[19]
`United States Patent
`4,351,760
`[11]
`
`Khanna et al.
`(451
`Sep. 28, 1982
`
`[56]
`
`References Cited
`
`260/112 R
`2130/112 B
`24/12 X
`250/112 R
`260/I21 X
`
`Us‘ PATENT DOCUMENTS
`4,111-.334 ll/1979 Ullman et al.
`Jaime.“ 3‘ 31'
`,
`.
`33:0
`4,230,305 10/1930 Singh et al.
`..
`4313.846
`3/1982 Khanna et al.
`Ex
`' —
`.
`
`
`
`'
`
`P '
`
`ABSTRACT
`[ST]
`Fluorescent antigen conjugates are provided compris-
`ing antigens covalently bonded to at least one 2,7-diaIi-
`phatic
`substituted-9-phenyl-6-hydroxy-3H-.xanthen-
`3-one, wherein the 1- and 8-positions are unsubstituted.
`Also provided are novel fluorescent compounds ab-
`sorlaing at yvave_lt;ngths i_n excess of 500_ nm,
`l1_aving
`actlve funcuonallttes for lmkmg to the antigen. Finally,
`methods are provided for analyzing antigens in serum,
`whereby serum interference is avoided.
`
`7 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2036
`
`Genentech Exhibit 2036
`
`IPR2016-00710
`
`[54] NOVEL ALKYL SUBSTITUTED
`FLUORESCENT COMPOUNDS AND
`POLYAMINO ACID CONJUGATES
`
`[75]
`
`31 -
`
`Inventors: Pyare Kharina, Mountain View;
`F- U“"'3"s -”“h"'°“= 5'0"‘ °f
`.
`
`.
`
`comm
`ml
`[21] Appl. No.: 73,153
`
`[22] Filed:
`
`SE11. 7. 1979
`
`[51]
`
`]m_ C]; _________________ __ A51]( 39/335; A61K 39/44;
`CUIG 7/00
`[52] U.S. Cl. .............................. 260/112 R; 23/230 B;
`260/I12 B; 260/112.5 R; 260/112.7; 260/ 121;
`424/8; 424/12; 424/35; 424/88; 435/7;
`435/ 188; 525/420; 549/388
`[58] Field of Surch ............. .. 260/112 R. 112 B, 121;
`424/35, 88; 435/188; 525/420
`
`

`
`1
`
`4,351,760
`
`NOVEL ALKYL SUBSTITUTED FLUORESCENT
`COMPOUNDS AND POLYAMINO ACID
`CONJUGATES
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`Fluorescent compounds find a wide variety of appli-
`cations. They find use in fluorescent
`immunoassays,
`histochernical staining, displays, inks, and the like. Of
`particular interest for the subject invention is the use of
`antigenic conjugates (includes receptor conjugates)
`with fluorescent compounds to be used in the determi-
`nation of a variety of ligands, both antigens and recep-
`tors. A substantial proportion of the ligands are assayed
`in physiological fluids. such as serum. where the serum
`can provide substantial background fluorescence. One
`way to diminish the background fluorescence resulting
`from naturally present fluorescers is to provide a fluo-
`rescent compound which absorbs at relatively long
`wavelengths. The compound should desirably have a
`large Stokes shift, be stable under conditions of the
`assay, be relatively free of non-specific interference,
`both from materials in solution and the compound to
`which the fluorescer is conjugated and to provide high
`quantum yields. In addition. for certain applications, it
`is desirable that
`the fluorescer be coupled with a
`quencher molecule, that is a molecule which is capable
`of absorbing the energy of the lluorescer in the excited
`state when within a predetermined distance, so that the
`fluorescer does not fluoresce.
`2. Description of the Prior Art
`A large number of lluorescein derivatives have been
`reported in the literature. The following are believed to
`be the most exemplary in relation to the subject inven-
`tion and are reported in conjunction with the Chemical
`Abstracts citation. The numbering is based on the par-
`ent molecule
`3',6'-dihydroxyspiro
`[isobenzofuran-
`l(3l-l),9’-(91-l)xanthen]~3~one.
`2',':"-di(n-hexyl) or di(n-heptyl)-4', 5'-dibromo-4,1
`dichloro- are reported as being prepared, CA. 31, I621;
`2',7’-di(n-hexyl)-, C.A. 31, 1621; 2',’7'-di(alkyl)-; C.A. 31,
`1388; 2','."-diethyl or 2','.-"—dibutyl-, CA. 27, 5056; 2’,'l"-
`dimethyl-, CA. 83,
`l89'l'2s; 2',4',5',7'-tetrabromo-5 or
`6-carboxy. CA. 63, l32l0h.
`SUMMARY OF THE INVENTION
`
`The subject compounds include novel fluorescent
`conjugates with members of specific binding pairs, li-
`gands and receptors, as well as the fluorescent precur-
`sors to the conjugates. The conjugates find a wide vari-
`ety of uses, particularly as reagents in immunoassays.
`The compounds are 2,Tr‘-dialiphatic-6-hydroxy-3H-xam
`then-3-ones, normally having at least two chloro sub-
`stituents, with the precursors having a linking group or
`functionality on a group, either aliphatic or aromatic,
`bonded to the 2- or 9-position of the xanthene.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`invention concerns fluorescent com-
`The subject
`pounds, which are analogs of iluorescein, being particu-
`larly
`2,7-dialiphatic
`substituted-9-substituted-6-
`hydroxy-3H-xanthen-3-ones. usually having at least two
`chloro substituents at other than the l,8-positions and
`having a functional group for linking to a member of a
`specific binding pair bonded to a hydrocarbon group
`substituted at the 2- or 9-position, particularly 9-posi-
`
`I0
`
`15
`
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`35
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`45'
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`SD
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`
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`
`2
`tion, as well as the conjugates of the fluorescent com-
`pound to the member of the specific binding pair. The
`conjugates find particular use as reagents in assays for
`members of specific binding pairs.
`The fluorescent precursors will have at least about 15
`carbon atoms, usually 21 carbon atoms, and usually not
`more than about 40 carbon atoms, usually having from
`about 22 to 36 carbon atoms. There will preferably be at
`least two chlorine groups at other than the 1,8-positions
`and may be as many as 6 chlorines. In addition to chlo-
`rine, the only other heteroatoms are bromine, chalce-
`gen, particularly oxygen and sulfur. and nitrogen, there
`being at least 4 heteroatoms and usually not more than
`20 heteroatoms, more usually not more than about 16
`heteroatoms and preferably not more than about 12
`heteroatoms. Of the heteroatoms other than chlorine,
`there will be at least 3 oxygens, more usually at least 5
`oxygens, and other than the oxygens which are part of
`the xanthene chromophore, are oxygens as non-oxo-
`carbonyl or oxy, particularly acid, ester or ether (nor-
`mally bonded solely to carbon and hydrogen); sulfur is
`normally present as sulfonyl,
`thioether or Inercapto;
`while nitrogen is normally present as amino or amido
`(bonded solely to carbon and hydrogen).
`The fluorescent compounds are further characterized
`by having absorption rnaxirna in 0.05 M phosphate
`buffer pH8 of at least about 500 nm, an extinction coeffi-
`cient in the same medium of at least about 65,000, more
`usually at least 70,000 and a Stokes shift in the same
`medium of at least about I0 run, more usually at least
`about 12 run.
`The 9-substituted-2,‘?-dialltylsubstituted xanthenes of
`this invention will for the most part have the following
`formula:
`
`0
`
`éo
`
`H
`
`Ltli
`
`H
`
`(ale
`
`pt’
`
`,,
`
`HO
`
`W
`
`wherein:
`
`pis an aliphatic group, normally aliphatic hydrocar-
`bylene (composed solely of carbon and hydrogen), satu-
`rated or unsaturated, branched or straight chain, partic-
`ularly alkylene, more particularly (CH;)¢, wherein a is
`of from 1 to 12, usually l to 6, more usually 1 to 4; p is
`normally of from I to 12, usually 1 to 6, more usually 1
`to 4 carbon atoms;
`the two tii‘s are the same or different, normally being
`the same, except when linking to ct, and are hydrogen,
`a non-oxo-carbonyl functionality or one of the tlfs may
`be a non-oxo-carbonyl linking functionality;
`L is a bond or divalent radical, usually an organic
`radical, of at least one carbon atom and not more than
`20. usually not more than 16, more usually not more
`than 10 carbon atoms. normally having an aliphatic or
`aromatic hydrocarbon chain, or combination thereof,
`wherein the aliphatic chain is usually of from about 2 to
`6 carbon atoms and the aromatic chain is of from about
`6 to 12, usually 6 to 10 carbon atoms; L normally has
`from 0 to 4, when aromatic, usually 1 to 4, more usually
`2 to 4 substituents. wherein the substituents may be
`halo, particularly chloro; non-oxo-carbonyl;
`thio,
`in-
`cluding inert sulfur acids, esters and amides; amino,
`particularly tert-amino or amido; and oxy, wherein the
`
`

`
`4,351,760
`
`3
`substituents are normally of from 0 to 4 carbon atoms,
`there being at least two carbon atoms between heteroat-
`oms bonded to saturated carbon atoms;
`ct is an organic compound, a member of a specific
`binding pair, either a ligand or receptor;
`,8 is 1, when at is covalently bonded to If: or iii’, and is
`otherwise 0; the covalent bond normally involves an
`amido, methylene sec-amino, ether, thioether or am
`link;
`lit is a group terminating in a heteroatom containing
`functionality when not bonded to ca, wherein the tenni-
`nal heteroatom containing functionality may be bonded
`directly to a carbon atom of L or through an oligomer
`of from 1 to 4 units. each unit of l to 4, usually 2 to 4
`carbon atoms, which units are amino acids,
`al-
`kyleneamino, or alkyleneoxy groups; the terminal func-
`tionality is normally oxo, including oxo-carbonyl and
`non-oxo-carbonyl; amino; oxy; thio; or active halogen;
`particularly non-oxo-carbonyl; and
`1: is one when ,8 is O and is otherwise on the average
`at least one and not more than the molecular weight of
`0'. divided by 500, usually divided by 1,000.
`Desirably, there are from 2 to 6 chloro substitucnts on
`the fluorescent group (in the brackets), bonded at other
`than the 1,8-positions of the xanthenone. Also, the 4,5-
`positions may be unsubstituted or one or both, usually
`both, substituted with bromo, chloro, or allcyl of from 1
`to 6, usually 1 to 3 carbon atoms.
`The lluorescer compound or conjugate with the or-
`ganic compound (ct) may be linked, covalently or non-
`covalently to a support. The conjugate may be bound
`either through the fluorescer or organic compound.
`The support will be described in greater detail subse-
`quently.
`For the most part, the compounds of this invention
`having a 9-phenyl will have the following formula:
`
`
`
`wherein:
`
`R is an aliphatic group of from 1 to 8, usually I to 6,
`more usually I to 4, and preferably 1 to 3 carbon atoms,
`which may be substituted or unsubstituted, aliphatically
`saturated or unsaturated, particularly alkyl or carbony-
`alltyl of from I to 6, usually 1 to 4 carbon atoms;
`Z is carboxy;
`I
`W is a bond or divalent radical having from 0 to 16,
`either 0 or usually I to 16 carbon atoms, more usually I
`to 8 carbon atoms and from 0 to 10, usually 2 to is het-
`eroatoms, which are chalcogen (oxygen and sulfur) or
`nitrogen, wherein chalcogen is present bonded solely to
`carbon (cry or oxo) and nitrogen is present bonded
`solely to carbon and hydrogen (amino and amido); car-
`bon is normally aromatic or aliphatic, particularly free
`of aliphatic unsaturation, having from 0 to 2 sites of
`ethylenic unsaturation; W is conveniently a monomer
`or oligomer of units of from I to 4 carbon atoms e.g.
`alkylene, aminoacid, oxyalkylene, aminoalkylene, etc.;
`
`4
`Y may be taken together with A to form an active
`functionality capable of forming a covalent bond with a
`heterofunctionality, such as amino, hydroxy, mercapto;
`that is with those functionalities present on A, when A
`is not taken together with Y; such heterofunctionality
`as, oxo- and non-oxo-carbonyl, oxy, thio, amino, active
`halo, active olefin, inorganic acyl group e.g. sulfonyl,
`etc. or acts as a linking functionality, being either meth-
`ylene or heteroatom containing;
`A, when not taken together with Y, is a member of a
`specific binding pair, which is
`ligand or receptor,
`wherein the ligand may be haptenic or antigenic, nor-
`mally being of from about 125 molecular weight to an
`indefinite upper limit, although for the most part, most
`ligands will be under 10 million molecular weight, more
`usually under 2 million molecular weight, with varying
`ranges depending upon the nature of the ligand or re-
`ceptor;
`m will he 0 to 3, more usually 0 to 2; and
`n will be 1 when Y and A are taken together and will
`otherwise be on the average 1 to the molecular weight
`of A divided by 500, more usually divided by 1,000, and
`more frequently divided by 2,000, wherein with specific
`binding pair members over 600,000 molecular weight A
`will normally be not greater than A divided by 5,000. In
`addition, there will usually be at least two chloro sub-
`stituts bonded on any of the available positions where
`no specific atom is indicated. Also, the 4,5-positions
`may be substituted as described previously. Further-
`more, either the conjugate or the fluorescer precursor
`may be bonded to a support of at least about 10,000
`molecular weight and up to an indefinite molecular
`weight.
`A preferred group of compounds will for the most
`part have the following formula:
`
`A.
`
`wherein:
`
`R’ is alkylene of from 1 to 6, usually 1 to 4, and prefer-
`ably 1 to 3 carbon atoms;
`D is hydrogen or carboxy; '
`Z’ is carboxy;
`m’ is 0 to 3, usually 0 to 2;
`Y’ may be taken together with A’ to form an active
`functionality which may be non-oxo-carbonyl, includ-
`ing the sulfur analog thereof, amino bonded to at least
`one hydrogen atom, mercapto, active ethylene, usually
`having an ct-carbonyl, halomethylcarbonyl, wherein
`halo is of atomic number 17 to 53, sulfonyl, or the like;
`when not taken together with A’, Y’ will be a linking
`functionality, either methylene or a heteroatom contain-
`ing linking functionality, usually being an amide, ester,
`ether or azc link;
`W’ is a bond or linking group of from 1 to 16, usually
`1 to 12, and preferably 1 to 8 atoms other than hydro-
`gen, which are carbon, nitrogen. oxygen or sulfur, pref-
`erably carbon, nitrogen and oxygen, there being from 0
`
`10
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`
`4,351,760
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`
`6
`R‘ is alkylene of from 1 to 6, usually I to 4, preferably
`1 to 2 carbon atoms;
`D1 is hydrogen or carboxy, preferably hydrogen;
`Z‘ is carboxy;
`E“ is hydrogen, alkyl of from 1 to 6, usually I to 3
`carbon atoms, or chloro;
`E1 is chloro;
`W1 is a bond or linking group of from I to 12, usually
`I to 8 atoms other than hydrogen, and generally I to 8,
`usually 1 to 6 atoms in the chain wherein the atoms are
`carbon, nitrogen, oxygen and sulfur, particularly car-
`bon, nitrogen and oxygen, wherein the carbon is all-
`phatic, the nitrogen is present as amido or amino, partic-
`ularly amino bonded solely to carbon. and oxygen and
`sulfur are bonded solely to carbon and are city or onto or
`the sulfur analogs thereof;
`W1 will generally be aliphatic, being saturated or
`unsaturated, normally saturated, having from 0 to 1 site
`of ethylenic unsaturation, alkylene or alkenylene of
`from 1
`to 3, usually 1
`to 4 carbon atoms, N-formyl
`amino acid or N-formyl poly(amino acid), where the
`terminal carboxy is derived from WA‘, amino, mer-
`capto, or the like;
`YUU are taken together to form a functionality for
`linking, wherein Y1A1 are bonded solely to carbon or
`nitrogen, with the proviso that when Y1 and A‘ are
`bonded to nitrogen, YIAI are carbonyl, including the
`nitrogen and sulfur analogs thereof and can be doubly
`bonded to nitrogen;
`YIAI can be non-oxo-carbonyl, haloacetyl, halogen
`of atomic no. 9 to 53, particularly chloro or bromo,
`maleirnido, rnercapto, amino, or inorganic acyl, having
`phosphorous or sulfur as the central atom;
`ml is 0 to 3, usually 0 to 2, there usually being not
`more than a total of 5 carboxyi groups in the molecule,
`usually not more than a total of 4 carboxyl groups. and
`preferably not more than about 2 carboxyl groups,
`other than Y1A1;
`x is 0 to 4, preferably 2 to 4, there generally being not
`more than a total of 6 chloro groups in the molecule,
`usually not more than a total of 4 chloro groups,
`wherein it plus ml is not greater than 4.
`For the most part, the compositions of this invention
`when bonded to ligand or support will have the follow-
`ing formula:
`
`1:!
`
`.
`
`wherein:
`E5’ is hydrogen or chloro;
`E3 is chloro;
`Z2 is carboxy;
`R3 is alkylene of from I to 6, usually 1 to 3, preferably -
`1 to 2 carbon atoms;'
`D3 is hydrogen or carboxy, preferably hydrogen;
`W3 is a bond or linking chain, when a linking chain
`being of from I to l2, usually of from 1 to 10, and pref-'
`
`5
`to 8 carbon atoms and 0 to B heteroatoms, with the
`number of carbon atoms and heteroatoms being at least
`1. wherein nitrogen will be bonded solely to hydrogen
`and carbon. and will be either amino or amido, oxygen
`and sulfur will be bonded solely to carbon as only (thio) S
`or oxo (thiono) and carbon is normally aliphatic and
`usually free of aliphatic unsaturation, generally having
`from 0 to I site of ethylenic unsaturation; W’ may be
`alkylene or alkenylene of from 1 to 8. usually 1 to 4
`carbon atoms, oxoalkylene or oxoalkenylene of from 1
`to 8, usually I to 4 carbon atoms, imino (NH), N-formyl
`amino acid or N-formyl poly(amino acid) e.g. glycine or
`polyglycine, there being from about 1 to 4 amino acids,
`with the terminal carboxy being Y’A’, or the like;
`if is I when Y’ and A’ are taken together and other-
`wise is on the average at least 1 to the molecular weight
`of A’ divided by 500, usually divided by 1,000, more
`usually divided by 2,000, and when A’ is over 500,000
`molecular weight, more usually divided by 5,000;
`there generally being not more than S carboxyl
`groups, usually not more than 4 carboxyl groups in
`total, and there being from 0 to 6 chloro groups, prefer-
`ably 2 to 5 chloro groups bonded to available carbon
`atoms; and
`A’ is a member of a specific binding pair, a ligand or
`receptor, wherein the ligand may be haptenic or anti-
`genic, and haptenic ligands will include compounds of
`interest such as drugs, hormones, pollutants, com-
`pounds of interest in processing, "agricultural chemicals,
`metabolites, and the like;
`antigens will primarily be proteins, polysaccharides
`or nucleic acids, individually or in combination with
`each other or other materials, such as in cells, viruses,
`phage, or the like. The haptens will normally be from
`about 125 to 2,000, more usually to 1,000 molecular
`weight, while the antigens will normally be from about
`2,000, more usually 5,000 molecular weight up to an
`indefinite molecular weight, usually not exceeding 10
`million, more usually not exceeding 2 million.
`The 4,5-positions are preferably unsubstituted or
`chloro-substituted.
`In addition, the above conjugate may be bonded to a
`support. Various supports may be employed, both solu-
`ble or insoluble, swellable or nonswellable. by aqueous
`or organic solvents, naturally occurring or synthetic,
`organic or inorganic, porous or nonporous, or the like.
`Various polymeric materials include vinyl polymers
`and copolymers, polysaccharides, silicones, glass, car-
`bon particles, such as graphite or charcoal, metals or
`metal compounds, poly(amino acids), nucleic acids or
`the like.
`For the most part, the fluorescent compounds of the
`subject invention employed for conjugation will have
`the following formula:
`
`10
`
`15
`
`20
`
`25
`
`'
`30
`
`35
`
`45
`
`55
`
`65
`
`wherein:
`
`

`
`4,351,760
`
`7
`erably of from about 1 to 8 atoms other than hydrogen,
`having from about 1 to I0, usually from about 1 to B,
`and preferably from about 1 to 6 atoms in the chain or
`spacer arm, wherein the atoms are carbon, oxygen,
`nitrogen and sulfur, particularly carbon, oxygen and
`nitrogen in the spacer arm, wherein oxygen and sulfur
`are bonded solely to carbon, as oxy or oxo, and nitrogen
`is bonded solely to carbon and hydrogen, namely amino
`and amido, wherein heteroatoms bonded to saturated
`carbon atoms are separated by at
`least
`two carbon
`atoms;
`W3 is particularly alkylene, carboxamidoalkylene,
`wherein alkylene is of from about I to 2 carbon atoms-
`(—C0NHC1.2—),, wherein a is in the range of from
`about 1 to 4, usually 1 to 3;
`thiocarbamyl,
`Y2 is non-oxo-carbonyl, carbamyl,
`methylene, amino, or thio, particularly a functionality
`having a non-oxo-carbonyl group or sulfur analog
`thereof;
`it! is 0 to 4;
`m3 is 0 to 3, preferably 1 to 2;
`n3 is 1 to the molecular weight of A3 divided by 500,
`usually divided by 1,000, more usually divided by 2,000,
`wherein when A3 is a ligand of between about 125 to
`2,000 molecular weight, n3 will generally be of from
`about 1 to 20, when A3 is a ligand of from about 2,000
`to 600,000 molecular weight, n3 will generally be in the
`range of about I to 100, more usually in the range of
`about 2 to 50; and
`A3 is a ligand of at least about 125 molecular weight
`and may be l0 million or more molecular weight, which
`is haptenic or antigenic, wherein haptens are from about
`125 to 2,000 molecular weight and antigens will gener-
`ally range from about 5,000 to 10 million molecular
`Weight, more usually from about 5,000 to 2 million
`molecular weight and frequently from about 5,000 to
`600,000 molecular weight, the ligand being a member of
`a specific binding pair, which comprises a compound
`having at least 1 determinant or epitopic site and a re-
`ceptor which is capable of recognizing the determinant
`site or A2 is a receptor of from about 10,000 to 1 million
`molecular weight.
`Finally, in some instances it may be desirable to have
`the fluorescent compound or the conjugate of the fluo-
`rescent compound with ligand, bonded to a support,
`where the linkage may be derived from either the fluo-
`rescent compound or the ligand, normally the ligand. In
`this situation, the linking group may be any convenient
`functionality which is present on the fluorescent com-
`pound or the ligand or a functionality which may be
`introduced, particularly on the ligand. These composi-
`tions will for the most part have the following formula,
`where the symbols are derived from the previous for-
`mula for the conjugate for the most part:
`
`
`
`wherein:
`
`8
`all of the symbols have been defined previously, ex-
`cept for:
`n3 which is at least 1 and up to the molecular weight
`of A3 divided by 500, usually 1000, more usually 1,500,
`with the proviso that when q is 0. I13 is 1;
`:2] which is 0 or 1;
`p which is at least 1 and of up to the molecular weight
`of the support divided by 500. more usually the molecu-
`lar weight of the support divided by l,000, wherein
`when the molecular weight of the support exceeds
`500,000, p will normally be not greater than the molecu-
`lar weight of the support divided by 5,000, more usually
`divided by 10,000; and
`Support intends a macromolecular support of at least
`about 10,000 molecular weight, which may be naturally
`occurring or synthetic, having a plurality of functionali-
`ties for linking e. g. carboxy, hydroxy, or amino, usually
`being a polymer, such as a polysaccharide or an addi-
`tion polymer; the support being bonded to the conju-
`gate by any convenient functionality remaining on A1
`or the conjugate in the brackets, the particular manner
`of linking not being a significant aspect of the subject
`invention. For example, if A3 is a poly(arnino acid),
`carboxylic groups on the support can be used for amide
`formation or maleimide groups may be introduced and
`linked to mercapto groups.
`Quite obviously, the compounds of the subject inven-
`tion can be modified so as not to be within the above
`formulas, without significantly affecting the properties
`of the compounds. For example, one or more of the
`acidic anionic groups could be esterified or amidified,
`or alkyl groups can be substituted on the phenyl, as well
`as other groups, such as cyano, nitro, or the like. How-
`ever, these changes will in most cases require additional
`synthetic steps which are not warranted by the degree
`of enhancement. if any, in the spectroscopic or chemical
`properties of the resulting product.
`Turning now to a consideration of the individual
`components of the subject compositions, the fluorescein
`derivatives will be considered first. The following is a
`list of illustrative fluorescein derivatives coming within
`the scope of the subject invention.
`TABLE I
`
`2,7-dirnethyl-4,5-dichloro-9-(2’,4',5'-tricarboxyphenyl)-
`6-hydroxy-3-xanthen-3-one
`2,7-diethyl-4,5-dichloro-9-(2',4’,5’-tricarboxy-3',6’-
`dichlorophenyl)-6-hydroity-3H-xanthen~3one
`2,7—dihexyl-9-(2',4’,5'-tricarboxyphenyl)-6-hydroxy-3H-
`xanthen-3-one
`2,?-dimethyl-4,5-dichloro-9-(2'-carboxy-4’-isothi-
`ocyanato-3',5'-dichlorophenyl)-6-hydroxy-3H-xan-
`then-3-one
`2,7-dimethyl-9-(2'-carboxy-4'-isocyanato-3’,5’,6’-tri-
`chlorophenyl)-6-hydroxy-3H-xanthen-3-one
`2,’?-dimethyl-9-(4’-carboxy-5’-carboxylphenyl)glycyl-
`glycylglycine amide-6-hydroxy-3H-xanthen-3-one
`2,7-di(carboxyrnethyl)—9-(4',5',-dicarboxy-2',3',6’-tri-
`chlorophenyl)-6-hydroxy-3H-xanthen-3—one
`2,7-di(carboxypropyl)-4,5-dichloro-9-(3',4'-dicarboxy-
`phenyl)-6-hydroxy-SH-xanthen-3—one
`2,'l-diethyl-9-(2’-carboxy-4’-amino—3’,5’-dichloro-
`phenyl)-6-hydroxy-31-I-xanthen-3-one
`2,?—dimethyl-9-(2'-carboxy-4‘-mercaptophenyl)—6-
`hydroxy-3H-xanthen-3-one
`2,7-dimethyl-9-(2‘-carboxy-4'-carboxymethylphenyl)-G
`hydroxy-3H-xanthen-3-one
`
`l0
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`

`
`9
`2,7-diinethyl-9-(2’-carboxy-4'-(4"-carboxybutyl)-
`phenyl)-6-hydroxy-3Hgxantl1en-3-one
`'
`2,?-dimethyl-4,5¥dichloro-9-(2',4‘-dicarboxy-5‘-(carbon
`amidomethylene)phenyl)-6-hydroxyg3H-xanthen-
`3-one
`'
`2,’?-dimethyl-4,5-dicl1loro«9(3'-carboxypropyI)-6-
`hydroxy-3H-xanthen—3-one.
`As indicated previously, the fluorescein derivatives
`of the subject invention will be conjugated with ligands
`and/or supports._ The following is a description of the
`applicable ligands.
`-
`Analyte
`The ligand ainalytes of this invention are character-
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poly(amino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations of assemblages include bacteria,
`viruses, -chromosomes; genes, mitochondria, nuclei, cell
`membranes, and the like.
`.
`For the most part, the polyepitopic ligand analytes
`employed in the subject invention will have a molecular
`weight of at least about 5,000, more usually at least
`about 10,000. In the poly(amino acid) category,
`the
`poly(aminoacids)ofinterestwillgenerallybefromabout
`5,000 to 5,000,000 molecular weight, more usually from
`about 20,000 to 1,000,000 molecular weight; among the '
`hormones of interest, the molecular weights will usually
`range from about 5,000 to 60,000 molecular weight.
`The wide variety of proteins may be oonsidered 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
`_
`\
`proteoglycans
`unclassified proteins, e. g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`on-Lipoprotein
`at-Acid glycoprotein
`on-Antitrypsin
`a1-Glycoprotein
`Transcortin
`4.63-Postalbumin
`Tryptophan-poor
`ct;-glycoprotein '
`cnx-Glycoprotein
`Tityroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin
`(Gc 1-1)
`(Gc 2-1)
`
`4,351,760
`
`10
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`S0
`
`55
`
`65
`
`(Ge 2-2)
`Haptogiobin
`(HP 1-1)
`(HP 2-1)
`(Hp 2-22)
`Ceruloplasmin
`Cholinesterase
`0.2-Lipoprotein(s)
`Myoglobin
`C-Reactive Protein
`0:2-Macroglobulin
`oz-HS-glycoprotein
`Zn-:13-glycoprotein
`az-Neuramino-glycoprotein
`Erythropoietin
`,8-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasrninogen
`B;-glycoprotein I
`_ B;-glycoprotein II
`Immunoglobulin G
`(IgG) or 7G-globulin
`Mol. formula:
`721:2-or 72%:
`Immunogiobulin A (IgA}
`or 'yA-globulin
`Moi. formula:
`
`(cc2n2)" or (a2A2)"
`Immunoglobulin M
`(IgM) or 'yM~globulin
`, Mo]. formula:
`012112)’ or (J&2?t2)5
`Imrnunoglobulin D(IgD)
`or 7D-Globulin ('yD)
`Moi. formula:
`(32112) 01' 5212)
`ilmmunoglobulin E (IgE)
`or 7E-Globulin ('yE)
`Mo]. formula:
`(52132) or (62712)
`Free 1'; and A light chains
`Complement factors:
`C’l
`C'1q
`C’lr
`C’ ls
`C'2
`C3
`131:4
`G213
`C4
`C5
`C'6
`.C.7.
`C8
`C9
`Important blood clotting factors include:
`
`BLOOD CLOTTING FACTORS
`
`International designation
`Name
`I
`Fibrinogen
`II
`Prothrombin
`lia
`Thrombin
`Ill
`Tissue th romboplastin
`V and VI
`Proaccelerin. accelerator
`globulin
`
`

`
`4,351,760
`
`11
`
`-continued
`BLOOD CLOTHNG FACTORS
`
` International designation Name
`VII
`Proconverlin
`VIII
`Antihernophilic globulin
`(AHG)
`Christmas factor,
`plasma thromboplastin
`component (PTC)
`Stuart-Prower factor,
`autoprothrombin III
`Plasma thromboplastin
`antecedent (PTA)
`Hagernann factor
`Fibrin-stabilizing factor
`
`IX
`
`X
`
`XI
`
`XII
`XIII
`
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`(parathrornone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`Erythropoietin
`Melanotropin
`(melanocyte-stimulating hormone; intermedin)
`Somatotropin
`-
`(growth hormone)
`Corticotropin
`(adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone
`(interstitial. cell—stimulating hormone)
`Luteomammotropic hormone
`(luteotropin, prolactin)
`Gonndotropin
`(chorionic gonadotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactcgen
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF, Somatotmpin-RF,
`GRF, FSI-I-RF, PIF, MIF
`Other polymeric materials of interest are rnucopoly-
`saccharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
`Species of Microorganisrns Hemoseneitin ‘Found in
`
`Stmpramccur pygenes
`Polyseeeharide
`Dipfococcus pncrrmombe
`Polyseecharide
`Nciuenia menfnginflis
`Polyseccharide
`Nerlneric gwrarrbcae
`Poly-saccharide
`Corynebactnrn.-m diphrherfee
`Poly-saccharide
`Acm:abac1'fl'r.r.9 mallet‘
`Crude extract
`Actirrabacilftrs wkiremarf
`Francrkeffc udarcnsir
`
`Pasteurelfa pesris
`Pasfeureffa pestrk
`Pnsneurefia mufrocfdn
`
`Lipopolysac-
`charide
`Polysaceharide
`.
`Folyaaccharide
`Capsular antigen
`
`10
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`12
`-continued
`Species of Microorganisms
`Hemosensitin Found in
`Bmcelfa aborrus
`Crude extract
`Haemcphifus influenzae
`Polysaccharide
`Hacmapkifus pertussis
`Crude
`Trcponemc refferi
`Poiysaccharide
`Veillonella
`Lipopolysac—
`_
`charide
`Erysipelothrix
`Polysaccharide
`Lirrerfa monacyrqgt-rues
`Puiysuccharide
`Chrcmohacteriurn
`Lipopolysaco
`charide
`Saline extract of"
`90% phenol
`extracted
`mycobacleria
`and polysac-
`chnride
`fraction of
`cells and
`' "tuberculin
`Polysaccharide
`folysaccharide
`LipopolySaC-
`charide.
`Pclysaccharitle
`Polysaccheride
`
`
`
`Mywbacredum tuberculosis
`
`Klensfella aemgenes
`Klehsfella cfoacce
`Salmonella Upkom
`:
`
`Salmonella rwhi-murfum.’
`Salmonella derby
`Safmonefla puflomm
`.§'F:.r'ge.f!a' dysenteriae
`Siaégellc flexneri '
`Crude, Poly-
`Sialgella sonnet’
`Crude extract
`Rickettsiae
`Polysaccharitle
`Candida albicuns
`
`Enramaebn nirrafyrica Crude extract
`
`_
`_
`Polysacchatide
`
`I The microo_1jganistns= which are assayed may be in-
`tact, lysed, ground or otherwise fragmented, and the
`resulting composition or portion, eg. by extraction,
`assayed. Microorganisms of interest include:
`Corynebacteria
`Caryrtebacterium dzptberfae
`Pneumococci
`
`Drjofaccccus pneumon foe
`Streptococci
`Streptococcus pyagenes
`Streptococcus salivarus
`Staphylococci
`Staphylococcus aureus
`Staphylococcus albus
`Neisseriae
`Ner'.r.s‘err'a meningmdrk
`Neisseria gonqrrheae
`
`Enterobacteriaciae
`.Elrc.r‘rer."c!H'a colt"
`Aerobacrer aemgenes
`Kfehsfelln pneumamhe
`Salmonella gpfrom
`Salmonella cfloieraexuir
`Salmonella grphimurium
`Sirigeflc dyrenrenhe
`Smgelfe scfimfrzir‘
`Sfu'geHa ambinotarda
`Skrfigefla flexneri
`SFn'ge.ffn' boydfi
`Skigefla Sonnet"
`Other enteric bacilli
`Proteus wan:-is
`Proteus mfmbffls
`Pmreus mcwgani
`fieudonwncs aenegfnescr
`Afcafigenes _faecc!tr
`F'r'bn‘o chafeme
`Hemoghilus-Bordetella group
`
`The colifonn
`bacteria
`
`The Sairnonellae
`
`The Shigellae
`
`Proteus species
`
`
`
`‘~....../\...._._—/\.,../\.,_/i
`
`

`
`4,351,760
`
`14
`
`5
`
`10
`
`Rlckeusla tsutsugamushi
`Rickerrsia burnetff
`Rfckerrsia quinrana
`Chlamydia (unclassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`Fungi
`Cryptacoccus neoformans
`Bfastomyres derm arfa':’s
`Hfsrapfasma capsularum
`C°“5df°d95 5’’'’-''‘'‘‘m-‘‘'
`_
`_
`Paracoccidfofdes brasiliensfs
`C"”d'd" 515359?‘
`.
`I
`.
`Aspergilfusfanirgarus
`is Mucar corymbqfér {Abs:a'm carymiufera)
`
`
`
`R1,,Mm mm
`gm,
`,v.-
`Rfiiflg: EH2:
`
`}
`
`P};
`“om”
`
`31,133
`
`13
`
`‘continued
`
`H‘”°”'”“‘ ”‘fl“‘”"“
`
`iii“:
`H: ,,,_,m,,,-,,,
`H. paminfiuenzae
`
`Horde-'e.I'."a pertussis
`
`B
`
`paste u,-ellae
`em-5
`page ,‘.;_:a
`Pastefirelfa fuiareusis
`ruce ac
`11
`Bmcefla meffrensfs
`3m,_.e;;a 350,,”
`B,““.115 ml,
`Aerobic Spore-forming Bacilli
`Bacffius anthmcis
`Baciilus subrfixic
`Bacfffus megarerium
`Baciffus versus
`A“af“:b:{ SP°b1;*'f"3_1'“1inE 35°51“
`as H mm :1: mum
`CIosm'd:'um mam’
`Clostridfum perfringens
`Cfmfridiflm 50".}-'1'
`Closrridfum sepzicum
`Closrridfum hilstofyricum
`Clastrfdfum terrfum
`Clostridfum bifenwenrans
`C10sm‘d='w_n spomgvm
`_
`_
`_
`MY°°b3°*e“a.
`Mycobacrerium tubgrcufosis harmms
`££$:::::-::::::;:,.
`Mycobacrerhtm Ieprae
`Mrycobacterfum paratulferrufosilv ‘
`Actmomycetes (fungus-like bactena)
`Actirwmyces ismelff
`Acifnomyces bow}:
`Actinorrfyces naeslmzdir‘
`Nocardm asrerafdes
`Nocardfa brasiliensils
`
`.
`T1" Smrochflef
`Trepanemc paiirdum
`Twpanemn pertenue
`
`_
`Spfrfffum minus
`Sn-gpiobactitus
`manffiflirmis
`.
`
`20
`
`25
`
`30
`
`45
`
`50
`
`55
`
`65
`
`Spomrrfchum schenkfi
`12
`ed
`-
`pggcfifii fagsfifia
`Fommm ,;,,.,,,afl-,3,-3
`C!aa‘ospan'um car