4,199,559
`[11]
`[191
`United States Patent
`
`[45] * Apr. 22, 1930
`Ullrnan et al.
`
`[54] FLUORESCENCE QUENCI-IING wrrn
`IMMUNOLOGICAL runs IN
`IMMUNOASSAYS
`
`[75]
`
`Inventors: Edwin F. Ullman, Atherton; Moshe
`Schwarzberg, Sunnyvale, 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] Appl. No.: 766,279
`
`[22] Filed:
`
`Feb. 7, 1977
`
`Related US. Application Data
`
`[63]
`
`Continuation-in-part of'Ser. No. 731,255, Oct. 12, 1976.
`which is a continuation-in-part of Ser. No. 591,386.
`Jun. 30, i9'.~'5, Pat. No. 3,996,345, which is a continua-
`tion-in—part of Ser. No. 497,167. Aug. 12, 191%, aban-
`cloned.
`
`Int. Cl.2 ........................................... .. G01N 33/16
`[51]
`[52] U.S. Cl. .................................... .. 424/8; 23/230 B;
`23/915; 424/12
`[58] Field of Search .................. .. 23/230 B; 424/8, 12;
`195/1035 R, 103.5 A
`
`[56}
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,899,293
`3,996,345
`
`Szczesniak
`8/1975
`12/1976 Ullman
`
`23/230 B
`424/12
`
`Primary Examiner—Sidney Marantz
`
`Attorney. Agent, or Firm—Bertram I. Rowland
`
`ABSTRACT
`[57]
`Imrnunoassays are provided employing antibodies and a
`fluorescer-quencher (F-Q) chromophoric pair, wherein
`one "or both of the chromophoric pair are bonded to
`antibodies. Depending on the particular ligand of inter-
`est, or whether antibodies are to be assayed, various
`reagent combinations can be employed. where the
`amount of quenching is directly related to the amount
`of ligand or antibody present in the assay medium.
`In carrying out the assay for ligands, the unknown and
`antiody 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) antiobodies specific for the ligand to which
`is bound the other of the F-112 pair or; finally, (3) a com-
`bination of a plurality of ligands bonded together
`through linking groups to a hub molecule, usually a
`polymer,
`in combination with antibody bound to the
`other of the EQ pair. The fluorescar is electronically
`excited, for example by irradiation with light at a wave
`length, absorbed by the fluorescing molecule and the
`amount of emitted light determined. By employing
`appropriate standards, the presence and amount of the
`ligand can be determined.-
`The technique for the determination of antibodies in a
`competitive mode is substantially the same except that
`ligand is added as a reagent.
`
`26 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2042
`Genentech Exhibit 2042
`
`IPR2016-00710
`
`

`
`1
`
`4,199,559
`
`'
`FLUORESCENCE QUENCHING WITH
`IMMUNOLOGICAL PAIRS IN IMMUNOASSAYS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`5
`
`This application is a continuation-in-part of applica-
`tion Ser. No. 731,255, filed Oct. 12, 1976, which is a
`continuation-in-part of application Ser. No. 591,386,
`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 effective for determining amounts ofmaterials 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.
`These techniques can also be employed for the deter-
`mination of antibodies by adding known amounts of
`ligand or known amounts of reagent (labeled ligand) or
`both.
`In developing imtnunoassays for ligands, one is lim-
`ited by the availability and properties of an appropriate
`receptor. However. as for the other reagents and the
`technique of measurement, there are a number ofdi.fi'er-
`ent considerations which make for a more accurate,
`convenient or commercially desirable assay. First, it is
`desirable that there be a minimum number of measure-
`ments of the various reagents. as well as transfers of the
`various reagents. Secondly, the equipment for measur-
`ing should be reasonably economical, so as to be acces-
`sible to a broad range of users. Thirdly, the reagts
`employed 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 case of
`training of tcchnicans, absence of health hazards, sensi-
`tivity, reproducibility, and applicability to a wide vari-
`ety of ligands.
`The subject invention is predicted on the phenome-
`non of energy transfer between two chromophores.
`When a fluorescing chromophor is irradiated with light
`absorbed by the chrornophore, the iluorescing chromo-
`phore can dissipate the energy of the absorbed light by
`emitting light of longer wavelength. that is, lluoyescing.
`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-
`
`2
`tors, that the fluorescer 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
`U.S. Pat. No. 3,709,868 is exemplary of a radioimmu-
`noassay. U.S. Pat. No. 3,690,334 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,
`Annual Review affiiachemirrry, 41, 843-868 (1972); and
`Stryer, Science. 1962. 526 (1968). Also of interest is
`co-pending application Ser. No. 402,693, filed Oct. 2,
`1973 U.S. Pat. No. 3,998,943.
`
`SUMMARY OF THE INVENTION
`
`A method is provided for determining the presence
`or amount ofan organic compound to which a receptor,
`usually antibody, is available or can be prepared or for
`the determination of antibodies. The organic compound
`will be hereinafter referred tons a ligand.
`In carrying out the assay,
`two chromophores are
`employed which are a fluorescenquencher 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 %ond chro-
`mophore can be introduced into the assay medium in
`different ways: (l) 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 I: 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 chromophcre 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 chrotnophore bonded to
`receptor for ligand-receptor from the other species. The
`latter method expands the versalitity 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.
`Where the determination of an antibody is involved,
`the same techniques can be employed as for the determi-
`nation of ligands, with the additional variation of add-
`ing ligand to the medium when no ligand analog or
`polyligand is employed.
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`4,199,559
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`3
`The various materials are brought together in an
`aqueous buffered medium. incubated and the fluorescer
`is electronically excited into an electronically excited
`state capable of emitting light. By determining the
`amount of emitted light, after incubation for a predeter-
`mined time interval or after the system has approached
`equilibrium, and comparing the results obtained with
`one or more known standards, the presence or amount
`of ligand or antibody 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
`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 fo competing with
`the ligand for the binding sites of a receptor, and diilers
`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 Inay 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.
`Chromophon-.—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 be electronically excited to an electron-
`ically excited state from which it returns to the ground
`state by the emission of light, for example. the chromo-
`phore is able to absorb light at one wavelength and emit
`light at a longer wavelength. Various means for trans-
`ferring energy to the fluorescer to electronically excite
`the fluorescer to an excited state capable of fluores-
`cence may be employed,
`the particular mode being
`primarily one of convenience. The quencher molecule
`is capable of inhibiting fluorescence, when Dwithin a
`short distance, usually less than about 100 A, 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 Chi and
`Chg.
`analog-
`(ligand
`analog-chromophore
`Ligand
`(Ch2)x)—ligand analog is covalently bound to one or
`more fluorescent molecules or quencher molecules.
`With small ligands, those below about 10,000 molecular
`weights, usually below about 2,000 molecular weight,
`
`4
`the ligand analog will usually be joined to fewer than 10
`chromophores, usually from 1 to 10 chromophores. not
`more than about I chromophore per 1,000 molecular
`weight. With a large ligand, at least 2,032! 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-C111 is bound to the ligand analog-
`(Cl12.)x«
`Polyfligand analog)-poly(chromophore)[poly(ligand
`analog]-poly(Ch1)]—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-Ch1 is bound to ligand analog, some Ch:
`groups will be present within quenching distance of
`Ch; groups.
`Poly(ligand analog)—ligand analog groups are
`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—Ch1 and receptor-Ch: in appropriate proportions.
`Receptor-chromophore (receptor-Ch; and receptor-
`Chg)——a receptor is a molecule which is capable of
`distinguishing an epitopic site and binding to‘sucl1 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 eomposition—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 lluorescer. The assay may
`be carried out competitively, where ligand analog com-
`petes with ligand for receptor. ligand analog being pres-
`ent as poIy(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. The assay may be carried out for
`
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`4, 1 99, 5'59
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`5
`antibodies, where unlabeled antibody to be assayed
`competes with labeled antibody for binding sites.
`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. poly(ligand ana-
`log)-poly(chromophore), polyfligand 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 Ana-
`log)-Poly(Chromophore)
`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
`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 usuallyllaviug 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 functionalitzies involved in the linking group will
`normally be non-oito carbonyl (including irnino and
`thionocarbonyl) oxy, amino (particularly tertiary amino
`or quaternary) or combinations thereof, e.g. amido,
`carbamyl, and arnidino.
`lluoresoer 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 s non-oito 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 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
`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-
`mides, ureas, guanidines and thioureas.
`Alternatively, the ligand and chromophore (Chi or
`Chi) may be linked to a hub molecule (polyfligand
`
`6
`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 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. 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 pemtit the introduction of the ligand and
`the chromophore molecules.
`Among proteins which can find use are albumius,
`globulins, proteoglycans. and the like; among polysac-
`charides are amylase. 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 chromphore molecules to ligand will
`generally be from about 0.05—20:1, more usually from
`about 0.5-20:1, preferably from about l-10:1, and more
`preferably from about 2—8:l.
`Where the chromophore is the lluoresoer molecule
`for the purposes of this invention, generally there will
`be at least about 0.5—20, more usually from about 1-10,
`and preferably from about 2-? iluorescing 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
`l-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.
`R.eceptor—Cbromophore
`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 chrotnophore to the antibody. Conveniently. the
`chromophore can have a non—oxo carbonyl functional-
`ity (including the nitrogen and sulfur analogs thereof]
`or active ct-halocarbonyl
`functionality.
`Illustrative
`functionalities for linking the chromophore to the anti-
`body include acyl halides, mixed anhydrides, imidate
`alkyl esters,
`isothiocyanate. chlorobromo- or iodoa-
`cetyl, and the like.
`The conditions for conjugation employ moderate
`temperatures 0° to 40° C., in aqueous media at moderate
`pl-I. Conjugation of cltromophores to protein is known
`in the art. The, et a1., Immunology, 18, 365 (l9?0); Ce-
`bra, et a].. J. Immunol., 95, 230 (1965); Goldman, Fluo-
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`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 tluorescer,
`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 chromophcre 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 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
`ligand.
`Po1y(Ligand Analog)
`The poly(ligand analog) differs from the ligand ana-
`log-chromophore
`and
`polyfligand
`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(]i-
`gand analog) as for
`the polyfligand analog)-poly(-
`chromophore). However,
`the ligand analog may be
`present inmuch 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 is that receptor molecules when bound to poly(li-
`gand analog) can come sufficiently close to allow the
`chrornophores 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 fluorescer to cause significant quenching.
`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 nrn, 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. Fifihly. the
`nucleus molecule should be inert to fuuctionalities 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 Pilak height. Therefore, the signal ob-
`tained would have to be averaged over a reasonable
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`8
`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
`medium, and proteins absorb light of wavelengths up to
`about 310 nm, the iluorescer will have substantial ab-
`sorption higher than 310 nm. normally higher than 350
`nm, and preferably higher than about 4-00 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-efficient 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
`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 san-
`thene dyes, which include the fluoresceins derived from
`3,6-dihydroxy-9-phenyl-xanthhydrol and rosamines and
`rhodamines, derived from 3,6-diamino-9-phenylxarm
`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-di':ne-
`thy}aminonaphthyl-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-isothiocyanatoacridine and acridine
`orange; N-(p-{2-benzoxazolyl)phenyl)maleimicle; ben-
`zoxadiazoles, such as 4chloro-7-nitroben.zo-2-oxa-l,3-
`diazole and 7-(p-methoxybenzylarnino)-4—nitrobenzo-2-
`oxa-l,3—diazole; stilbenes, such as 4-dimethylamiuo-42
`isothiocyanatostilbene
`and
`4-dimethylamino-45
`maleimidostilbene; N,N'-dioctadecyloxacarbocyanine
`p-toluenesulfonate; pyrenes, such as 8-hydroxy-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 fluorescing molecules.
`These dyes, either have active functionalities or such
`funtionalities may be readily introduced.
`Similar considerations involved with‘ the iluorescer
`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 charactcristics of the dye may vary from being free
`
`
`
`

`
`9
`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 tluorscing 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
`most part, the significant factor oonceming 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 com-
`pound to a compound havng 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
`Immunochemistry, Charles C. Thomas, Springfield,
`Illinois, 1967.
`The non-polymeric ligands ofinterest will normally
`be of from about Hi 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-
`orns 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, carboni-
`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-
`ter-ion.
`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 of ring structure, or because of
`sha