United States Patent [19]
`
`Ullman et al.
`
`[11]
`
`[45]
`
`4,261,968
`* Apr. 14, 1981
`
`[54]
`
`[751
`
`FLUORESCENCE QUENCHING WITH
`IMMUNOLOGICAL PAIRS IN
`IMMUNOASSAYS
`
`Inventors: Edwin F. Ullman, Atherton; Moshe
`Sehwarzherg, Palo Alto, both of
`Calif.
`
`OTHER PUBLICATIONS
`
`Forsum. 1., Immunological Methods, vol. 2, 1972, pp.
`l83—l95.
`
`Primary Exam1'ner—Anna P. Fagelson
`Attorney, Agent, or FIrm—-Bertram I. Rowland
`
`I73]
`
`Assignee:
`
`Syva Company, Palo Alto, Calif.
`
`[57]
`
`ABSTRACT
`
`[ ‘ J
`
`[21]
`
`[22]
`
`[60]
`
`[51]
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`[52]
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`[53]
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`[56]
`
`Notice:
`
`The portion of the term of this patent
`subsequent to Nov. 13, 1996, has
`been disclaimed.
`I
`App}. No.: 37,802
`
`Filed:
`
`May 10, 1979
`
`Related US. Application Data
`Division of Ser. No. 731,255, Oct. 12, 1976, Pat. No.
`4,174,383, which is a continuation of Ser. No. 591.386,
`Jun. 30, 1975, Pat. No. 3,996,345, which is a continua-
`tion-in-part of Ser. No. 497,167, Aug. 12, l9T4, aha:-1»
`cloned.
`
`Int. C1.-‘~ ................... .. GOIN 33/53; G01N 33/68;
`-
`GOIN 33/74; GOIN 33/94
`(3.5. CI. .................................... .. 424/8; 23/230 B;
`250/302; 424/1; 424/1; 424/11; 424/12;
`424/13; 435/‘:
`'
`Field of Search ......................... .. 424/7, 8, 12, 13;
`-
`23/230 13; 435/7; 250/302
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`$160,016
`4,174,384»
`
`7/1979 Ullman
`ll/1979 Ullrnan et al.
`
`424/I3 X
`................... .. 424/i2 X
`
`Immunoassays are provided employing antibodies and
`fluorescer-quencher (F-Q) ehromophoric pair, wherein
`one or both of the chromophoric pair are bonded to
`antibodies. Depending on the particular ligand of inter-
`est, various reagent combinations can be employed,
`where the amount of quenching is directly related to the
`amount ofligand present in the assay medium.
`In carrying out the assay, the unknown and antibody
`specific for the ligand of interest to which is bound one
`of the F-Q pair, are combined in an aqueous buffered
`medium. Depending on the protocol, different assay
`reagents are employed in the aqueous buffered medium:
`(1) ligand analog bonded to the other of the F-Q pair;
`(2) antibodies specific for the ligand to which is bound
`the other of the F-Q pair or; finally, (3) a combination of
`a plurality of ligands bonded together through linking
`groups to_ a hub molecule, usually a polymer, in combi-
`nation with antibody bound to the other of the F-Q pair.
`The composition is irradiated with light at a wave-
`length, absorbed by the fluoreseing molecule and the
`amount of fluorescence determined. By employing ap-
`propriate standards,
`the presence and amount of the
`ligand can be determined.
`'
`
`4 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2051
`
`Genentech Exhibit 2051
`
`IPR2016-00710
`
`

`
`1
`
`4,261,968
`
`FLUORESCENCE QUENCHING WITH
`IMMUNOLOGICAL PAIRS IN TMMUNOASSAYS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a divisional of application Ser. No.
`731,255,
`filed Oct.
`12,
`1976, now U.S. Pat. No.
`4,174,383, which is a continuation of application Ser.
`No. 591,386, filed June 30, 1975 now U.S. Pat. No.
`3,996,345, which is a continuation-in-part o_f application
`Ser. No. 497,167, filed Aug. 12, 1974, now abandoned.
`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
`techniques 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
`EMIT ®, and hemagglutination (HI). These techniques
`are effective for determining amounts of materials in the
`range of 10-5 to 10-10 M or less.
`These techniques 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 the amount of the compound of interest which is
`present.
`‘
`In developing immunoassays, one is limited by the
`availability and properties of an appropriate receptor.
`However, as for the other reagents and the technique of
`measurement, there are a number of different consider-
`ations which make for a more accurate, convenient or
`commercially desirable assay. First, it is desirable that
`there be a minimum number of measurements of 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 employed
`should be relatively stable, so as to be capable of storage
`and shipment. Fourthly, the method should not be sub-
`ject to significant
`interference from other materials
`which may be adventiously present in the sample to be
`assayed. Other considerations are ease of training of
`technicans, absence of health hazards, sensitivity, repro-
`ducibility, and applicability to a wide variety of ligands.
`The subject invention is predicated on the phenome-
`non of energy transfer between two chromophores.
`When a flourescing chromophor is irradiated with light
`absorbed by the chromophore, the fluorescing chromo-
`phore can dissipate the energy of the absorbed light by
`emitting light of longer wavelength, that is, fluorescing.
`If another chromophore is within less than 100 A of the
`fluorescer and absorbs light at the wavelength of emis-
`sion, there is a probability, depending upon other fac-
`tors, that the lluorescer will transfer to the other chro-
`mophore the energy which would otherwise have been
`emitted as light, in effect, quenching the fluorescer.
`2. Description of the Prior Art
`
`5
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`25
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`35
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`40
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`45
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`
`2
`U.S. Pat. No. 3,709,868 is exemplary of a radioimmu-
`noassay. U.S. Pat. No. 3,690,834 is exemplary of a 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,
`Amman’ Review offifochemisrry, 41, 843-868 (1972); and
`Sayer, Science. 162, 526 (1968). Also of interest is co-
`pending application Ser. No. 402,693, filed Oct. 2, 1973.
`SUMMARY OF THE INVENTION
`
`' A method is provided for determining the presence
`or amount of an organic compound to which a receptor,
`usually antibody, is available or can be prepared. The
`organic compound will be hereinafter referred to as a
`ligand.
`I
`two chromophores are
`In carrying out the assay,
`employed which are a lluorescer-quencher pair. The
`amount of lluorescer within quenching -distance of
`quencher is affected by the amount of ligand present in
`the assay medium.
`-
`-
`One chromophore 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 chromo-
`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 ligand.
`Where the ligand has only one independent epitopic
`site (monoepitopic), usually one chromophore will be
`covalently. bonded to a receptor for ligand, and'the
`other chromophore will be provided as covalently
`bonded to a ligand analog or a combination of poly(li-
`gand analog) and the chromophore covalently bonded
`to receptor for ligand.
`Where the ligand has a plurality of independent epi-
`topic sites (polyepitopic), the modes indicated above
`may be 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 chro-
`mophore is bonded to receptor for the ligand-receptor
`from one species and the other chromophore 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 procedures, 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 bullered 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 determined.
`
`

`
`3
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Definitions
`
`4,261,968
`
`Ligand--an organic molecule or assemblage, nor-
`mally greater than l00 molecular weight and having at
`least one functionality, normally polar, for which a
`receptor is either naturally available or can be prepared.
`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 molecule or in
`having a linking group which has been introduced in
`place of one or more atoms originally present in the
`ligand. The ligand analog precursor is the compound
`employed for conjugating ligand or ligand analog to
`another molecule, e.g. chromophore.
`Assemblage--a combination of organic molecules
`bound together by other than covalent bonds, generally
`having molecular weights 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, nor-
`mally joined together by weak bonds, such as polar
`bonds or disulfide bonds, which under the conditions of
`the system are capable of being in equilibrium with the
`individual entities.
`Chron1ophore—a fluorescer or quencher molecule;
`in the subject invention, the fluorescer and quencher are
`interrelated. The fluorescer molecule is a chromophore
`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 100 A, of the fluorescer
`molecule, by accepting the energy which would other-
`wise be emitted as fluorescent light. As far as the mole-
`cule or composition to which the chromophores are
`joined, in most instances, the fluorescer and quencher
`will be interchangeable, although there will frequently
`be some preference. Therefore, for purposes of general-
`ity, the two molecules will be referred to as chromo-
`phores, and individually referred to as Chi and Ch}.
`Ligand
`analog-chromophore
`(ligand
`analog-
`(Ch2),_-)—-ligand analog is covalently bound to one or
`more fluorescent molecules or quencher molecules.
`With small ligands, those below about 10,000 molecular
`weight, usually below about 2,000 molecular weight,
`the ligand analog will usually be joined to fewer than 10
`chromophores, usually from I to 10 chromophores, 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 10,000 molecular weight,
`a plurality of chrornophores may be covalently bound
`to ligand analog. The number of chromophores 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 chromo-
`phores to insure a substantial amount of quenching
`when receptor-Ch]
`is bound to the ligand analog-
`(Ch2)x-
`Polyfligand analog)-poly(chromophore)[poly(ligand
`analog)-poly(Ch2)]—ligand analog and chromophore
`are bonded to a high molecular weight (as compared to
`the ligand analog and chromophore) water soluble
`
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`4
`polyfunctionalized hub or nucleus molecule, to provide
`a plurality of ligand analog groups and chromophore
`groups spaced on the surface of the molecule, so that
`when receptor-Chi is bound to ligand analog, some Ch;
`groups will be present within quenching distance of
`Ch; groups.
`are
`analog groups
`Polyfligand analog)——ligand
`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 analogs per unit area for quenching to occur
`when the poly(ligand analog) is saturated with recep-
`tor-Ch. and receptor-Ch; in appropriate proportions.
`Receptor-chromophore (receptor-Chi and receptor-
`Ch2)—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 104, frequently, in excess of l0‘5. 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 receptors
`will be antibodies to which one or more, usually at least
`two or more, chromophore groups will be bound.
`Receptor composition——receptor composition is a
`homogeneous or heterogeneous composition capable of
`specific non—covalent binding to ligand and ligand ana-
`log ‘and includes anti-ligand (a composition which spe-
`cifically recognizes the ligand) and a combination of
`anti-ligand and anti(anti-ligand) (a composition which
`specifically recognizes the anti-ligand).
`General Statement of the Invention
`
`The method is predicated on the employment of two
`chromophores which form a fluorescer-quencher pair.
`One of the chromophores is covalently bonded to a
`composition (receptor) which specifically recognizes or
`binds to a ligand. The other chromophore is covalently
`bonded to ligand analog or receptor. When the two
`chromophore containing compositions are introduced
`into the assay medium. 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 com-
`petes with ligand for receptor, ligand analog being pres-
`ent as poly(ligand analog) or covalently bonded to
`chromophore. The assay may also be carried out non-
`competitively with ligands having a plurality of epi-
`topic sites, where receptor having each of the chromo-
`phores binds to ligand.
`
`Compositions
`
`Depending upon the particular protocol employed
`and the ligand of interest, one or more of the following
`reagent compositions will be employed in the assay
`medium: ligand analog-chromophore, polyfligand ana-
`log)-poly(chromophore), poly(ligand analog), one or
`two receptors and one or two receptor-chromophores.
`The first composition to be considered will be the li-
`gand analog-chromophore.
`
`Ligand Analog-Chromophore and Poly(Ligand
`Analog)-Poly(Chrornophore)
`
`The ligand analog-chrornophore 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-
`
`

`
`20
`
`5
`log for the most part will-be haptenic, rather than anti-
`genic, and generally be less than about 10,000 molecular
`weight, more usually less than about 6,000 molecular
`weight, and frequently in the range of about 125 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
`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 vari-
`ous haptenic or low molecular weight ligands will be
`discussed subsequently.
`The linking group will normally have not more than
`about 10 atoms in the chain between the ligand and the
`chromophore. more usually having either a bond or
`from about 1 to 6 atoms in the chain. The atoms for the
`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 irnino and
`thionocarbonyl) oxy, amino (particularly tertiary amino
`or quaternary) or combinations thereof, e.g. amido,
`carbamyl, and amicino.
`lluorescer or
`either
`The
`two chromophores,
`quencher, will normally have either an amino or alco-
`hol function 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 10,000
`molecular weight and not more than onechromophore
`group per 1,000 molecular weight, more usually not
`more than one chromophore group per 2,000 molecular
`weight. The considerations concerning the number of 40
`chromophores conjugated to the ligand have been pre-
`viously enumerated. The linking groups will be as pre-
`viously described. Usually, the ligand will be an anti-
`genic polypeptide or protein having a plurality of amino
`groups. Active halogen or non-oxo carbonyl (including
`nitrogen and sulfur analogs) can be used for conjugation
`to form a covalent bond or amides, amidines, thionoa-
`Inides, ureas. guanidines and thioureas.
`Alternatively, the ligand and chromophore (Ch; or
`Chg) may be linked to a hub molecule (poly(ligand
`analog}-polytjchrornophore). The hub molecule or nu-
`cleus 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 dispersible in an aqueous medium to
`provide a stable dispersion, where the dispersible mate-
`rial does not interfere with the absorption or irradiation
`of light. The nucleus molecule may be a naturally oc-
`curring material, a modified naturally occurring mate-
`rial, or synthetic. lncluded among nucleus molecules
`are polypeptides, proteins, polysaccharides, synthetic
`polymers, and the like. The nature of the hub molecule
`may be widely varied, so long as it is sufficiently func-
`tionalized to permit the introduction of the ligand and
`the chromophore molecules.
`
`6
`Among proteins which can find use are albumins,
`globulins, proteoglycans, and the like: among polysac-
`charides are amylose, cellulose. agarose, dextrans, or
`the like, either as obtained or partially degraded; among
`synthetic polymers. polyvinylalcohol, acrylates, co-
`polymers thereof or the like may be employed.
`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 mo-
`lecular weight.
`The ratio of chromophore molecules to ligand will
`generally be from about 0.05—20:l, more usually from
`about 0.5—20: 1, preferably from about l—l0:l, and more
`preferably from about 2—8:l.
`Where the chromophore is the lluorescer molecule
`for the purposes of this invention, generally there will
`be at least about 0.5—20, more usually from about I-10,
`and preferably from about 2-7 fluorescing molecules
`per ligand molecule. Where the chromophore is the
`quencher molecule, the number of quencher molecules
`per ligand will generally be from about 05-20, more
`usually from about 1-20, and preferably from about
`2-15 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 functional
`groups on the nucleus molecule.
`
`Receptor-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 at-halocarbonyl
`functionality.
`Illustrative
`functionalities for linking the chromophore to the anti-
`body include acyl halides, mixed anhydrides, irnidate
`alkyl esters, isothiocyanate, chloro- bromo- or iodoa-
`cetyl, and the like.
`The conditions for conjugation employ moderate
`temperatures 0° to 40° C., in aqueous media at moderate
`pH. Conjugation of chromophores to protein is known
`in the art. The, et al., Immunology, 18, B65 (1970); Ce-
`bra, et a]., J. Immunol., 95, 230 (1965); Goldman, Fluo-
`rescent Antibody Methods, Academic Press, New York
`(1968).
`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 fluorescer,
`the average number of chromophore groups per anti-
`body will be from about 1 to 20, usually 2 to 15 and
`more usually 2 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 epitopic sites,
`
`4,261,968
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`
`4,261,963
`
`7
`the receptor composition is not homogeneous. That is,
`the receptor will have antibodies which recognize dif-
`ferent epitopic sites. In referring to receptor, it is in-
`tended to include all the antibodies which are capable of
`specifically binding to any of the epitopic sites of the 5
`ligand.
`
`Poly(Ligand Analog)
`
`8
`analog of interest. A high extinction co-efficient is desir-
`able, greatly in excess of 10, preferably in excess of 103,
`and particularly preferred in excess of 104. A good
`quantum yield should be available in the aqueous me-
`dium for the fluorescer. As a matter of convenience. the
`absorption peak of the fluorescer should not vary signif-
`icantly with variation in the ligand.
`A number of different lluorescers are described in the
`articles previously noted; namely, Stryer, supra, and
`Brand, et aI., supra.
`_
`One group of lluorescers having a number of the
`desirable properties described previously are the inan-
`thene dyes, which include the fluoresceins derived from
`3,6-dihydroxy»9-phenyl-xanthhydrol and rosamines and
`rhodamines, derived from 3,6-diamino-9-phenylxanb
`hhydrol. The rhodamines and fluoresceins have a 9-0-
`carboxyphenyl group, and are derivatives of 9-o-car-
`boxyphenylxanthhydrol.
`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
`naphthylamino
`compounds
`are
`l-dime-
`thylan1inonaphthy1-5-sulfonate, l-anilino-8-naphthalene
`sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate.
`-Other dyes include 3~phenyl-7-isocyanatocoumarin,
`acridines, such as 9-isotliiocyanatoacridine and acridine
`orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; ben--
`zoxadiazoles, such as 4-chloro-7-nitrobenzo-2-oxa-1,3-
`diazole and 7-(p-methoxybenzylamino)—4-nitrobenzo-2-
`oxo—l,3-diazole; stilbenes, such as 4-dimethylamino-42
`isothiocyanatostilbene
`and
`4-dimethylamino-41
`maleimidcstilbene; N,N'-dioctadecyloxacarbocyanine
`p-toluenesulfonate; pyrenes, such as 8-hyclroxy-l-,3,6-
`pyrenetrisulfonic acid, and l-pyrenebutyric acid, mero-
`cyanine 540, rose bengal, 2,4-diphenyl-3{2H)-furanone,
`as well as other readily available lluorescing molecules.
`These dyes, either have active functionalities or such
`functionalities may be readily introduced.
`Similar considerations involved with the fiuorescer
`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 fluoresoer. Good overlap of the fluo-
`rescer emission and quencher absorption is desirable.
`It should he 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 coellicients of "the
`same order or higher than those set forth for absorption
`by the lluorscing molecule.
`
`Ligand
`
`As indicated, the ligand will vary widely, normally
`having a molecular weight of at least 110, more usually
`at least 125 with the maximum molecular weight unlim-
`ited, although usually not exceeding 10 million. For the
`
`.
`
`20
`
`35
`
`45
`
`50
`
`The poly(ligand analog) differs from the ligand ana-
`and
`polyfligand
`analog)-poly(- 10
`log-chromophcre
`chromophore) in that no chrornophore is present, only
`ligand analog. The same types of nucleus molecules and
`the same degree of conjugation apply for the poly(li-
`gand analog) as for
`the" poly(ligand analog)-poly(-
`chromophore). However,
`the ligand analog may be :5
`present in much higher ratio than the hub nucleus can
`accommodate receptor. Therefore, while a minimum
`number of ligand analog groups are essential, the maxi-
`mum number is one of expedience. The significant fac-
`tor is that receptor molecules when bound to poly(1i-
`gand 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 bear on the choice. While it is not essential
`that the nucleus molecule be water soluble, in most 25
`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 lluorescer to cause significant quenching. 30
`Thirdly, the nucleus molecule should not fluoresce at
`the emission wavelengths of the fluorescer when irradi-
`ated with the exciting light. Therefore, any significant
`absorption by the nucleus molecule should be below
`about 520 nm, preferably below about 450 nm.
`The nucleus molecule should be highly functional-
`ized, ‘preferably with amino or hydroxyl groups, al-
`though other reactive functionalities are also useful, e.g.
`carboxy. Fourthly,
`the nucleus molecule should be
`stable under conditions of storage and use. Fifthly, the
`nucleus molecule should be inert to functionalities pres-
`ent in the chromophore and ligand, other than the func-
`tionality 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 molecular passing through
`the light beam of the fluorometer could provide a sud-
`den increase in the peak height. Therefore, the signal
`obtained would have to be averaged over a reasonable
`period of time. Large molecules will also result in in-
`creased scatter, but the scatter could be compensated
`for by an appropriate optical system. Preferably, for the 55
`most part, molecules will be employed which are less
`than about 10 million molecular weight, more prefera-
`bly from about 30,000 to 1,000,000 molecular weight.
`Chromophore
`Since antibodies are normally present in the assay
`medium, and proteins absorb light of wavelengths up to
`about 310 nm, the fluorescer will have substantial ab-
`sorption higher than 3l0 nm, normally higher than 350
`nm, and preferably higher than about 400 nm. The 65
`choice of fluorescer will also be governed by the partic-
`. ular ligand of interest. The lluorescer should absorb
`light at a higher wavelength than the ligand or ligand
`
`

`
`9
`most part, the significant factor concerning a ligand is
`that a receptor can be made to the ligand or is available.
`Normally, receptors can be made for most organic com-
`pounds having a polar functionality. Compounds for
`which antibodies can be formed by bonding the oom-
`pound to a compound to a compound having antigenic
`properties are referred to as haptens. Those compounds
`which elicit antibody formation without chemical mod»
`ification are referred to as antigens. See Kabat, et al.,
`Experimental Immunochemistry, Charles C. Thomas,
`Springfield. lll., 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 acylic, 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, carbon-
`ylic acids, esters and amides, amines (primary, second-
`ary,
`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.
`Heterocyclic rings which are present include pyrrole,
`pyridine, piperidine, indole, thiazole, piperazine, pyran,
`coumarin, 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, fre-
`quently artificial, categories, either by the presence of a
`particular functionality or ring structure, or because of
`sharing a particular function or because of being recog-
`nized as a class.
`The first class of compounds of interest are those
`having an amino group, either as a heterocyclic mem-
`her, or as a functionality on an aliphatic chain. These
`compounds will normally be of from about 110 to 300
`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 from a benzene ring.
`The first group of compounds of interest are the alka-
`loids and the metabolites of those alkaloids which are
`ingested. The first group of important alkaloids are
`alkaloids of the morphine group. Included in this group
`are morphine, codeine, heroin, morphine glucuronide
`and the like.
`The next group of alkaloids are the cocaine alkaloids,
`which includes, particularly as metabolites, benzoyl
`ecgonine and ecgonine.
`Another group of alkaloids are the cinchona alkaloids
`which includes quinine.
`The isoquinoline group of alkaloids includes mesca-
`line.
`
`10
`
`I5
`
`20
`
`25
`
`35
`
`40
`
`45
`
`S0
`
`55
`
`The henzylisoqui