United States Patent
`
`[.9]
`
`[in
`
`3,996,345
`
`
`
` Ullman et al. - [45] Dec. 7, 1976
`
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`[54] FLUORESCENCE QUENCHING WITH
`IMMUNOLOGICAL PAIRS IN
`
`[751
`
`IMMUNOASSAYS
`lnventors: Edwin F. Ullman, Atherton; Moshe
`schwa.-zberg, P310 Alto, both of
`Ca1if_
`
`_
`AS5189‘-3°? S3/"3 C°mP3“3’v P310 Alma Cam
`[73]
`Filed:
`[22]
`June 30, 1975
`[21] APPL N0-3 591,336
`
`Related U.S. Application Data
`.
`_.
`_
`
`.
`
`$:7nfiI,n::;:lno;‘:g.art of Ser. No. 497,l67. Aug. I2.
`
`[63]
`
`[52] U-S- CL ............................. .. 424/12; 23/230 B;
`195/1035 R; 250/112 B; 252/301-16
`CNN 21/00: GOIN 33/16
`Int Cl-2
`[51]
`[53] Field Of S9fil'¢h ------------------ -- 23/230 B; 424/12;
`195/ 103-5 R; 260/1 12 3; 252/30l.16
`
`we
`
`‘
`3,90l,654
`3,935,074
`
`UNITED STATES PATENTS
`8/i975 Gross ............................. .. 23/230 B
`
`.. 23/230 R X
`I/I976
`Rubenstein ............
`
`Primary Examiner—Morri_s O. Wolk
`Assistant Examiner—Sidney Marantz
`Attorney, Agent, or Firm—Townser1d and Townsend
`
`[57]
`
`ABSTRACT
`
`Immunoassays employing antibodies and a fluorescer-
`quencher (F—Q) chromophoric pair, wherein one or
`both of the qhromophoric pa_1r are bfmded to antibod-
`1es._ Depending on ltahe parttcularbligandl of énteaest,
`various reagent com matnons can e emp oye , w ere
`the amount of quenching is directly related to the
`amount of ligand present in the assay medium. In carry-
`ing cilutlthe ajssafy: tthe utnlt<nov;/In and Sntibgdy spefcgic
`or t e xgan o in eres
`o W1C IS oun one o
`e
`F—Q pair, are combined in an aqueous buffered me-
`dium. Depending on the protocol, different assay re-
`agents are employed in the aqueous buffered medium:
`( l ) ligand analog bonded to the other of the F—Q pair;
`
`El?) a3:iboclfi<:ls1s|::t:éfic f:0l' theF1ig';1lnd(t;))which igbopnd
`
`acom ma ion
`pair or; ma y,
`e
`e 0 er 0
`of a plurality of ligands bonded together through link-
`ing groups to a hub molecule. usually a polymer,
`in
`combination with antibody bound to the other of the
`F—Q pair. The composition is irradiated with light at a
`wavelength, absorbed by the fluorescing molecule and
`:.';;;::;':::::::;?.:::::°::::S:s::;";:°;f;:;::i:?::§
`figand can be date.-mi‘;-.ed_
`
`38 Claims, No Drawings
`
`
`
`‘mm Genentech Exhibit 2004
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2004
`
`IPR2016-00710
`
`

`
`I
`
`3,996,345
`
`2
`
`FLUORESCENCE QUENCI-IING WITI-I
`IMMUNOLOGICAI, PAIRS IN IMMUNOASSAYS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation-in-part of applica-
`tion Ser. No. 497,167, filed Aug. 12, 1974, now aban-
`doned.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`There is a continuing need for rapid sensitive meth-
`ods for determining minute amounts of organic com-
`pounds. A number of techniques have been developed
`toward this end. Among the commercially available
`techinques are radioimmunoassay, spin-labeled immu-
`noassay, for which reagents are sold under the trade-
`mark FRAT, homogeneous enzyme immunoassay, for
`which reagents are sold under the trademark I:'Ml'l‘,
`and hernagglutination (HI). These techniques are ef-
`fective for determining amounts of materials in the
`range of 10"“ to l0“°M or less.
`These techinques all involve the ability of a receptor
`molecule, usually an antibody, to be able to recognize
`a specific spatial and polar organization of a molecule.
`Except for hemagglutination, the techniques depend
`upon providing a reagent which can compete with the
`molecule being assayed for the receptor. By being able
`to distinguish between the reagent which is bound to
`receptor and reagent which is unbound, one can deter-
`mine thc amount of the compound of interest which is
`present.
`In developing irnmunoassays, one is limited by the
`availability and properties of an appropriate receptor.
`However, as for the other reagents and the techinque of
`measurement, there are a number of different. consider-
`ations which made for a more accurate, convenient or
`commercially desirable assay. First, it is desirable that
`there be a minimum number or measurements or the
`
`various reagents, as well as transfers of the various
`reagents. Secondly,
`the equipment
`for measuring
`should be reasonably economical, so as to be accessible
`to a broad range of users. Thirdly, the reagents em-
`ployed should be relatively stable, so as to be capable
`of storage and shipment. Fourthly, the method should
`not be subject to significant interference from other
`materials which may be adventiously present in the
`sample to be assayed. Other considerations are ease of
`training of technicians absence of health hazards, sensi-
`tivity, reproducibility, and applicability to a wide vari-
`ety of ligands.
`The subject invention is predicated on the phenome-
`non of energy transfer between two chromophores.
`When a fluorescing chromophor is irradiated with light
`absorbed by the ehromophore, the fluorescing chromo-
`phore can dissipate the energy of the absorbed light by
`emitting light of longer wavelength, that is, tluorcscing.
`If another ehromophore is within less than 100A of the
`fluorescer and absorbs light at the wavelength of emis-
`sion, there is a probability, depending upon other fac-
`tors, that the fluorescer will transfer to the other chro-
`mophore the energy which would otherwise havebeen
`emitted as light, in effect, quenching the fluorescer.
`DESCRIl"I‘I()N OF "l'l*lF, PRIOR ART
`
`is-: oxeinplary of :1 r_adioim-
`l"-lo. 3,’/09,868.
`l.’at.
`1.1.3.
`munoassay. US. Pat. No. 3,690,834 is exemplary of a
`
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`spin immunoassay. U.S. Pat. Nos. 3,654,090 and
`3,817,837 are exemplary of enzyme immunoassays.
`Articles of interest include an article by Ludwig Brand
`and James R. Gohlke, entitled, Fluorescence Probes
`for Structure, Annual Review of Biochemistry, 41,
`843-868 (1972); and Stryer, Science, 162, 526 ( 1968).
`Also of interest
`is co-pending application Ser. No.
`402,693, filed Oct. 2, 1973.
`SUMMARY OF THE INVE.N'l‘ION
`
`A method is provided for determining the presence
`or amount of an organic compound to which a recep-
`tor, usually antibody,
`is vailable or can be prepared,
`The organic compound will be hereinafter referred to
`as a ligand.
`two chromophores are
`In carrying out the assay,
`employed which are a fluorescer-quencher pair. The
`amount of fluorescer within quenching distance of
`quencher is affected by the amount of ligand present in
`the assay medium.
`One ehromophore is introduced into the assay me-
`dium covalently bonded to a receptor composition
`which specifically binds to the ligand. The second chro-
`mophore can be introduced into the assay medium in
`different ways: (1) covalently bonded to a receptor
`composition which is the same or different from the
`receptor composition conjugated to the first ehromo-
`phore, but in both instances specifically binds to the
`ligand, and in the presence or absence of polyligand; or
`covalently bonded to ligand analog, where the ligand
`analog can compete with ligand for the receptor com-
`position. The choice of modes of introduction will de-
`pend to a significant degree on the number of indepen-
`dent epitopic or haptenic sites present in the ligan(|.
`Where the ligand has only one independent epitopic
`site (monoepitopic), usually one ehromophore will be
`covalently bonded to a receptor for ligand, and the
`other ehromophore will be provided as covalently
`bonded to a ligand analog or a combination of poly(li—
`gand analog) and the ehromophore covalently bonded
`to receptor for ligand.
`Where the ligand has a plurality of independent epi-
`topic sites (polyepitopic), the modes indicated above
`maybe used in addition to the following modes. In one
`mode, the two chromophores are individually bonded
`to receptor for ligand. In another mode, receptor for
`ligand is obtained from different species and one ehro-
`mophore is bonded to receptor for the ligand-receptor
`from one species and the other ehromophore bonded
`to receptor for ligand—receptor from the other species.
`The latter method expands the versatility of the subject
`assay in allowing for common reagents for a wide vari-
`ety of assays, simplifies purification pI‘()(,7I:(ll.lI'eS, and
`allows for the determination of the presence of assem-
`blages, as distinct from the component parts.
`The various materials are brought together in an
`aqueous buffered medium, incubated and irradiated
`with light absorbed by the fluorescer molecules. By
`determining the amount of fluorescence, after incuba-
`tion for a predetermined time interval or after the sys-
`tem has approached equilibrium, and comparing the
`results obtained with one or more known standards, the
`presence or amount of ligand can be deterrnined.
`l)F,SCRIl’TlON OF THE SPECIFIC FMBODIMENTS
`Definitions
`Ligand--a.n organic molecule or assemblage, nor-
`mally greater than 100 molecular weight and having at
`
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`3,996,345
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`4
`
`3
`least one functionality, normally polar, for which a
`receptor is either naturally available or can be pre-
`pared.
`Ligand analog——a mono- or polyvalent radical a sub-
`stantial proportion of which has the same spatial and
`polar organization as the ligand to define one or more
`determinant or epitopic sites capable of competing
`with the ligand for the binding sites of a receptor, and
`differs from the ligand in the absence of an atom or
`functional group at the site of binding to another mole-
`cule or in having a linking group which has been intro-
`duced in place of one or more atoms originally present
`in the ligand. The ligand analog precursor is the com-
`pound employed for conjugating ligand or ligand ana-
`log to another molecule, e.g. chromophore.
`Assemblage--a combination of organic molecules
`bound together by other than covalent bonds, generally
`having molecular weight exceeding 600, usually ex-
`ceeding l,000 and may be 1,000,000 or more, for
`which receptor is either naturally available or can be
`prepared; an illustrative assemblage is an antigen and
`antibody) or, a molecule prepared from two discrete
`entities, normally joined together by weak bonds, such
`as polar bonds or disullidc bonds, which under the
`conditions of the system are capable of being in equilib-
`rium with the individual entities.
`Chromophore—--a fluorescer or quencher molecule;
`in the subject invention, the fluorescer and quencher
`are interrelated. The tluoresccr molecule is a chromo-
`phore which is able to absorb light at one wavelength
`and emit light at a longer wavelength. The quencher
`molecule is capable of inhibiting fluorescence, when
`within a short distance. usually less than about IOOA. of
`the fluorescer molecule. by accepting the energy which
`would otherwise be emitted as fluorescent light. As far
`as the molecule or composition to which the chromo-
`phores are joined. in most‘ instances. the fluorescer and
`quencher will be interchangeable. although there will
`frequently be some preference. Therefore. for purposes
`of generality. the two molecules will be referred to as
`chromophores. and individually referred to as Ch, and
`Cllg.
`analog-
`(ligand
`analog-chromophore
`Ligand
`(Ci1g)J.]—lig3t‘lCl analog is covalently bound to one or
`more fluorescent molecules or quencher molecules.
`With small ligands. those below about l0,0l}U molecu-
`lar weight, usually below about 2,000 molecular
`weight, the ligand analog will usually be joined to fewer
`than 10 chromophore-s, usually from I
`to 10 chromo-
`phores, not more than about I chromophore per 1.000
`molecular weight. With a large ligand, at least 2,000
`molecular weight, usually at least about l0.000 molec-
`ular weight, a plurality of chromophores may be cova-
`lently bound to ligand analog. The number of chromo-
`phores present will be limited by the number which
`may be introduced without masking too many epitopic
`sites of the ligand and the desire to have a sufficient
`number of chromophores to insure a substantial
`amount of quenching when receptor—Ch.
`is bound to
`the ligand analog—{Chg),.
`lP0ly(li-
`Polyljllgancl
`analog)-polylchrornophorel
`gand analog)-poly(Chg)]-—ligand analog and chrome-
`phore are bonded to a high molecular weight (as com-
`pared to the ligand analog and chromophore} water
`soluble polyfunctionalized hub or nucleus molecule, to
`provide a plurality of ligand analog groups and chrome-
`phore groups spaced on the surface of the molecule, so
`that when receptor-Ch, is bound to ligand analog. some
`
`Ch. groups will be present within quenching distance of
`Chg groups.
`are
`Polytligand analog)—ligand ‘ analog groups
`bonded to a high molecular weight (as compared to
`ligand analog) water soluble polyfunctionalized hub or
`nucleus molecule, so that there are a sufficient number
`of ligand attalogs per unit area for quenching to occur
`when the polyfligand analog) is saturated with recep-
`tor-Ch. and receptor—Ch, in appropriate proportions.
`Receptor—cl1romophore (receptor~Ch. and receptor-
`Ch;-)—a receptor is a molecule which is capable of
`distinguishing an epitopic site and binding to such site.
`Usually receptors will have binding constants in excess
`of 10‘. frequently, in excess of 10‘. For the most part.
`receptors are antibodies. although enzymes. nucleic
`acids, and certain globulins, may also act as receptors.
`In the subject invention, for the most part. the recep-
`tors willbe antibodies to which one or more,‘ usually _at
`least two or more, chromophore groups will be bound.
`Receptor composition——receptor composition is a
`homogeneous o_r heterogeneous composition capable
`of specific non-covalent binding to ligand and ligand
`analog and includes a composition which specifically
`recognizes the ligandlanti-ligand} and a combination
`of anti-ligand and a composition which specifically
`recognizes the anti-ligand (anti(anti—ligand)).
`General Statement of the Invention
`
`The method is predicated on the employment of two
`chromophores which form a fluorescer~quench_er_ pair.
`By having a composition (receptor) which specifically
`recognizes or binds to a ligand to which one of the
`chromophores is covalently bonded. and having the
`second chromophore bonded to ligand analog or recep-
`tor. the amount of ligand present in the assay _solution
`will affect the amount of quencher within quenching
`distance of fluorescer. The assay may be ‘carried out
`competitively. where ligand analog competes with li-
`gand for receptor. ligand analog being present as po_ly{-
`ligand analog) or covalently bonded to cl'ti'ornophore_.
`'1'he assay may also be carried out non-competitively
`with ligands having a plurality of epitopic sites, where
`receptor having each of the chromophores binds to
`ligand..
`Compositions
`Depending upon the particular protocol emploved
`and the ligand of interest. one o_r more of the following
`reagent compositions will be emploved in the assay
`medium: ligand analog-chromophore. poly( ligand ana-
`log)-po|y(chromophore), poly(ligand analog). one or
`two receptors and one or two receptor-ehromophores.
`The first composition to be considered will be the li-
`gand analog-chromophore.
`
`Ligand Analog-Chromophore and Poly(Ligand
`Analog )-Po|y(Cl'tromophore)
`
`The ligand anaIog—chromophore may be subdivided
`into two groups. The first group is where the ligand
`analog-chromophore has a single ligand analog and a
`single chromophore joined together by a relatively
`short linking group. In these instances, the ligand ana-
`log for the most part will be haptenic, rather than anti-
`genic, and generally be less than about l0.000 molecu-
`lar weight. more usually less than about 6.000 molecu-
`lar weight. and frequently in the range of about I25 to
`1.000 molecular weight. excluding the linking group
`employed for linking to the chromophore. For the most
`part, the ligand analog will differ from the ligand in
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`3,996,345
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`5
`having a particular functionality replaced by a bond, a
`hydrogen replaced by a bond, or a short carbon chain
`replaced by a bond (by bond, it is intended to include
`multiple bonds, as well as single bonds) to join to the
`linking group for
`linking to the chromophore. The
`various haptenic or low molecular weight ligands will
`be discussed subsequently.
`The linking group will normally have not rrrore than
`about 10 atoms in the chain between the ligand and the
`clrrornoplrore, more usually having either a bond or
`from about 1 to 6 atoms in the chain. The atoms for the
`
`6
`Normally, there will be not less than about one conju-
`gate (ligand analog or chromophore) molecule per
`50,000 molecular weight, more usually not less than
`about one conjugate molecule per 25,000 molecular
`weight, and usually not more than about one conjugate
`molecule per 1,000 molecular weight, more usually not
`more than about one conjugate molecule per 2,000
`molecular weight.
`The ratio of chromphorc molecules to ligand will
`generally be from about 0.05-—20: l, more usually from
`about 0.5—20:l, preferably from about
`lv--10:], and
`more preferably from about 2-8:].
`Where the chromophore is the fluorescer molecule
`for the purposes of this invention, generally there will
`be at
`least about (l.5~2(), more usually from about
`l—-l 0, and preferably from about 2-7 fluorescing mole-
`cules per ligand molecule. Where the chromophore is
`the quencher molecule, the number of quencher mole-
`U..1‘u.u,
`cules per ligand wil. generally be from about “ 5 ”"
`more usually from about
`l—20, and preferably from
`about 2—l5 per ligand molecule.
`The conjugates to the hub molecule will have the
`same type of linking group as was employed for joining
`the chromophore to the ligand. The particular choice
`of functionality will depend upon the available func—
`tional groups on the nucleus molecule.
`Reccptor—Chromophore
`Since in most instances the receptor is antibody, the
`present description will refer to antibody as exemplary
`of receptor. Antibodies have a number of active amino
`groups which can be used for covalently conjugating
`the chromophore to the antibody. Conveniently,
`the
`chromophore can have a non--oxo carbonyl functional—
`ity (including the nitrogen and sulfur analogs thereof]
`or active whalocarbonyl
`functionality.
`Illustrative
`functionalities for linking the chromophore to the anti-
`body include aeyl halides, mixed anhydridcs, imidate
`alkyl esters,
`isothiocyanate, chlorobromo- or io(loa~
`cetyl, and the like.
`The conditions for conjugation employ rnodcratc
`temperatures 0° to 40° C, in aqueous media at moder-
`ate pH. Conjugation of chrornophorcs to protein is
`known in the art. The, et al., Immunology, 18, 865
`(1970); Cebra, et al., J.
`lmmunol., 95, 230 (1965);
`Goldman, Fluorescent Antibody Methods, Academic
`Press, New York (1968).
`A
`The number of chromophore groups which are con-
`jugated to the antibody may be varied over a relatively
`broad range, depending on the chromophore involved.
`There will be at least one chromophore group per anti
`body, and usually on the average, from about 2 to 30,
`more usually from about 3 to 25 chromophore groups
`per antibody. Where the chromophore is
`the fluo--
`rescer, the average number of chromophore groups per
`'1 antibody will be from about 1 to 20, usually 2 to 15 and
`more usually 7.. to 10. Where the chromophore is the
`quencher, the average number of chromophore groups
`per antibody will be from about 2 to 30, usually 3 to 25,
`and more usually 5 to 25.
`It should also be noted that when antibodies are pre»
`pared for a ligand having a plurality of cpitopic sites,
`the receptor composition is not homogeneous. That is,
`the receptor will have antibodies which recognize dif-
`ferent cpitopic sites. In referring to receptor, it is in-~
`tended to include all the antibodies which are capable
`of specifically binding to any of the cpitopic sites of the
`ligarid.
`l’oly(Ligand Analog)
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`most part will be carbon, oxygen, nitrogen and sulfur,
`particularly carbon, oxygen, and nitrogen.
`The functionalities involved in the linking group will
`normally be non—oxo carbonyl (including imino and
`thionocarbonyl)
`oxy,
`amino (particularly tertiary
`amino or quaternary) or combinations thereof, e.g.
`amido, carbamyl, and amidino.
`or
`tluorescer
`either
`The
`two
`chromophores,
`quencher, will normally have either an amino or alco-
`hol funetion for reacting with a non—oxo carbonyl func—
`tion (including the nitrogen and sulfur analogs thereof)
`or have a non—oxo carbonyl function, which can be
`reacted with an amine or alcohol functionality.
`Where the ligand is of at
`least 2,000 molecular
`weight, a plurality of chromophore groups may be
`bound to the ligand. Usually, there will be at least one
`chromophore group per 20,000 molecular weight,
`more usually at
`least one chromophore group per
`l0,000 molecular weight and not more than one chro-
`mophore group per l,000 molecular weight, more usu—
`ally not more than one chromophore group per 2,000
`molecular weight. The considerations concerning the
`number of chromophores conjugated to the ligand have
`been previously enumerated. The linking groups will be
`as previously described. Usually, the ligand will be an
`antigenic polypeptide or protein having a plurality of
`amino groups. Active halogen or nonoxo carbonyl (in;
`cluding nitrogen and sulfur analogs) can be used for ,
`conjugation to form a covalent bond or amides, ami-
`dines, thionoarnides, urcas, guanidincs and thioureas.
`Alternatively, the ligand and chromophore (Ch, or
`Chg) may be linked to a hub molecule (poly(ligand
`ana|og)—poly(chromophore). The hub molecule or nu—
`clcus molecule can be employed with advantage for a
`variety of reasons. The nucleus molecule will generally
`be a polymeric molecule of relatively high molecular
`weight, normally in excess of 20,000 molecular weight,
`frequently 60,000 molecular weight, and may be 10
`million or higher. The nucleus molecule will normally
`be water soluble or dispcrsible ir1 an aqueous medium
`to provide a stable dispersion, where the dispersible
`material does not interfere with the absorption or irra-
`diation of light. The nucleus molecule may be a natu-
`rally occurring material, a modified naturally occurring
`material, or synthetic. Included among nucleus mole-
`cules are polypeptides, proteins, polysaccharides, syn-
`thetic polymers, and the like. The nature of the hub
`rrrolecule may be widely varied, so long as it
`is suffi-
`ciently functionalized to permit the introduction of the
`ligand and the chromophore molecules.
`Among proteins which can find use are alburnins,
`globulins, proteoglycans, and the like; among polysac-
`charides are amylosc, cellulose, agarose, dcxtrans, or
`the like, either as obtained or partially degraded;
`aniong synrlietic polymers, polyviriylaleoliol, arzrylates,
`copolymcrs thereof or the like may be employed.
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`The poly(ligand analog) differs from the ligand ana-
`log-ehromophore
`and
`poly(ligand
`analog)-poly(-
`Chromophore) in that no chroinophore is present, only
`ligand analog. The same types of nucleus molecules
`and the same degree of conjugation apply for the poly(—
`ligand analog) as for the poly(ligand analog)—poly(—
`chromophore). However,
`the ligand analog may be
`present in much higher ratio than the hub nucleus can
`accomodate receptor. Therefore, while a minimum
`number of ligand analog groups are essential, the maxi-
`mum number is one of expedience. The significant
`factor is that receptor molecules when bnunt] to polyt-
`ligand analog) can come sufficiently close to allow the
`chromophores to come within quenching distance.
`In choosing a nucleus molecule, a number of consid-
`erations will bcar on the choice. While it is not essential
`that the nucleus molecule be water soluble,
`in most
`instances, it will be desirable. In any event, the nucleus
`molecule or composition will be capable of stable dis-
`persion in an aqueous medium. Secondly, the nucleus
`molecule should not absorb light at the emission wave-
`length, of the fluorescer to cause significant quenching.
`Thirdly, the nucleus molecule should not fluorescc at
`the emission wavelengths of the fluorcsscer when irradi-
`ated with the exciting light. Therefore, any significant
`absorption by the nucleus molecule should be below
`about 520nm, preferably below about 45{lnn1.
`The nucleus molecule should be highly functional-
`ized, preferably with amino or hydroxyl groups, al-
`though other reactive functionalities are also useful,
`c.g, carboxy. Fourthly, the nucleus molecule should be
`stable under conditions of storage and Lise. Fifthly, the
`nucleus molecule should be inert
`to functionalities
`present in the chromophorc and ligand, other than the
`functionality for linking. Finally, the nucleus molecule
`should not interfere with the immunoassay, for exam-
`ple, by having naturally occurring receptors which may
`be present in physiological fluids which are studied.
`While any size of molecule may be employed, very
`large molecules or cells will create practical problems.
`For example, a very large molecule passing through the
`light beam of the fluorometer could provide a sudden
`increase in the peak height. Therefore, the signal ob-
`tained would have to be averaged over a reasonable
`period of time. Large molecules will also result in in-
`creased scatter, but the scatter could he cornpensated
`for by an appropriate optical system. Preferably. for the
`most part, molecules will be employed which are less
`than about 10 million molecular weight, more prefer-
`ably from about 30,000 to 1,000,000 molecular weight.
`Chromophore
`Since antibodies are normally present in the assay
`medium, and proteins absorb light ofwavelengths up to
`about 310nm, the fluorescer will have substantial ab-
`sorption higher than 310nm, normally higher than
`350nm, and preferably higher than about 400nm. The
`choice offluorescer will also be governed by the partic-
`ular ligand of interest. The fluorescer should absorb
`light at a higher wavelength than the ligand or ligand
`analog of interest. A high extinction co-efficient
`is
`desirable, greatly in excess of l0, preferably in excess
`of 103, and particularly preferred in excess of 104. A
`good quantum yield should be available in the aqueous
`medium for the fluorescer. As a matter of convenience,
`the absorption peak of the fluorescer should not vary
`significantly with variation in the ligand.
`A number of different fluorescers are described in
`the articles previously noted; namely, Stryer, supra,
`and Brand, et al., supra.
`
`I0
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`S5
`
`60
`
`65
`
`3,996,345
`
`8
`One group of fluorescers having a number of the
`desirable properties described previously are the min-
`thene dyes, which include the fluoresceins derived
`from 3,6-diliydroxy—9—phenyl-xanthhydrol and rosa-
`mines and rhodamines, derived from 3,6—dianiino—9—
`phenylxanthhydrol. The rhodamines and fluoresceins
`have a 9-0-carboxyphenyl group, and are derivatives of
`9-o—carboxyphenylxanthhydrol.
`These compounds are commercially available with
`substituents on the phenyl group which can be used as
`the site for bonding or as the bonding functionality. For
`example, amino and isothiocyanate substituted fluores-
`cein compounds are available.
`Another group of fluorescent compounds are the
`naphthylamines, having an amino group in the alpha or
`beta position, usually alpha position. Included among
`the
`naphthylainino
`compounds
`are
`l-dime-
`thylaininonaphthj,/l—5—sulfonate,
`l-anilino—8—naphtha-
`lent: sulfonate and 2—p—toluidinyl-6—naphthalene sulfo—
`nate.
`
`Other dyes include 3-phenyl-7—isocyanatocoumarin,
`acridines, such as 9-isothiocyanatoacridine and acri-
`dine orange; N—[p-(2-bcnzoxazolyl)phenyl)malcimide;
`benzoxadiazoles, such as 4—chloro—7—nitrobenzo-2-oxa-
`[,3-diazole
`and
`7-(p-methoxybenzylamino)-IL
`1iitrobenzo—2-oxa--1,3-diazole;
`stilbenes,
`such as 4-
`dirnethylarnirto—4'—isothiocyanatostilbene and 4—dime-
`thylamino—4'—rnaleimidostilbene; N,N’—dioctadecylox-
`acarbocyanine p—toluenesulfonatc; pyrenes, such as
`8—hydroxy-l ,3,6-pyrenetrisulfonic
`acid,
`. and
`l—
`pyrenebutyric acid, mcrocyanine 540,
`rose biengal,
`2,4-diphcnyl-3t2lH)-furanone, as well as other readily
`available lluorescing molecules. These dyes, either
`have active funclionalitics or such functionalities may
`be readily introduced.
`Similar considerations involved with the fluorescer
`molecule are applicable to the quenching molecule,
`except that a good fluorescent quantum yield is not
`required where fluorescence of the fluorescer is being
`measured. An additional consideration for the quench-
`ing molecule is that it has its absorption at an emission
`wavelength of the fluorescer. Good overlap of the fluo-
`rescer emission and quencher absorption is desirable.
`It should be noted that both the absorption and emis-
`sion characteristics of the dye may vary from being free
`in solution and being bound to a protein or ligand.
`Therefore, when referring to the various ranges and
`characteristics of the dyes, it is intended to indicate the
`dye as employed and not the dye which is unconjugated
`and characterized in an arbitrary solvent. In the area of
`overlap between fluorescence and quenching,
`the
`quencher should have extinction coefficients of the
`same order or higher than those set forth for absorption
`by the fluorscing molecule.
`Ligand
`As indicated, the ligand will vary widely, normally
`having a molecular weight of at least 1 10, more usually
`at least 125 with the maximum molecular weight unlim-
`ited, although usually not exceeding 10 million. For the
`most part, the significant factor concerning a ligand is
`that a receptor can be made to the ligand or is avail-
`able. Normally, receptors can be made for most or-
`ganic compounds having a polar functionality. Com-
`pounds for which antibodies can be formed by bonding
`the compound to a compound having antigenic proper-
`ties are referred to as haptens. Those compounds which
`elicit antibody formation without chemical modifica-
`tion are referred to as antigens. Sec Kabat, et al., Ex-
`
`

`
`9
`
`3,996,345
`
`lmmunochemistry, Charles C. Thomas,
`perimental
`Springfield, Illinois, 1967.
`_
`The non-polymeric ligands of interest will normally
`be of from about 125 to 2,000 molecular weight. These
`compounds involve a wide variety of compounds of
`varying structure,
`functionality,
`and physiological
`properties. The compounds may be acyclic, alicyclic or
`heterocyclic, both mono— and polycyclic. The heteroat—
`oms involved include oxygen, nitrogen, sulfur, halogen
`(fluorine, chlorine, bromine and iodine) boron, phos-
`phorous, metal cations of Groups IA and 2A of the
`Periodic Chart, transition metals, and the like.
`The functionalities include alcohols, ethers, carbox-
`ylic acids, esters and amides, amines (primary, secon-
`dary, tertiary and quaternary) halo, nitrilo, mercapto,
`and the like. Normally, the compounds will be Com-
`posed solely of carbon, hydrogen, oxygen, nitrogen,
`halogen and phosphorous, particularly carbon, hydro-
`gen, oxygen, and nitrogen and where salts are involved,
`the appropriate metal counterion or ammonium coun-
`terion.
`
`Hctcrocyclic rings which are present include pyrrole,
`pyridine, pipcridine,
`indolc,
`thiazole, piperazine, py—
`ran. cournarin, pyrimidine, purine, triazine, imidazole,
`and the like.
`Because of the wide variety of compounds which can
`be determined in accordance with the subject assay,
`the different groups will be broken down into various,
`frequently artificial, categories, either by the presence
`of a particular functionality or ring structure, or be-
`cause of sharing a particular function or because of
`being recognized as a class.
`The first class of compounds of interest are those
`having an amino group, either as a heterocyclic mem-
`be_r, or as a functionality on an aliphatic chain. These
`compounds will normally be of from about ll0 to 800
`molecular weight, more usually of about 125 to 650
`molecular weight. These compounds frequently have
`an amino group separated by 2 to 3 aliphatic carbon
`atoms trom a benzene ring.
`The first group of compounds of interest are the
`alkaloids and the metabolites of those alkaloids which
`are ingested. The first group of important alkaloids are
`alkaloids of the morphine group. lncluded in this group
`are morphine, codeine, heroin, morphine glucuronide
`and the like.
`
`5
`
`ll)
`
`i5
`
`25
`
`30
`
`35
`
`41)
`
`45
`
`The next group of alkaloids are the cocaine alkaloids,
`which includes, particularly as metabolites, benzoyl
`ccgonine and ecgonine.
`Another group of alkaloids are the cinchona alka— ,
`loids which includes quinine.
`The isoquinoline group of alkaloids includes mesca--
`line.
`The benzylisoquinoline alkaloid group includes pa-
`paverine.
`,
`The phthalide isoquinoline alkaloid group includes
`narcotine, narceine, and cotarnine.
`The indolopyridocoline alkaloid group includes yo-
`hirnbine and reserpine.
`The crgot alkaloid group includes ergotamine and
`lysergic acid.
`Other groups of alkaloids are slrychnine alkaloids,
`pyridine alkaloids, piperidine alkaloids, pyrrolizidine
`alkaloids, and the like.
`The alkaloids of primary interest are those which
`come within the category of drugs of abuse, such as
`morphine, cocaine, mcscaline, and lyscrgic acid, which
`may be