[11]
`[45]
`
`4,174,384
`* Nov. 13, 1979
`
`Wachsmith, Histochem. vol. 38, No. 4. 1974. pp.
`339-350.
`Cohen. The J. of Immunology. vol. 93, 1967. pp.
`I43-149.
`
`Kahat. Bxptl. In1munochem.. C. C. Thomas, Spring-
`field, III.. 2nd ed.. 1961, pp. 809-811.
`Williams, Methods in Immunol. & Immunoohe1n., Aca-
`demic Press. N.Y., vol. III, 1971, pp. 435-453.
`
`Primary E.xann'ner—Anna P. Fagelson
`Attorney. Agent. or Firm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Immunoassays are provided employing antibodies and a
`fluoreacer-quencher (F-Q) chrotnophoric 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 of ligand present in the assay medium.
`In carrying out the assay, the unknown and antibody
`specific for the ligand ofinterest 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 F41 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-112 pair.
`The composition is irradiated with light at a wave-
`length. absorbed by the fluorescing molecule and the
`amount of fluorescence determined. By employing ap-
`propriate standards. the presence and amount of the
`ligand can be determined.
`
`United States Patent [19]
`Ullrnan et al.
`
`[54] FLUORESCENCE QUENCHING WITH
`IMMUNOLOGICAL PAIRS [N
`IMMUNOASSAYS
`
`[75]
`
`Inventors: Edwin F. Ullman, Atherton; Moshe
`Schwarzlrerg. Palo Alto. both of
`Calif.
`
`[73] Assignee:
`
`Syva Company. Palo Alto, Calif.
`
`[ * ] Notice:
`
`The portion of the term of this patent
`subsequent to Dec. 7. 1993. has been
`disclaimed.
`
`[21] App]. No.: 131,255
`
`[22] Filed:
`
`Oct. 12, 1916
`
`Related US. Application Data
`
`[63]
`
`l9'r'5. Pat.
`Continuation of Ser. No. 591,336, Jun. 30,
`No. 3,996,345, which is a continuation-in-part of Ser.
`No. 497.167. Aug. 12. 1974. abandoned.
`
`[51]
`
`Int. (21.1 ................... .. com 21/as; oom 31/00;
`com 33/1.5; omo mo
`[52] U.S. Cl. ................................... .. 42-vs; 23/230 R;
`23/230 B; 435/7; 250/302; 260/ I 12 11; 260/112
`13; 424/1; 424/7; 424/1 1; 424/12; 424/13
`[53] Field of Search ........... .. 23/230 R. 230 B; 424/1.
`424/7, 8, 11, 12, 13; 260/112 R, 112 B;
`195/ 103.5 A, 103.5 R; 250/302
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`ll/1916 Harte42-1-/IZX
`3,992,531
`I2/1976 Ullrnan
`424/12
`3.995.345
`3.993.943 12/ma uuman424/12
`
`
`
`OTHER PUBLICATIONS
`
`Winkler. Biochemistry. vol. 8. Jun. 1969. pp. 2586-2590.
`
`4 Claims, No Dravringa
`
`SANOFI V. GENENTECH
`SANOFI v. GENE(cid:49)TECH(cid:3)
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2039
`EXHIBIT 2039
`
`
`

`
`1
`
`4,174,334
`
`FLUORESCENCE QUENCHING WIT]-I
`IMIVIUNOLOGICAL PAIRS IN IJVIMUNOASSAYS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`5
`
`I0
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`15
`
`25
`
`30
`
`35
`
`This application is a continuation of application Ser.
`No. 591,336, filed June 30, 1975. now U.S. Pat. No.
`3,996,345, which is a continuation-in-part of 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 efiective for determining amounts of materials in the
`range of 10-5 to li]—“lM 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 imniunoassays, one is limited by the
`availability and properties of an appropriate receptor.
`However, as for the other reagents and the technique of
`measurernent, 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 ofstorage
`and shipment Fourthly. the method should not be sub-
`ject to significant
`interference from other materials
`which may be advcntiously present in the sample to be
`assayed. Other considerations are case of training of 50
`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 chrotnophores.
`When a tluorescing 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.
`lf_another chromophore 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 lluorescer will transfer to the other chro-
`mophore the energy which would otherwise have been
`emitted as light, in effect, quenching the fluoroescer.
`2. Description of the Prior Art
`U.S. Pat. No. 3,709,363 is exemplary of a radioirumu-
`noassay. U.S. Pat. No. 3,690,833 is exemplary of a spin
`immunoassay. US. Pat. Nos. 3,654,090 and 3,817,337
`
`45
`
`55
`
`65
`
`2
`are exemplary of enzyme immunoassays. Articles of
`interest include an article by Ludwig Brand and James
`R. Gohllte, entitled, Fluorescence Probes for Structure,
`Annual Review offlicchemisny, 41, 343-868 (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 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.
`In carrying out the assay, two chromophores are
`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 chromophore is introduced into the assay me-
`dium covalently bonded to a receptor composition
`which specifically binds to the ligand. The second chro-
`ruophore 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 chrontophore will be
`covalently bonded to a receptor for ligand, and the
`other chrornophore will be provided as covalently
`bonded to a ligand analog or a combination of poly(li-
`gand analog) and the chromophorc 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 chrontophore 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 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 determined.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Definitions
`
`Ligand—an organic molecule or assemblage, nor-
`mally greater than 100 molecular weight and having at
`
`
`
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`
`4,174,384
`
`4
`are
`Poly(ligand 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 analogs per unit area for quenching to occur
`when the polytfligand analog) is saturated with recep-
`tor-Chi and receptor-Chg in appropriate proportions.
`Receptor-chromophore (receptor-Ch: and receptor-
`Chz)-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 I04, frequently. in excess of 105. 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 sna-
`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 iluorescer. The assay may
`be carried out competitively, where ligand analog corn-
`petes with ligand for receptor, ligand analog being pres-
`ent as polyfligand 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, polyaigand ana-
`log)-poly(chromophore), poly(ligand_anslog), one or
`two receptors and one or two receptor-chromophores.
`The first composition to he considered will be the li-
`gand analog-chromophore.
`
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`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 sitw 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.
`Asse:nhlage—a. combination of organic molecules
`bound together by other than covalent bonds. generally
`having molecular weights exceeding 600, usually eat-
`ceeding 1,000 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.
`Chromophore—a fluoresccr or quencher molecule;
`inthe subject invention, the fluorescer and quencher are
`interrelated. 'I‘he 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 Chg.
`Ligand
`analog-chromophore
`(ligand
`analog-
`(Ch;_),;)—1igand 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 1 to 10 chromophores, not
`more than about 1 chromophore per 1.000 molecular
`wght. With a large ligand, at least 2,000 molecular
`weight, usually at least about 10,000 molecular weight.
`a plurality of chromophores 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-Ch1 is bound to the ligand analog-
`(Chm.
`Polyfligand analog)-poly(chromophore)[poly(ligand
`analog)-po1y(Ch2}]-ligand analog and chromophore
`are bonded to a high molecular weight (as compared to
`the ligand analog and chromophore) water soluble
`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-Ch: is bound to ligand analog. some Ch|
`groups will be present within quenching distance of
`Ch; groups.
`
`SS
`
`Ligand Analog-Chromophore and Poly(Ligsnd
`Analog)-Poly(Ch.romophore)
`
`The ligand analog-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 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
`
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`
`4,174,334
`
`.
`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 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 have 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, partic-
`ularly carbon, oxygen. and nitrogen.
`The functionalities involved in the linking group will
`normally be non-oxo carbonyl (including iniino and
`thionocarbonyl) oxy, amino (particularly tertiary amino
`or quaternary) or combinations thereof, e.g. amido,
`carbamyl, and arnidino.
`lluorescer or
`either
`The
`two chromophores,
`quencher, will normally have either an amino or alco-
`hol function for reacting with a non-onto 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 chrornophore 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 one chromophore
`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
`cltromophores 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 he used for conjugation
`to form a covalent bond or amides, arnidines, thionoa-
`mides, areas, guanidines and thioureas.
`Alternatively. the ligand and chromophore (Ch1 or
`Chg} may be linked to a hub molecule (po1y(1igand
`analog)-poly(chromophore). 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 docs 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. Included 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.
`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.
`
`5
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`Normally, there will be not less than about one conju-
`gate (ligaud 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 chromphore molecules to ligand will
`generally be from about 0.05—20:l. more usually from
`about 05-20:]. preferably from about I-10:], and more
`preferably from about 2-8:1.
`Where the chromophore is the lluorescer molecule
`for the purposes of this invention, generally there will
`be at least about 05-20, more usually from about 1-10,
`and preferably from about 2-7 lluorescing molecules
`per ligand molecule. “There the clrtromophore is the
`quencher molecule. the number of quencher molecules
`per ligand will generally be from about 0.5—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-CHROMOPI-[ORE
`
`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. Co-nvenitly. the
`chromophore can have a non-oxo carbonyl functional-
`ity (including the nitrogen and sulfur analogs thereoi)
`or active ct-halocarbonyl
`functionality.
`Illustrative
`functionalities for linking the chromophone to the anti-
`body include acyl halides. mixed anhydrides. imidate
`alkyl esters, isothiocyanate. chloro-. brorno- 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, 13, 865 (1970); Ce-
`bra, ct 2.1., 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 chrotnophore 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,
`the receptor composition is not homogeneous. That is,
`the receptor will have antibodies which recognize dif-
`ferent epitopic tes. 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
`ligand.
`
`
`
`

`
`7
`
`POLY(LIGAND ANALOG)
`
`4,174,384
`
`The poly(ligand analog) differs from the ligand ana-
`log-chromophore
`and
`poly(ligand
`analog)-poly(-
`chromophore} in that no chromophore 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 polyfligand analog)-poly(-
`cltromophore). 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 fac-
`tor ia that receptor molecules when bound to poly(li-
`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
`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.
`Thirdly, the nucleus molecule should not fluoresce at
`the emission wavelengths of the tluorescer 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-
`ieed, preferably with amino or hydroityl groups, al-
`though other reactive fiinctionalities 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 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 be compensated
`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 prefera-
`bly from about 30.000 to 1,000,000 molecular weight.
`CHROMOPHORE
`
`Since antibodies are normally present in the assay
`niedium, and proteins absorb light of wavelengths up to
`about 310nm, the lluorescer will have substantial ab-
`sorption higher than 310 nm, normally higher than 350
`um, and preferably higher than about 400 nm. The
`choice of fluorescer 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—eflicient is desir-
`able, greatly in excess of 10, preferably in excess of 103.
`and particularly preferred in excess of 10‘. A good
`quantum yield should be available in the aqueous me-
`dium for the fluorescer. As a matter of convenience. the
`
`20
`
`25
`
`30
`
`35
`
`55
`
`65
`
`8
`absorption peak of the fluorescer should not vary signif-
`icantly 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.
`One group of lluorescers having a number of the
`desirable properties described previously are the xen-
`thene dyes, which include the fluoresceins derived from
`3,6-dihydroxy-9-phenyl-xanthhydrol and rosamines and
`rhodarnines. derived from 3,6-diamino-9-phenylxant-
`hhydrol. The rhodamines and tluoresceins 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-
`thylarninonaphthyl-5-sulfonate. 1-anilino-8-naphthalene
`sulfonate and 2-p—toluidinyl-6-naphthalene sulfonate.
`Other dyes include 3-phenyl-T-isocyanatocoumarin.
`acrldines, such as 9-isothiocyanatoacridine and acridine
`orange; N-(p-(2-benzoxazolyl)phyl)maleimide; ben-
`zoxadiazoles. such as 4—chloro-7-nitrcbenzo-2-oxa-1,}
`diazole and 7-(p-methoxybenzylamino)-4—nitrobenzo-I
`oxa-1,3-diazole; stilbenes, such as 4-dimethylamino-4%
`isothiocyanatostilbene
`and
`4-dimethylamino-45
`msleimidostilbene; N,N‘-dioctadecyloxacarbocyanine
`p-toluenesulfonate; pyrenes, such as li-hydroxy-l,3,6-
`pyrenetrisulfonio acid. and I-pyrenebutyric acid. mero-
`cyanine 540, rose be-ngal, 2,4-diphenyl-3(2H)-furanone,
`as well as other readily available iluorescing molecules.
`These dyes, either have active functionalities 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 lluorescer. Good overlap of the Hun-
`rescer emission and quencher absorption is desirable.
`It should be noted that both the absorption and s-
`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 coeificients of the
`same order or higher than those set forth for absorption
`by the fluorsciug molecule.
`LIGAND
`
`As indicated, the ligand will vary widely, normally
`having a molecular weight of at least 110, more usually
`at least 12.5 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 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 corn-
`
`
`
`

`
`4,114,334
`
`9
`pound to a compound having antigenic properties are
`referred to as haptens. Those compounds which elicit
`antibody formation without chemical modification are
`referred to as antigens. See Kabat, et al., Experimental ’
`Immunochcmistry, Charles C. Thomas, Springfield,
`Illinois, 1967.
`The non-polymeric ligands of intermt 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, chloride, bromine and iodine) boron, phos-
`phorous, metal cations of Groups IA and 2A of the
`Periodic Chart, transition metals, and the like.
`The functionuiities include alcohols, ethers. carbon-
`ylic acids, esters and amides, amines (primary. second-
`ary, tertiary and quaternary) halo. nitrilo, rnercapto,
`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.
`
`10
`
`20
`
`10
`The alkaloids of primary interest are those which
`come within the category of drugs of abuse, such as
`morphine, 'coca.ine,-'mesca]ine,' and lysergic acid, which
`may beanslyzed for the compo_und_or its metabolite.
`depending on the physiological fluid which is analyzed
`for its presence.
`A number of synthetic drugs mimic the physiological
`properties, in part or in whole, of the naturally occur-
`ring drugs of abuse. Included among these drugs are
`methadone, meperidine, amphetamine, methamphet-
`amine, glutethirnide, diphenylhydantoin, and drugs
`which come within the category ofben.zdiazocyclohep-
`lanes, phenothiazines and barbiturates.
`Drugs of interest because of their physiological prop-
`erties are those which are referred to as catecholamines.
`
`Among the catecholamines are epinephrine, ephedrine,
`L-dopa, and norepinephrine.
`Other drugs of interest are the tranquilizer Meproba-
`mate, Tegritol and succinimid, such as Ethoxsumide.
`Other compounds of interest are tetrahydrocannabi-
`nol. cannabinol. and derivatives thereof, primarily com-
`pounds derived from marijuana, synthetic modifications
`and metabolites thereof.
`Another group of compounds of significant interest
`are the steroids. The steroids include estrogens, gusto-
`gens. androgens, adrenocortical honnones. bile acids,
`cardiotonic glycoids, algycones, saponins and sapoge-
`mus.
`
`Another class of compounds are th