate dehydrogenase
`1.1.3 With 01 as acceptor
`4. glucose oxidase
`9. galactose oxidasc
`1.2 Acting on the CH-NH; group of donors
`1.43 With 0; as acceptor
`.
`2. L-amino acid oxidase
`
`30
`
`35
`
`50
`
`55
`
`_
`3. D-amino acid oxidase
`1.6 Acting on reduced NAD or NADP as donor
`1.6.99 With other acceptors
`diaphorase
`
`65
`
`Of particular interest are the dehydrogenases, illus-
`trated by malate dehydrogenase and glucose-6-phos-
`phate dehydrogenase, the hydrolases, such as ,3-ga]ac-
`tosidase and lysozyme, and peroxidases.
`In the next group of compounds are the mercaptan
`compounds, which may have a mercaptan group natu-
`rally or may have a mercaptan group introduced. With
`a naturally occurring compound of interest having a
`plurality of rnercaptau groups,
`it will frequently be
`necessary to deactivate such groups by functionalizing
`them with permanent or removable groups. Usually, the
`functionalization will be by the formation of a thioether.
`'I'l1erefore, when the compound of interest has a plural-
`ity of mercaptan groups present, the compound of inter-
`est will be treated with a reagent which will react with
`the rnercapto groups to prevent their subsequent reac-
`tion in the conjugation and a mercaptan group then
`introduced synthetically to provide the unique site of
`conjugation.
`Similar considerations are involved with the poly-
`amino functionalized compound. Where the polyarnino
`
`

`
`4,328,311
`
`' 11
`functionalized compound has one or more active mer-
`capto groups, these may be deactivated prior to intro-
`duction of the ct-haloalkyl carbonyl compound.
`The compounds of interest will generally have either
`hydroxyl or amino functionalities or both as sites for
`conjugation to a disulfide linkage. The linking'function-
`ality will normally be an ester, amide or ether. Where a
`plurality of functionalities are present in the molecule of
`interest, which functionalitles may react with the disul-
`fide compound, it will frequently be necessary to pro-
`tect the other functionalities with removable groups
`prior to conjugation of the disulfide containing com-
`pound. In some instances, a mercapto group will be
`naturally present, and this can serve as the site for link-
`ing without synthetic introduction of a mercaptan.
`For the most part, the rnercapuo compounds and their
`precursors will have the following formula:
`
`12
`pound may be bonded to a convenient polypeptide or
`protein, desirably a relatively large number of haptenic
`compounds being conjugated to the polypeptide or
`protein.
`A third possibility is the conjugation of a label to a
`polypeptide or protein for use in an immunoassay for
`determination of the polypeptide or protein. Labels can
`take many forms. One class of labels is fluorescent com-
`pounds. Another class of labels is labels having enzy-
`rnatically labile bonds. In this instance, the labile bond
`may be bonded distant from the thio ether linkage or
`may be part of the linkage between the compound of
`interest and the polypeptide or protein. Thus, the link
`between the compound of interest and the polypeptide
`or protein may be cleaved by an enzyme. These types of
`compounds
`include coenzymes,
`fluorescent
`com-
`pounds, chemiluminescent compounds, chemical cata-
`lysts, electron transfer agents, dyes, and the like.
`The first group of compounds are monoepitopic li-
`gands, which are generally involved in assays for their
`determination. These include drugs which are used for
`therapeutic purposes, naturally occurring physiological
`compounds, metabolites, pesticides, pollutants, and the
`like.
`Included among drugs of interest are the alkaloids.
`Among the alkaloids are morphine alkaloids, which
`includes morphine, codeine, heroin, dextrornethorphan,
`their derivatives and metabolites; cocaine alkaloids,
`which includes cocaine and benzoyl ecogonine, their
`derivatives and metabolites; ergot alkaloids, which in-
`cludes the diethylamide of lysergic acid; steroid alka-
`loids;
`iminazoyl alkaloids; quinazoline alkaloids;
`iso-
`quinoline alkaloids; quinoline alkaloids; which includes
`quinine and quinidine; diterpene alkaloids, their deriva-
`tives and metabolites.
`The next group of drugs includes steroids, which
`includes the estrogens, gestrogens, androgens, an-
`drenocortical steroids, bile acids, cardiotonic glycosides
`and aglycones, 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 lactan-is having from 5 to
`6 annular members, which include the barbiturates, e.g.
`phenobarbital and secobarbital,diphenylhydantoin, and
`their metabolites.
`
`The next group of drugs is aminoalkylbenzenes, with
`alkyl of from 2 to 3 carbon atoms, which includes the
`amphetamines, catecholamines, which includes ephed-
`rine, L-dopa, methyldopa, epinephrine, narceine, pa-
`paverine. their metabolites and derivatives.
`The next group of drugsis benzheterocyclics which
`include benzothiadiazides, oxazepam, chlorpromazine,
`tegretol, imipramine, their derivatives and metabolites.
`the heterocyclic rings being azepines, diazepines,
`thiadiazines, and phenothiazines.
`The next group of drugs is purines, which includes
`theophylline, caffeine, theobromine, their metabolites
`and derivatives.
`
`The next group of drugs includes those derived from
`marijuana, which includes cannabinol and tetrahydro-
`cannabinol.
`The next group of drugs includes the vitamins such as
`A, B, C, D, E and K.
`The next group of drugs is prostaglandins, which
`differ by the degree and sites of hydroxylation and
`unsaturation.
`
`10
`
`15
`
`20
`
`'25
`
`30
`
`35
`
`'45
`
`SO
`
`55
`
`65
`
`EM(Q},;TSU
`
`wherein:
`E—the compound of interest, which will be discussed
`in more detail subsequently, which may be modified by
`introduction of an hydroxyl or amino functionality, by
`protection of one or more reactive functionalities with
`protective groups, desirably removable, or in any other
`manner appropriate to the purpose of its intended use;
`M—O, NH
`Q—C= W, wherein W is 0, NH or S, particularly 0,
`T—a linking group having "at least one carbon atom
`and not more than 9 atoms, other than hydrogen, usu-
`ally one to four atoms, which are carbon, oxygen, nitro-
`gen and sulfur, preferably carbon, the. terminal atoms
`being carbon atoms, any oxygen is present as oxy ether
`or oxo, particularly non-oxo carbonyl; nitrogen is pres-
`ent as arnido or bonded ‘solely to carbon and hydrogen
`e.g. amino, primary, secondary or tertiary; and sulfur is
`present as thiono or tbioether; the number of heteroat-
`oms being in the range of zero to 4, usually zero to 2; T
`is preferably hydrocarbon, more preferably alltylene,
`there being not more than one site of aliphatic unsatura-
`tion, either branched or straight chained, preferably
`straight chained, particularly methylene or polymethyl-
`ene (CH2):,, where b is from 1 to 4;
`S—sulfur
`U—H, alkylthio of from one to four carbon atoms,
`particularly methyl, or an alkali metal cation of atomic
`number 3 to 19;
`'
`'a—-zero or I.
`
`Where E has a natural occurring mercaptan group, M
`and T are taken together to form a single bond, a is zero
`and U is hydrogen.
`'
`'
`-Depending upon the purpose of the conjugate, the
`mercaptan_containing compound may vary widely. Of
`particular interest are conjugates to enzymes which are
`intended for use in immunoassays. Of greater interest,
`are those enzyme conjugates which are employed in a
`homogeneous enzyme immunoassays as described in
`U.S. Pat. No. 3,817,337. In these conjugates, when anti-
`body binds to the mercapto compound conjugated to
`the enzyme, there is a substantial reduction in the enzy-
`n1atic‘activity. Therefore, it is quite advantageous once
`one has developed a particular distribution of active
`sites for a particular enzyme, that the mercapto com-
`pounds will bond substantially to the same sites, regard-
`less of the nature of the mercapto compound.
`Another use for the conjugates is for forming anti-
`genic products for haptens, so as to be able to-produce
`antibodies. In this situation, any non-antigenic com-
`
`

`
`4,328,311
`
`13
`The next group of drugs is antibiotics, which includes
`rnacrolides, aminoglycosides,
`,8~lactams, etc. such as
`penicillins, ethambutol, isoniazid, vancomycin, methe-
`namine mandelate, chloromycetin, actinomycetin, tetra-
`cyclines,
`terrarnycin, cephalosporins, erythromycin,
`rifampin, clindarnycin the aminoglyoosides such as
`streptomycin, gentamicin, tobramycin, amikacin, karm-
`mycin, neomycin, nalidixic acid, nitrofurantoin, colisti-
`rnethate, lincomycin, amphotericin B, flucytosine, their
`metabolites and derivatives.
`The next group of drugs is the nucleosides and nucle-
`otides, which include ATP, NAD, FMN, adenosine,
`guauosine, thymidine, uridine and cytidine with their
`appropriate sugar and phosphate substituents.
`The next group of drugs is miscellaneous individual
`drugs which include methadone. phenoxybenzaminc
`and related haloalkylamines, tolamol, sotalol, guane-
`thide, rneprobamate, serotonin, rneperidine, chlorcycla-
`zine, chlorphenirarnine, amitriptyline, nortriptyline,
`lidocaine, procaineaniide, acetylprocainearnide, pro-
`panolol, griseofulviu, butyrophenones, antihistamines.
`methotrexate, aminopterin, anticholinergic drugs, such
`as atropine. their metabolites and derivatives.
`The next group of compounds is amino acids and
`small peptides which include polyiodothyronines e.g.
`thyroxine, and triiodothyronine, oriytocin, ACTH, an-
`giotensin, endorphin, met- and leu-enkephalin,
`their
`metabolites and derivatives.
`
`Metabolites related to diseased states include sper-
`mine. galactose, phenylpyruvic acid, and porphyrin
`type 1.
`Among pesticides of interest are polyhalogenated
`biphenyls, phosphate esters,
`thiophosphates, carba-
`mates, polyhalogenated sulfenamides, their metabolites
`and derivatives.
`
`Electron transfer labels will for the most part be
`either metal complexes or aromatic compounds.
`Compounds of interest are ligands, particularly hap-
`tenic ligands, bonded to poiy(amino acids), particularly
`antigen and enzymes. These compounds will have for
`the most part the following formula:
`
`Y
`II
`
`r'
`ll
`
`EEM(Q)dTSCH2C((A)3t(D)r!:(c)m)mgJTPA-A
`
`wherein all of the symbols have been defined previ-
`ously except:
`PAA—-poly(amino acid); and
`j——a number on the average of at least one and not
`more than the mo