CA1108987A - Chemically induced fluorescence immunoassay - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A competitive protein binding method is provided for the determination of an analyte which is a member of an immunological pair consisting of ligand and receptor for the ligand. A chemiluminescent source is employed comprised of one or more individual members, one chemiluminescent source member being conjugated to one of the members of the immuno-logical pair, so as to provide chemiluminescence adjacent to the site of conjugation. A quencher molecule is conjugated to a member of the immunological pair. When the members of the immunological pair bind, the quencher molecule is brought within quenching distance of the chemiluminescent source so as to inhibit the emission of light by the chemi-luminescent source. The amount of analyte present in the assay medium affects the amount of binding between the members of the immunological pair which results in quenching of the chemiluminescence. By observing the light emitted from the assay medium, either from the chemiluminescent source or the quencher, the change in light emission in relation to the concentration of analyte present in the assay medium can be used to determine the amount of analyte present in the assay medium. By employing standards having known amounts of analyte, the amount of analyte in an unknown sample can be quantitatively determined. Reagent kits can be provided having predetermined amounts of the reagents, so as to substantially optimize the sensitivity of the assay.

Description

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This invention relates to a method of determininy in an assay solution the presence of an anal~-te and to a ~diagnostic kit useful in the method.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of mater-ials which may be readily and accurately determined, as well as the methods for determination. One broad category of techniques involves the use of an oryanic receptor which is able to specifically bind to a particular spatial and polar ~o organization of another molecule. For the most part, these compounds are antibodies, which are able to distinguish between the compound or composition of interest, and other compounds of analogous structure. By virtue of the binding ; of the receptor to a labeled ligand, one is able to dis-tinguish between labeled ligand which is bound to receptor and unbound labeled ligand.
The observed effect of binding by the receptor will depend upon the label. In some instances, the binding of the antibody merely provides for a differentiation in molecular weight between bound and unbound labeled ligand.
In other instances, the presence of the receptor may affect the nature of the signal obtained from the label, so that the signal varies with the amount of the receptor bound to labeled ligand. A further variation is that the receptor is -; 25 labeled and the liyand unlabeled. Where receptors are labeled with two different labels which interact when in close proximity, the amount of ligand present affects the degree to which the labels on the receptor may interact.
In developing an assay, there are many consi~
derations. One consideration is -the signal response to ~2-.
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changes in the concentration of analyte. A second consi-deration is the ease with which the protocol for the assay may be carried out. A third considerationis the variation in interference from sample to sample. Ease of preparation and purification of reagents, a~ailability of equipment, ease of automation, and interaction with ligands, are additional considerations, which do not exhaust the various concerns in developing a useful assay.
There is therefore a continuing need for new and accurate techniques which can be adapted for a wide spectrum of different ligands or be used in specific cases where other methods may not be readily adaptable.
U.S. Patent No. 3,709,868 issued January 9, 1975 to Spector is exemplary of a radioimmunoassay. U.S. Patent No. 3,960j834 issued ~une 1, 1976 to Umezawa et al is ememplary of a spin immunoassay. U.S. Patent No~ 3,654,090 issued April 4, 1972 to Schuurs et al and German Auslengung-sschrift 2,223,385 are exemplary of enzyme imrnunoassays.
Artiales of interest include an article by Ludwig Brand and James R. Gohlke, Annual Review of Bioche ~ , . .
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1 41, 843~868 (1972) and Stryer, Science, 162, 526 (1968).

2 Smith, FEBS Letters 77, 25, (1977) describes a fluorescent

3 in~unoassay, where thyroxine is bound to a ~luorescer and

4 quenches the fluorescer, the quenching being reversed by binding of antibody to thyroxine. See also, Ullman et al, 6 J. Biol. Chem. 251, 4172 (1976).
7 An excellent review of chemiluminescence may be 8 found in McCapra, Quarterly Reviews 20, 485 (1966) 11 A competitive protein binding assay is provided 12 having as an analyte a member of an immunological pair which 13 consists of ligand and receptor for the ligand. The assay 14 is predicated on the presence of the analyte in an assay medium affecting the degree to which a chemiluminescenc~
16 source is quenched by energy transfer to a quencher, at 17 relatively long distances. By conjugating the chemiluminescence 18 source or where the chemiluminescence source requires a 19 plurality of component~, one component of the chemiluminescence source, with a member of the immunological pair and conjugating 21 a ~uencher with a member of ~he immunological pair, reagents 22 can be prepared which when combin0d in the assay medium will 23 pxovide varying degrees of light emission, depending upon 24 the amount of analyte present in the assay medium.
In particular, the chemiluminescence source or 26 component thereof and the quencher may be conjugated to 27 either the ligand or the receptor and the resulting reayent 28 combined in an aqueous, normally buf~ered medium at a mild ., ... ,. : .:.
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~LS38~387 1 temperature, and the amount of light emitt~d deter~ined. By 21 comparison with assay media having known amounts of analyte, 3 la quantitative rela~ionship can be developed between the 41 quanta of emitted light and the amount of analyte in the B¦ assay medium.
61 Kits can be provided, where the reagents are 71 included in premeasured amounts, 50 that they may be used 81 directly or may be readily diluted to assay reagent solutions 9¦ to provide concentrations which substantially optimize the 10¦ sensitivity and performance of $he assay.

1`21 13¦ In accordance with the subject invention, chemi-14 luminescence is employed to provide a signal related to the amount of analyte in an assay medium. The analyte is a 1~ member of an immunological pair which includes ligand and 17 receptôr. By con~ugating the chemiluminescenee sour~ or-18 where the source is co~prised of more than one component, 19 one component of the chemiluminescence source, with a member of the immunological pair and a quencher with a member of 21 the immunological pair, the presence of analyte affects the 22 amount of quenchex which is within quenching distance of the 23 conjugated chemlluminescence source. By combining the 24 chemiluminescence source xeagent and the quencher reagent 2~ where the two labels are on different molecules, and 26 additional immunological pair members, as required, 27 with the analyte in an as6ay medium, including any ancillary 28 xeagents nece6sary for the chemiluminescence, and determining 29 the amount of light emitted from the assay medium, at a 31 particular wavelength or a range of wavelengths fxom the ~' 5 .
, assay medi~lm, in relation to an assay medium having a known amount of analy-te, the amount of anal~yte in the sample can be determined.
The method is predicated on the observation that -when a dye is within a limited distance from a chemilumines-cer in the excited state, the chemiluminescer may transfer its eneryy to the quencher without collision and without emitting radiation. The quencher may then emit radiation of a higher wavelength than the chemiluminescer or may lose the energy by radiationless decay. One can conjugate the member of the chemiluminescence source and the receptor to either ligand or receptor, so that when the two conjugates are brought together the amount o~ quencher within quenching distance of the chemiluminescer is affected by the amount of analyte present in the assay medium. The nature and amount of light emitted from the assay medium will therefore be a function of the analyte present in the assay medium. sy performing assays with known amounts of analyte, one can ; develop a quantitative relationship between the amount of analyte in the assay medium and the amount of radiation emitted from the assay medium at one or more wavelengths.
Thus, more specifically, the invention is a method for determining in an assay solution, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair con-sisting of ligand and antiligand, ~aid ligand having at least one epitopic site and said antiligand being capable of specifically binding to said epitopic site of said ligand;
wherein a light emitting reciprocal pair are employed as labels, said light emitting reciprocal pair consistiny of a .' ' .' ' . ' '', , ' ' . ' .

3~7 chemiluminescence source having at least one cornponen~
and a quencher capable of quenching the light emitted by said chemiluminescence source without collision, said labels being conjugated to members of said immunological pair to form a chemiluminescence label conjugate and a quencher label conjugate, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium; said method comprising:
~. combining in an aqueous medium to ~orm an assay solution:
1. said sample;
2. said chemiluminescence label conjugate;
3. said quencher label conjugate;
~- any additional components of said chemiluminescence source;
with the proviso that:
a. when said analyte is monoepitopic ligand and neither of said labels are conjugated to ligand, poly(ligand analog) is included in said assay solution;
b. when said analyte is polyvalent antiligand for monoepitopic ligand, poly(ligand analog)-label or poly(ligand analog) or the combination of quencher label ligand and chemiluminescer label ligand is included in said assay solution and when said analyte is monovalent antili-gand, poly(ligand analog)-label is included in said assay solution;
c. when said analyte is antiligand for polyepi-topic ligand and neither label is conjugated to ligand, ligand is included in said assay solution;

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d. when said analyte is polyepitopic ligand and both the quencher label and the chemiluminescer label are bonded to ligand, antiligand is included in said assay solution;
wherein said ligand analog has at least one epi-topic site common with said ligand and capable of competing with ligand for antiligand; and s. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.
De~initions ~ nalyte - the compound or composition to be measured, which may be a ligand which is mono - or polyepi-topic, antigenic or haptenic, a single or plurality ofcompounds which share at least one common epitopic site or a receptOr.

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1 Ligand - any compound for which a receptor naturally 2 exis~s or can be prepared.
3 Ligand analog - a modi~ied ligand which can 4 compete with the analogous ligand for receptor, the modifica~
~ tion providing means to join to a label or to a hub nucleus~
6 Poly(ligand analog) - a plurali~y of ligand 7 analogs joined together covalently~ normally to a hub 8 nucleus, to provide a compound having a plurality of epitopic 9 sites capable of competing with the analogous ligand for receptor.
11 Label - either a component of a chemiluminescence 12 source or a quencher dye, which form a light emitting reciprocal 13 pair, where the quencher dye has a high transition probability 14 of absorbing energy from the chemiluminescence source.
~ ~hemiluminescer label - a compound which by 16 itself or in combination with other compounds produces 17 a molecule in an electronically excited state, which molecule 18 can decay to a lower energy state by the emission of light and 19 the total process resul-ts in a chemical chang~ in one or more of the compounds.
21 ~ ; ~uencher - a molecule capable of inhibiting 22 the chemiluminescent emission of light, when within a short ~3 but non-colliding distance, usually less than about lOOA, 24 of the chemiluminescer molecule, by accepting the energy 2~ which would otherwise be emitted as chemiluminescent light.
26 In effect, the quencher need not be the nearest neighbor to 27 the chemiluminescer ~o ef~ect quenching.
2B Label-conjugate - the label, either a compound o~
29 the chemiluminescence source or the quencher, is bonded, eithQr by a bond or linking chain, to a member of the immuno-31 logical pair but not both to the same molecule. The conjugate 32 wili have at lea~t one label and may have a plurality o~

1 labels bonded to -the member of the il~nunological paix or a 2 plurality of such members bonded to the label or a plurality 3 of ligands and labels i.e. poly(ligand analog)-polylabel.
4 In particular, where an enzyme i5 the component of the chemiluminescence source employed as the label, a plurality 6 of ligand analogs may be conjugated to the enzyme to form a 7 poly(ligand analog) label.
8 Receptor - any compound or composition capable of 9 recognizing a particular spatial and polar organization of a molecule i.e. epitopic site. Illustrative receptors 11 include naturally occurring receptors, antibodies, enzymes, 12 lectins, Fab fragments and the like. The receptor may be 1~ monovalent or polyvalent in receptor sites usually polyvalent 14 e.g. antibodies. For any specific liyand, the receptor will be referred to as "antiligand". The receptor-an-tiligand-and 16 its reciproca1 lig~nd form an immunological pair.
17 Poly(ligand analog)-label - a composition in which 18 a plurality of ligand analogs and one or a plurality of lQ labels are bonded together whereby the ligand analog and label are in juxtaposition, so that when receptor is bound 21 to ligand analog, label on the labeled receptor is in within 22 quenching distance of the reciprocal label. Where an enzyme 23 is part of the chemiluminescence source and the ligand is 24 haptenic, a plurality of ligand analogs may be bonded to the enzyme. Alternatively, a plurality of ligand analogs and 26 one or more labels may be conjugated to a water soluble 27 polyfunctionalized hub nucleu6.
28 Assa~
29 The subject assay is carried out in an aqueous, normally homogeneous, zone normally, but not necessarily at 31 a moderate pH, generally close to optimum assay sensitivity.
32 The assay z~ne for the determination of analyte is prepared . ~ 7 1 by employing in an appropriate assay solution, usually 2 buffered, the unknown sample, which may have been subject to 3 prior treatment, the chemiluminescer labeled reayent and the 4 quencher labeled reagent (includes poly(ligand analog)-polylabel), and as appropriate ligand or antiligand~
6 The presence of antiliyand or ligand in combination 7 with a predetermined amount of antiligand in the assay 8 ¦medium controls the degree to which the quencher comes 9 within quenching distance of the chemiluminescer.
There are four basic variations in the preparation 11 of the quencher and chemiluminescer reagents. The our 12 variations are:
13 (1) chemiluminescer conjugated to ligand as 14 chemiluminescer labeled ligand and quencher conjugated to receptor as quencher labeled antiligand;
16 (2)-quencher conjugated to ligand as quencher 17 labeled lig~n~ and chemiluminescer conjugated to receptor as 18 chemiluminescer conjugated antiligand; and 19 (3)-chemiluminescer-conjugated to receptor as chemiluminescer labeled antiligand and quencher conjugated 21 to receptor as quencher labeled antiligand.
22 (4) chemiluminescer conjugated to ligand as 23 chemiluminescer labeled ligand and quencher conjugated 24 to ligand as quencher labeled ligand.
With the first two combinations, when the reagents 26 are combined, the quencher will be in quenching distance of ~7 the chemiluminescer. The presence of analyte, either 2~ ligand or antiligand, will serve to reduce the amount of 29 energy transfer between the chemiluminescer and quencher by diminishing the number of quencher molecules within quenching 7 ~, 1 ¦distance of the chemiluminescer. In the third combination, 2 la polyepitopic ligand (includes poly(ligand analog)) must be 3 ¦added for either antiliyand or monoepitopic ligand as analyte.
4 ¦Where the ligand is polyepitopic, increased quenchirlg will

5 ¦be observed as the concentration of the polyepitopic ligand increases to a maximum quenching, followed by ~ecreased 7 ¦quenching as the concentration of polyepitopic ligand 8 ¦continues to increase. Thus, a biphasic response is obtained, 9 Iso that one must know on which portion of the curve one is 10 ¦operating in order to obtain a discrete result. By contrast, 11 ¦with poly(ligand analog), the presence of monoepitopic 12 ¦ligand will serve to diminish quenching. With receptor as 13 ¦analyte, increasing concentrations of receptor will also 14 ¦serve to diminish quenching.
15 ¦ Where the chemiluminescer and the quencher are 16 ¦both c~njugated to ligand, an assay for either ligand or 17 ¦ polyva~ent antiligand may be perfQrmed. Where the assay is 18 ¦ for ligand, the two label-conjuyates are employed in conjunc-lg ¦ tion with antiligand which brin~s the chemiluminescer and quencher together into quenching distancP of each other.
21 ¦ The addition of ligand reduces the amount of chemiluminescer 22 and label which are within quenching distance. For the 23 determination of antiligand the two label-conjugates are 24 employed. With increasing amounts of antiligand, there will be a decrease of chemiluminescence to a minimum and then an 26 increase aB the concentration of antiligand increases. If 27 one is uncextain as to which portion of the biphasic curve 28 is involved, one or more sample dilutions will indicate the 29 particular concentration~
It should be understood, that in referring to 31 quenching, all that is intended is that there be transfer of 321 ~ ~o 3B~3~ ¦

~ energy from the chemiluminescer to the quencher. The 2 result of this transfer will be that light of a single or 3 range of wavelengths which might otherwise have been emitted 4 by the chemiluminescer will be transferred to the quencher, which may then fluoresce, emitting light of a higher wavelength ~ than the energy absorbed. Depending upon the quantum efficiency 7 of emission of the chemiluminescer, the efficiency of energy 8 transfer from the chemiluminescer to the quencher, and the ~ quantum efficiency of emission of the quencher, as well as the wavelength range which is monitored, one may observe 11 greater or lesser amounts of light due to the quenching.
12 Therefore, when referring to quenching, it is not intended 13 that there necessarily be a diminution of the signal which 14 ¦is observed. In fact, if one is observing the light emitted 15 ¦by the quencher, increasing quenching will result in an 16 ¦incre~ gly large signal.
17 1 A special situation e~ists with small haptens, 18 ¦those of from about 125 to 2000 molecular weight. With 19 ¦these haptens a substantially reduced chemiluminescence 20 ¦ can be achieved i.é. quenching without quencher bonded 21 ¦ to receptor, particularly where the receptor is an antibody.
22 1 While the reduction in signal will not be as great as when 23 ¦ quencher is conjugated to receptor, a sufficient reduction 24 ¦ may be achieved to have an acceptable assay. Except for 25 ¦ using receptor without quencher, the assay will be per-26 ¦ formed in the same manner, reading the light emitted by 28 ~¦ the chemil nescer.

32 ll ~ 7 1 ¦ In carrying out the assay, an aqueous medium will 2 ¦normally be employed. Other polar solvents may also be 3 ¦employed, usually oxygenated organic solvents of from one to 41 six, more usually from one to four carbon atoms, including ~¦ alcohols, ethers and the like. Usually, these cosolvents 61 will be present in less than about 40 weight percen-t, more 71 usually in less than about 20 weight percent.
8 The pH for the medium will usually be in the range 9 from about 5 to 12, more usually in the range from about 7 to 10, and when enzymes are employed as part of the chemi-11 luminescence source, 7 to 9. Various buffers may be used to 12 achieve the desired pH and maintain the pH during the deter-13 mination. Illustrative buffers include borate, phosphate, 14 carbonate, Tris, barbital and the like. The particular buffer employed is not critical to this invention, but in 16 l in idual assays, o~e b~ffer may be preferred over an~ther.

~ ~ $7 1 ¦ Moderate temperatllres are norrnally ernployed for 21 carrying out the assay and usually constant temperatures 31 during the pericd of the assay ~ill be employed. The 41 temperatures will n~rmally range ~rom about 10 to 50C, more 51 usually frGm about 15 to 40C.
~¦ The concentration of analyte which may be assayed 71 will generally vary from about 10 ~ to 10 15M, more usually 8 from about 10 6 to 10 13M. Stated another way, the concentration 9 ranges of interest will generally be from about 10 3 to 10 14g/ml.
In àddition to the concentration range of analyte 11 of interest, consideraticns such as whether the assay is 12 qualitative, semi-quan~ative or quantitative, the equipment 13 employ~d, and the characteristics of the reagents will 14 normally determine the concentration of the reagents. While the concentration of analyte will determine the range of 16 concentrations of the other reage~ts~ normally to optimize 17 the sensitivity of the assay, -ndividual reagent concentra-18 tions will be determined empirically. Since the binding 19 constant and binding profile of re~eptors will vary, for example, with antibodies from bleed to bleed, each new batch 21 of antibodies may r~quire different concentration ratios for 22 the different reagents.
23 ~ormally, for mono- and polyepitopic ligand analytes, 24 the concentration of antiligand based on binding sites will be about equal to the minimum concentrakion of interest 26 based on binding sites and not n~ore than about 50 times the 27 maximum concentration of interest based on binding sites, 28 usually about 1 to 10 times, and more usually about 1 to 3 ~9 times the maximum concentration of interest based on binding sites.
31 For polyepitopic ligand receptor analytes, the 32 equivalent ratios of labeled ligand cr ligand to receptor ......... .

~ 7 1 analyte will generally be in the range of about 0.01 times 2 the minimum concentration of interes~ and not more than 3 about 100 times the maximum concentration of interest based 4 on binding sites. The labele~ receptor employed in conjunc-tion with the labeled ligand or ligand will generally be

6 present in from about O.Ol to lO0 times the concentra~ion of

7 ligand or labeled ligand based on bindiny si~es.

8 For polyepitopic ligand analytes, where labeled

9 ligand is employed, the concentration of labeled lisand will generally be not less than about 10 4, more usually not less 11 than about 10 2 times the minimum concentration of interest 12 and usually in the range of about equal to the minimum 13 concentration of interest cnd not exceedin~ a~,out the maximum 14 concentration of interest. The ratio of labeled receptor will generally be not less than about O.l times the concentra-16 tion of labeled ligand based on binding sites and not greater 17 than about lO0 times the concentration of labeled ligand 18 based on binding sites.
19 For monoepitopic ligand anc,lytes and monoepitopic ligand receptor analytes, when employing labeled ligand 21 (includes poly(ligand analog)-label), the concentration of 22 labeled ligand based on binding sites will usually be not 23 less than 10 4 times the minimum concentration of interest, 24 more usually not less than 10 2 times the minimum concentra-tion of interest and usually in the range of about the 26 minimum concentration of interest to the maximum concentra-27 tion of interest. When poly(ligand analog) is employed with 28 labeled antiligand, the concentration of poly~ligand analog~
29 will fall within the same ranges as indicated for the labeled ligand and the concentration of antiligand has been indicated 31 previously.

r~t~3~7 I ¦ The order of addition of the ~arious reayents may 2 ¦vary widely, depending upon whether an equilibrium or rate 31 measurement i5 involved, the nature of the reagents, the 4 ¦rate at which equilibrium is achieved between the ligand 51 and antiligand, and the nature of the chemiluminescence 61 source. Where the chemiluminescence source has a pluxality 71 of components, with one of the components being a label, the 81 chemiluminescence can be initiated at any time hy the 9¦ addition of the other components of the chemiluminescence

10¦ source. In those situations where the chemiluminescence

11 ¦ source involves more than one component, the labeled reagents

12¦ and the unknown may be combined simultaneously, followed by

13¦ the addition of the other components of the chemiluminescence

14 ¦ source. Alternatively, one could combine the analyte with

15¦ the labeled antiligand, followed by the addition of labeled

16 ligand, as a~ropriate, followed by the addition of the

17 remaining components of the chemiluminescence source. The

18 various additions may be interrupted by incubation. In

19 those instances where the chemiluminescence source is a single component, normally the labeled receptor will be 21 combined with the analyte, followed by the addition of the 22 labeled ligand, as appropriate.
23 Depending on the mode employed, equilibrium or 24 nonequilibri~m, the rate of binding of the antiligand to ligand and labeled ligand and the relative concentrations of 26 the ligand, labeled ligand and labeled antiligand, one or 27 more incubation steps may be involved. Normally, times 3~ l~

~1~SJE39B7 1¦ between additions may vary from a ~ew seconds to many hours, 21 usually not exceeding 16hrs, more usually not e~ceeding 31 6hrs. Usually, incubation times will vary from about 41 0.5min to lhr, more usually from about 0.15min to 30min.
51 Since the ultimate result will be dependent upon the result 6¦ obtained with standard(s) treated in substantially the same 71 manner, and when possible in the identical manner ths 8¦ particular mode and periods of time are not critical, so 9 long as significant reproducible differentiations are obtained with varying concentrations of analyte.
11 Depending upon the choice of assay protocol, the 12 equipment employed and the concentration of analyte involved, 13 assay volumes may be as small as about 1~1, more usually 14 being about 25~1, and will usually not exceed 5ml, more usually not exceeding 2ml.
1~ The assay measurement will depend upon counting 17 the quanta of light emitted from the assay medium. Various 18 instruments may be used, such as scintillation counters, 19 photocells or the like, which are capable of measuring light at a single or over a range of wavelengths.
21 Materials . .__ 22 The primary components in the subject assay for ~3 analyte, which may or may not be employed in every case are:

24 labeled ligand (includes poly (ligand analog)-label); labeled antiligand; ligand; antiligand; and additional components as 27 required for the chemilurniinescence source.

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1 ¦ Analyte 2¦ The ligand analytes of this invention are characterized 31 by being monoepitopic or polyepitopic. The polyepitopic 41 ligand analytes will normally be poly(amino acids) i.e.
~ polypeptides and proteins, polysaccharides, nucleic acids, 6 and com~inations thereof. Such combinations o~ assemblages 7 include bacteria, viruses, chromosomes, genes, mitochondria, 8 nuclei, cell membranes, and the like.
9 For the most part, the polyepitopic ligand analytes employed in the subject invention will have a molecular 11 weight of at least about 5,000, more usually at least about 12 lO,000. In the poly(amino acid) category, the poly(amino ~3 acids) of interest will generally be from about 5,000 to 14 5,000,000 molecular weight, more usually from about 20,000 to l,000/ nno molecular weight; among the hormones of interest, 16 the mol~c~lar weights will usually range from about 5,000 to 21 l 60,00 lecular weight.

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l The wide variety of proteins may be considered as 2 to the family of proteins having similar structural ~eatures, 3 proteins having particular biological functions, proteins 4 related to specific microorganisms, particularly disease causing microorganisms, etc.
6 The following are classes of proteins related by 7 structure:
8 protamines 9 histones albumins 11 globulins 12 scleroproteins 13 phosphoproteins 1~ mucoproteins 1~ chromoprQteins 16 ~ Qrc~eins 17 nucleo.rroteins Zl ~ lyco~rotein~

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32 i 3~3~387 1 ¦ unclassi~ied proteins, e.y. somakotropin, 2 ¦ prolactin, insulin J pepsin ¦ A number of proteins found in the human plasma ~re 4 ¦important clinically and include:
5 ¦ Prealbumin ~¦ Albumin 7 ¦ al-Lipoprotein ~¦ ~l-Acid glycoprotein 9 ¦ al-Antitrypsin 10¦ al-Glycoprotein 11 1 Transcortin 12¦ 4.6S-Postalbumin 13¦ Tryptophan-poor 14 ¦ al-glycoprotein alx~Glycoprotein 16 Thyroxi~-binding globulin 17 Inter-~trypsin-inhibitor 18 Gc-globulin 19 (Gc l-l) . .
(Gc 2-l) .
21 (Gc 2-2) 22 Haptoglobin 23 (Hp l-l) 24 (Hp 2-l) ~Hp 2-2) 26 Ceruloplasmin 2~ Cholinesterase 28 a2 Lipoprotein(s) 29 .

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33~ 1 ~ ¦ ~2-Macroglc:)bulin 2 ¦ ~2-Hs-glycopro~ein 3 ¦ Zn-a2-glycoprotein 41 . a2-Neuramino-glycoprotein ~¦ Erythropoietin 61 ~-lipoprotein 71 - Transferrin 8 ¦ Hemopexin 9 ¦ Fibrinogen 10 ¦ Plasminogen 11 ¦ ~2-glycoprotein I
12 I . ~2-glycoprotein II
13¦ Immunoglobulin G .
14 ¦ (IgG) or ~G-globulin 15 ¦ Mol. formula:
16 ~Y2K2 or Y2 2 .
17¦Immunoglobulin A (IgA) 18 ¦or yA-globulin 1~ ¦Mol. formula: -

20¦(a2K2) or (~2~2)n .

21 ¦I~nunoglobulin M

22 ¦(IgM) or yM-globulin

23 ¦Mol. formula:

24 ~2K2) or (~2~2)

25 ¦Immunoglobulin D(IgD)

26 1or yD-Globulin (~D)

27 ¦Mol. formula:
29 ¦¦ (621~2) or 162~2) . ¦

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32 I . 19 " I . ' ' 1 Immunoglobulin E (IgE) 2 or yE-Globulin (yE~
3 Mol. formula:
4 (~2K2) or (~2 2) Free K and r light chains 61 Complement factors: -C'l 8l C'lq 9~ C'lr I0¦ C'ls 11 ~ C'2 .
12l C'.3 13¦ ~lA
a2 51 C'4 51 C'5 71 C'6 , 81 C'7 91 C'8 2~ ~ C' 271 . , 3~1 .' ' '.

1'L~ 9~7 1 ¦ Important blood clotting ~actors include:

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61 International designation Name 7 ~
I I Fibrinogen 8 I II Prothrombin I IIa Thrombin 9 ¦ III Tissue thromboplastin V and VI Proaccelerin, accelerator 0¦ globulin l VII Proconvertin -11 ¦ VIII Antihemophilic globulin (AHG) l IX Christmas factor, 12¦ plasma thromboplastin ! component (PTC) 13¦ X Stuar-t~Prower factor, .
I . autoprothrombin III
14i XI Plasma thromboplastin - antecedent (PTA) 151 XII Hagemann factor l XIII Fibrin-stabilizing factor 161 . ~
171 .

I8¦ Important protein hormones include:
lgl Peptide and Protein Hormones 201 Parathyroid hormone 21 ¦ (parathromone) 22¦ Thyrocalcitonin 231 Insulin 241 Glucagon 251 Relaxin 26¦ Erythropoietin .
~71 Melanotropin

28 (melanocyte-stimulatiny

29 hormone; intermedin) 32 ~ 21 . ..

1 ¦ Somatotropin 2 ¦ (growth hormone) 3 ¦ Corticotropin 4 ¦ (adrenocorticotropic hormone) ~¦ Thyrotropin 61 Follicle-stimulating hormone 71 Luteinizing hormone (interstitial cell stimulating 9 hormone) Luteomammotropic hormone 11 (luteotropin, prolactin) 12 Gonadotropin 13 (chorionic gonadotropin) 14 Tissue Hormones Secretin 16 Gastrin 17 Angiotensin I and II
18 Bradykinin 19 Human placental lactogen 20 Peptide Hormones from the Neurohypophysis .
21 Oxytocin 22 Vasopressin 23 Releasing factors (RF) 24 CRF, LRF, TRF, Somatotropin-RF, 2~ GRF, FSH-RF, PIF, MIF
2~ Other polymer:ic materials of interest are mucopoly-27 saccharides and polysaccharides.

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~.'L'J.~3~7 .
1 Illustrative antiyenic polysaccharides derived 2 from microorganisms are as follo~s:

4 Species of Microorganisms Hemosensitin Found in 5 Streptococcus pyogenes Polysaccharide 6 Diplococcus pneumoniae Polysaccharide - 7 Neisseria meningitidis Polysaccharide Neisseria gonorrhoeae Polysaccharide 8 Corynebacterium diphtheriae Polysaccharide 9 Actinobacillus mallei; Crude extract Actinobacillus whitemori 10 Francisella tularensis Lipopolysaccharide Polysaccharide 1 Pasteurella pestis 12 Pasteurella pestis Polysaccharide 13 Pasteurella multocida Capsular antigen Brucella abortus Crude extract 14 Haemophilus. influenzae Polysaccharide .
Haemophilus pertussis Crude 15 Treponema reiteri Polysaccharide Veillonella Lipopolysaccharide 1~ Erysipelothrix Polysaccharide Listeria monocytogenes Polysaccharide 17 Chromobacterium ~ Lipopolysaccharide Mycobacterium tuberculosis Saline extract of 90%
phenol extracted 18 mycobacteria and poly-saccharide fraction o~.
19 . cells and tuberculin .
20 Klebsiella aerogenes Polysaccharide 1 Klebsiella cloacae Polysaccharide 2 Salmonella typhosa Lipopolysaccharide, 22 PolysaccharLde 23 Salmonella typhi-murium; Polysaccharide Salmonella derby 24 Salmonella pullorum Shigella dysenteriae Polysaccharide Shigella flexneri 26 Shigella sonnei Crude, polysaccharide Rickettsiae Crude extract 27 ~andida albicans Polysaccharide 28 Entamoeba histolytica . Crude extract 31 .

.

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~ 7 .' .

I The microorganisms which are assayed may be intact, 2 lysed, ground or otherwise fragmented, and the resulting 3 composition or portion, e.g. by extraction/ assayed. Micro-4 organisms of interest include:
Coryne~acteria 6 Corynebacterium dlptheriae 7 . .
8 Pneumococci 9 Diplococcus pneumoniae la 11 Streptococci 12 Streptococcus pyogenes 13 Streptococcus salivarus Staphylococci 16 S~.. yl~ococcus aureus l7 St~phylocoFcus albus 21 .
~2 . .

24 .
2~ . . .
26 .

28 .
~9 . .
31 .

, 1 Neisseriae 2 Neisseria meningitidis 3 Neisseria gonorrheae Enterobacteriaciae Escherichia coli 7 Aerobacter aerogenesJ The coliform bacteria 8 Klebsiella pneumoniae 9 Salmonella typhosa Salmonella choleraesuis~ The Salmonellae 11 Salmonella typhimurium J
12 Shigella dysenteriae ~3 Shigella schmitzii ¦
14 Shigella arabinotardaThe Shigellae Shi~ella ~lexneri 16 Shiy~.la boydii 17 S~.igella Sonnei -_ 1~
1g Other enteric bacilli Proteus vulgaris 21 Proteus mirabilis ~ Proteus species 22 Proteus morgani J
23 Pseudomonas aeruginosa 24 Alcaligenes faecalis 26 I rio cholerae 28 . -2g . ' .

.

, ~ l 1 ¦ ~emnph;~ Rordetella groUP
2 ¦ Hemophilus influenzae, H. ducreyi 3 ¦ H. hemophilus 41 H~ aegypticus ~¦ H. paraiufluenzae 61 Bordetella pertussis ~¦ Pasteurellae 9¦ Pasteurella pestis l~¦ Pasteurella tulareusis 12¦ Brucellae 13 Brucella melitensis 14 Brucella abortus .
Brucella suis 16 . . .
17 Aerobic Spor.e-~nrming Bacilli 18 B~cilllls anthracis 19 Bacillus subtilis Bacillus megaterium 21 Bacillus cereus 22 . .
23 Anaerobic Spore-forming Bacilli 24 Clostridium botulinum Clostridium ketani 26 Clostridium perfringens 27 Clostridium novyi 28 Clostridium sept.icum .'.' . ..

1 Clostridium histolyticum ~ Clostridium tertium 3 Clostridium bifermentans 4 Clostridium sporogenes .
Mycobacteria 7 Mycobacterium tuberculosis hominis 8 ~ycobacterium bovis 9 Mycobacterium avium Mycobacterium leprae 11 Mycobacterium paratuberculosis 13 Act.inomycetes (fungus-like bacteria) 14 Actinomyces israelii 1~ .~.ctinomyces bovis 16 Actinomyces naeslundii 1~ Nocardia asteroides l8 Nocardia brasiliensis lg , , , ~0 The Spirochetes .
~1 Treponema pallidum Spirillum minus 22 Treponema pertenue Streptobacillus moniliformis 23 Treponema carateum 24 Borrelia recurrentis .
Leptospira icterohemorrhagiae 26 Leptospira canicola 3~ 27 ,' .
'' '.' ~ ,, ' ~ 89~3~
¦~coplasmas 2 ¦ r~lycoplasma pneumoniae 3 l 4 ¦ Other ~athogens ¦ Listeria monocytogenes ~ ¦ Erysipelothrix rhusiopathiae 7 ¦ Streptobacillus moniliformis 8 ¦ Donvania yranulomatis 9 I Bartonella bacilliformis 10 I .
11 ¦ Rickettsiae (bacteria-like parasites) 12¦ Rickettsia prowazekii 13¦ Rickettsia mooseri t41 Rickettsia rickettsii 15¦ Rickettsia conori ~61 Rick~t~.sia australis 17¦ Rickettsia sibiricus 18¦ Rickettsia akari 19¦ Rickettsia tsutsugzmushi 20¦ Rickettsia burnetii 21 ¦ Rickettsia quintana 231 Chlamydia (unclassifiable parasites bacterial/viral) 241 Chlamydia agent~ (naming uncertain) 251 , ~6¦ Fungi 271 Cryptococcus neoformans 28 Blastomyces dermatidis 3~ 28 . . . , ', ,'. . ',.'' 1 ¦ Histoplasma capsulatum 2 ¦ Coccidioides immitis ¦ Paracoccidioides brasiliensis 4 ¦ Candida albicans ~ ¦ Aspergillus fumigatus 6 ¦ Mucor corymbifer (Absidia corymbifera) 7 ¦ Rhizopus oryzae . ¦ Rhizopus arrhizus J Phycomycetes Rhizopus nigricans 10 I Sporotrichum schenkii 11 ¦ Fonsecaea pedrosoi 12¦ Fonsecaea compacta 13¦ Fonsecaea dermatitidis 14 ¦ Cladosporium carrionii 1~¦ Phialophora verrucosa 16¦ Aspergillus nidulans 17¦ Madurella mycetomi .
I8¦ Madurella grisea 19¦ Allescheria boydii ¦ Phialosphora jeanselmei Microsporum gypseum 22¦ Trichophyton mentagrophytes .
23¦ Keratinom~ces ajelloi 241 . M~crosporum canis 25¦ Trichophyton rubrum 26 Microsporum andouini ~7 28 Viruses 31 .
32 . ~9 . .
, , '' ' ' ~ 7 1 Adenoviruses I .
2 1 Herpes viruses I
¦ Herpes simplex 41 Varicella (Chicken pox) ¦ Herpes Zoster (Shingles) 61 . Virus B
71 Cytomegalovirus 81 . .
9l Pox Viruses I
0¦ Variola (smallpox) .
~1 ¦ Vaccinia 12 1 Poxvirus bovis 13¦ Paravaccinia 4¦ Molluscum contagiosum 1~1 . .
61 Picornaviruses i 17¦ Poliovirus.
18¦ Co~sackievirus 19 ¦ ' Ec~loviruses 2~1 Rhinoviruses .
211 . . . - .

22¦ Myxoviruses 231 Influenza (A, B, and C) 241 . Parainfluenza (1-4) .
251 Mumps Virus 26¦ Newcastle Disease Virus 271 Measles Virus 28¦ ~inderpest Virus .311 .' I
. I
' . I . , , ' ', 1'1~8~8~
. ~ ¦ Canine Distemper Virus 2 ¦ Respiratory Syncytial Virus .
¦ Rubella Virus 4 I .
~; ¦ Arbovlruses 61 Eastern Equine Eucephalitis Virus 7 ¦ Weskern Equine Eucephalitis Virus 8 ¦ Sinabis virus 9¦ Chikugunya Virus 10¦ Semliki Forest Virus 11 ¦ Mayora Virus 12 ¦ St. Louis Encephalitis Virus 13¦ California Encephalitis Virus 1~ ¦ Colorado Tick Fever Virus 15 ¦ Yellow Fever Virus 16 Dengue Virus 17 . .
Reoviruses 18 ~
19 Reovirus Types 1-3 21 Hepatitis ~2 Hepatitis A Virus 23 Hepatitis B Virus _mor Viruses 26 ~auscher Leukemia Virus 27 Gross Virus 28 Maloney Leukemia Virus Allergens 3~ .

.,.' , ..,.,, , ;"
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~ 7 1 ¦ The monoepitopic ligand analytes will generally be 21 from about 100 to 2,000 molecular weight, more usually from ¦ 125 to 1,000 molecular weight. The analytes of interest 4¦ include drugs, metabolites, pesticides, pollutants, and the 51 like. Included among drugs of interest axe the alkaloids.
61 Among the alkaloids are morphine alkaloids, which includes 7 morphine, codeine, heroin, detromethorphan, their derivatives 8 and metabolites; cocaine alkaloids, which includes cocaine 9 and benzoyl ecgonine, their derivatives and metabolites;
ergot alkaloids, which includes the diethylamide of lysergic 11 acid; steroid alkaloids; iminazoyl alkaloids; quinazoline 12 alkaloids; lsoquinoline alkaloids; quinoline alkaloids;
13 which includes quinine and quinidine; diterpene alkaloids, 14 their derivatives and metabolites.
The next group of drugs includes steroids, which 16 includes the estrogens, gestrogens, androgens, andrenocortical 17 steroids, bile acids, cardiotonic glycosides and aglycones, 18 which includes digoxin and digoxigenin, saponins and sapogenins, 19 their derivatives and metabolites. Also included are the steroid mimetic substances, such as diethyl stilbestrol.
21 The next group of drugs is lactams having from 5 22 to 6 annular members, which include the barbitlrates, e.g.
~3 phenobarbital and secobarbital diphenylhydantoin, and their 24 metabolites.
The next group of drugs is aminoalkylbenzenes, 26 with alkyl of from 2 to 3 carbon atoms, which includes the 27 amphetamines, catecholamines, which includes ephedrine, L-28 dopa, epinephrine, narceine, papaverine, their metabolites 29 and derivatives.
The next group of drugs is benzheterocyclics which 31 include oxazepam, chlorpromazine, tegretol, imipramine, their iL11~89~37 1 ¦ derivatives and metabolites, the heterocyclic rings being 2 ¦ azepines, diazepines and phenothiazines.
31 The next group of drugs is purines, which includes 41 theophylline, caffeine, their metabolites and derivatives.
31 The next group of drugs includes those derived from 61 marijuana, which includes cannabinol and tetrahydrocannabinol.
71 The next group of drugs includes the vitamins such 8¦ as A, B, C, D, E and K.
9¦ The next group of drugs is prostaglandins, which 10¦ differ by the degree and sites of hydroxylation and unsaturation.
11¦ The next group of drugs i5 antibiotics, which 12¦ include penicillin, chloromycetin, actinomycetin, tetra-13 cycline, terramycin, their metabolites and derivatives.
14 The next group of drugs is the nucleosides and nucelotides, which include ATP, NAD, FMN, adenosine, guanosine, 16 thymidine, and cytidine with heir appropriate sugar and 17 phosphate substituents.
18 The next group of drugs is miscellaneous individual 19 drugs which include methado~e~ meprobamate, serotonin, meperidine, amitriptyline, nortriptyline, lidocaine, procaine-21 amide, acetylprocaineamide, propanolol, griseofulvin, 22 butyrophenones, antihistamines, anticholinergic drugs, such 23 as atropine, their metabolites and derivatives.
24 The next group of compounds is amino acids and small peptldes which include polyiodothyromines e.g. thyroxine, 26 and triiodothyronine/ oxytocin, ACTH/ angiotensin, gentamycin, 27 met- and leu-enkephalin their metabolites and derivatives.
28 Metabolites related to diseased states include 29 spermine, galactose, phenylpyruvic acid, and porphyrin type 1.

~ 3~3g~7 1~ Among pesticides of interest are polyhalogenated 21 biphenyls, phosphaee esters, thiophosphates, carbamates, 31 polyhalogenated sulfenamides, their metabolites and derivatives.
4¦ For receptor analytes, the molecular weights will 61 generally range from 10~000 to 2xl06, more usually from 61 10,000 to 106. For immunoglobulins, IgA, IgG, IgE and IgM, 71 the molecular weights will generally vary from about 160,000 8 ¦to about 106. Enzymes will normally range from about 10,000 9 ¦to 600,000 in molecular weight. ~atural receptors vary lQ ¦widely, generally being at least about 25,000 molecular 11 ¦weight and may be 106 or higher molecular weight, including 12 ¦such materials as avidin, thyroxine binding globulin, thyroxine 13 ¦ binding prealbumin, transcortin, etc.
14 ~ Label 15 ¦ Quencher 16 ¦ The quencher molecule is a chromophore which absorbs 17 ¦ light in the wavelength band emitted by the chemiluminescer.
1~ ¦Preferably, the quencher will absorb light at a wavelength 14 ¦ close to the emission maximum wavelength of the chemiluminescer.
20 ¦ What is desired, i5 that there be a high efficiency of energy ~1 ¦ transfer when the quencher is in relatively close juxta-22 ¦ position to the chemiluminescer source. Normally, the 23 ¦ quencher will absorb light at greater than about 350A, more 24 usually at greater than about 400A. Various chromophores which may be employed as quenchers include the xanthene 26 dyes, which include the fluoresceins derived from 3,6-27 dihydroxy-9-phenylxanthhydrol and rosamines and rhodamines, 28 derived from 3,6-diamino-9-phenylxanthhydrol. The rhoda-29 mines and fluoresceins have a 9-o-carboxyphenyl group and are derivatives of 9-o-carboxyphenylxanthhydrol.

32 ~ 34 ~ 313~3~7 1 These compounds are co~nercially available with 2 substituents on the phenyl group which can be used as the 3 site for bonding or as the bonding functionality. For 4 example, amino and isothiocyanate substituted fluorescein compounds are available.
6 Other dyes which may be used as quenchers include 7 3-phenyl-7-isocyanatocoumarin, acridines, such as 8 9-isothiocyanatoacridine and acridine orange; N-(_-(2-9 benzoxazolyl)phenyl)maleimide; benzoxadiazoles, such as 4-IO chloro-7-nitrobenzo-2-oxa-1,3-diazole and 7-(~-methoxybenzylamino) Il 4-nitrobenzo-2-oxa-1,3-diazole; stilbenes, such as 4-dimethylamino 12 4'-isothiocyanatostilbene and 4-dimethylamino-4~-maleimidostilbene 13 N,N'-dioctadecyloxacarbocyanine p-toluenesulfonate; pyrenes, 14 such as 8-hydroxy-1,3,6-pyrenetrisulfonic acid, and 1-pyrenebutyric acid; merocyanines e.g. merocyanine 540, rose 16 bengal, 2,4-diph~ny-1-3(2H)-furanone; cyanines; anthiaquinones;
17 porphyrines; triarylmethanes; as well as other readily I8 available dyes which are capable of quenching. These dyes, I9 either have acti~e functionalities for conjugation or such functionalities may be readily introduced.
21 It should further be noted that the absorption and 22 emission characteristics of the dye may vary from being free 23 in solution and being bound to a protein or ligand. Therefore, when referring to the varlous wavelength ranges and 2~

.

, : , ~ ~ ~f~ 7 1 ¦ characteristics of the dyes, it is intended to indicate the 2 1 dyes as employed and not the dye which is unconjugated and 3 ¦ characteristized in an arbitrary solvent. In the area of 4 ¦ overlap between the chemiluminescer and quencher, it is ~¦ desirable that the quencher should have a high transition 61 probability.
7 ¦ Finally, the "blue fluorescent proteins" and/or 8 ¦"green fluorescent proteins" normally associated with 9¦ certain bacterial luciferases e.g. the luciferase from 10¦ Photobacterium fisheri, may also be used as a quencher.
11 1 Chemiluminescer 12¦ The chemiluminescent source may have a single 13¦ component or a plurality of components, usually two or three 14 components. While it is feasible that there be a single molecule which is thermally labile and on decomposition 16 chemiluminesces, such as certain dioxetanes, for a number of 17 reasons the use of these molecules will not be commercially 18 practical. While one could prepare reagents and maintain 1~ them at sufficiently low temperatures, so that the rate of decomposition was acceptably slow and then warm the reagent ~1 immediately prior to use, such technique will generally be 22 inconvenient, even thcugh it does have some parallel with 23 radioimmunoassay. Therefore, for the most par~, the 24 ¦¦chemi1 nescence source wi11 have at least two components 3~ 36 ~ 7 , l and the major portion of the discussion will be directed to 2 this situation.
3 For purposes of convenience~ the chemiluminescence source will be divided into two categories: those which do not involve the intermediacy of enzyme catalysis; and those 6 which do involve enzyme catalysis.
7 Consideriny chemiluminescence sources which do 8 not involve enzyme catalysis, only those sources can be 9 employed which chemiluminesce under conditions which either do not inhibit the binding of the receptor to the ligand, or 11 degrade the receptor and ligand at an unacceptable rate 12 during the period of measurement. While ordinarily 13 chemiluminescent sources which are dependent upon nonaqueous 14 solvents and strong basic conditions, greater than pHll, will not be useful, techniques can be employed involving rapid 16 injection cr flow tec~niques where the modulated emission is 17 substantially completed before the protein is denatured and 18 significant disassociation occurs. After injection of base, 19 one would observe a burst o~ l~ght which could be measured.
A diverse numbe~ of families of compounds have 21 been found to provide chemiluminescence under a variety of 22 conditions. One family of compounds are 2,3-dihydro-1,4-23 phthalaæinediones. The most popular compound is luminol, 24 which is the 5-amino compound. Other members of the family include the 5-amino-6,7,8-trimethoxy and the dimethylamino-26 [ca]benz analog. These compounds can be made to luminesce 27 with alkaline hydrogen peroxide or calcium hypochlorite and 28 base. Another f~mily of compounds are the 2,4,5-triphenyl-29 imidazoles, with lophine as the common name for the parent product. Chemiluminescent analogs include para-dimethylamino 31 and -methoxy substituents.

, . ' :

9al7 1 ¦ The next group of cheniiluminescent compounds are ~¦ indolen-3-yl hydroperoxides, precurscrs thereto a~d derivatives 31 thereof.
41 The next group of compounds are the bis-9,97-~¦ biacridinium salts, of which ll~cigenin, N,N'-dimethyl-9,9'-61 biacridinium dinitrate is illustrative. These COmPOUndS
71 chemiluminesce upon combination with alkaline hydrogen 81 peroxide.
9¦ The next group of compcunds are acridinium salts which are substituted in the g position. Particular substituents 11 are carboxylic ester~, particularly the aryl esters, acyl 12 substituents, particularly benzoyl, and cyano. Alkalire 13 hydrogen peroxide is employed to induce chemiluminescence.
14 Another group of compounds are various acyl peroxy esters and hydroperoxides, which may be formed in 16 situ in combination with comp~oun~s such as 9,lO-diphenyl-17 anthracene.
18 Another source of chemiluminescence is hydroperoxides 19 eOg. tetralin hydroperoxide -in combination with metal complexes, particularly porphyrins and phthalocyanines, where the 21 metals are iron and zinc.
22 Preferred systems are those which provide a satis-23 factory quantum efficiency of emission from the chemiluminescer ~4 at a pH at or below ll, preferably at or below lO, and, furthermore, rely on a catalyst which may be conjugated to a 26 member of the immunological pair. Where the system does not 27 involve the catalyst, the compound which decompcses with 28 the emission of light will be conjugated to the member of 29 the immunological pair. In these circumstances, the number of chemiluminescent molecules will be limited to those which 31 are conj~gated.

9~7 1 The next group of compounds are based on chemi-2 luminescers which chemiluminescence under enzymatic catalysis.
3 Primarily, ~here are two groups of enzymatically catalyzed 4 chemiluminescers. The first group are those cornpounds which chemiluminesce in combination with alkaline hydrogen peroxide.
(~ /7) By employing a peroxidase~ e.g. horseradish peroxidase or 7 catalase, in combination with hydrogen peroxide and the 8 chemiluminescer, chemiluminescence can be achieved. Illustrative 9 systems include 2,3-dihydro-1,4-phthalazinediones.
tO The second enzymatic source of chemiluminescence 11 is based on luciferins and ~h~ir analogs and luciferases.
12 Labeled Ligand 13 The ligands may be divided into two categories, 14 haptens which are generally from 125 to 5,000 molecular weight, more usually from about 125 to 2,000 molecular 16 weight and more parti~ularly from about 125 to 1,000 molecular 17 weight and antigens which will generally be not less than 18 about 2,000 molecular weight, usually not less than about 19 5,000 molecular weight and whe~ present as part of a cell, virus, chromosome or the like, may have molecular weights in 21 excess of 10,000,000. ~or the most part, the antigens of 22 diagnostic interest ~ill generally be less than about 1,000,000 23 molecular weight, more usually less than about 600,000 24 molecular weight and more particularly will range from about 10,000 to 350,000 molecular weight.
26 The label will either be a quencher, which will 27 generally be from about 125 to 1,000 molecular weight or a Z8 component of the chemiluminescent source, which may be a 29 small molecule of from abouk 125 to 1,000 molecular weight or a large molecule, such as an enzyme or hemin which will 31 range from about 10,000 to 250,000 molecular weight, more 32 usually from about 10,000 to 150,000 molecular weight.

., ~ . . . ..

9~7 1 ¦ Dependiny upon the nature of the ligand and the 21 label, the character of the labeled ligand will vary widely.
31 The first labeled ligand which will be considered is the 41 quencher-hapten reagent. Since both molecules are small, ~¦ there will normally be a 1 to 1 ratio with a relatively 61 short linkiny group between the two parks of the quencher-71 hapten.
81 Where an antigen is involved, there may be one or 9 more quenchers conjugated to the antigen. Generally, there 1~ will be at least about one quencher molecule per 100,000 11 molecular weight, more usually at least about one quencher 12 molecule per 50,000 molecular weight and generally not more 13 than about one quencher molecule per 1,090 molecular weight, 14 more usually not more than about one quencher molecule per 2,000 molecular weight. With antigens in the range of about 16 10,000 to 300,000 molecular weight~ the number of quencher 17 molecules will generally be in the range of about 2 to 30, 18 more usually from about 2 to 20, and preferably from about 2 19 to 16.
Where the component of the chemiluminescent source 21 which is conjugated is a small molecule, that is having a ~2 molecular weight in the range of about 125 to 1,000, there 23 will normally be a one to one ratio of chemiluminescer 24 component to hapten in the chemiluminescer-hapten reagent.
2~ With antigens, the number of small chemiluminescent components 26 will generally be n~t le55 than 1, usually not less than 1 27 per 100,000 molecular weight, more usually not less than 1 28 per 50,000 molecular weiyht and generally not moxe than 1 3tl37 per 1,000 molecular weight, more usually not more khan 1 per 2,000 molecular weight.
Where antigens and large (over 10,000~ chemilumi-nescent source components are involved, the ratios will vary widely, with either a plurality of the chemiluminescent source component bonded to the antigenic ligand or a plurality of ligands bonded to the chemiluminescent source component.
~,enerally, the ratio o~ antigen to chemiluminescent source component will be in the range of about 0.05 to 20, more usually in the range of about 0.01 to 10, where both the chemiluminescent source and the antigen have molecular weights in the range of about 10,000 to 300,000.
The nature of the linking group will vary widely depending upon the particular materials which are being joined. ~enerally, linking groups will vary from a bond to abou* 20 atoms in the chain which are carbon, oxygen, nitrogen and sulfur, particularly carbon, oxygen and nitro gen where the nitrogen is substituted solely with carbon or is neutral, where it may be substituted with both carbon and hydrogen e.g. amido. ~ wide variety of linking groups are set forth in our U.S. Patent No. 3,817,837, issued June 18, 1974.
Whereby a poly(ligand analog)-label is involved, a hub nucleus may be employed, which is normally a water soluble polymer and conveniently a poly(amino acid) or polysaccharide.
Usually, the hub nucleus will be from about 25,000 to ~00,000 molecular weight, more usually from about 30,000 .

~41-.
:

~ 9~37 1 ¦ to 300,000 molecular weight. Enzymes to which ligand analog 2 ¦ may be linked will generally vary from about 15,000 to 31 300,000 molecular weight. The same linking groups as discussed 41 above may be employed for linking the label and the ligand analog to the hub nucleus or enzyme.
6 The functionalities involved in the linking will 7 normally include alkylamine, amide, amidine, thioamide, 8 urea, thiourea and guanidine. Illustrative functionalities 9 involved in linking are carboxylic acids in conjuction with diimides, mixed anhydrides with carbonate monoesters, 11 aldehydes in conjunction with reductants e.g. borohydrides, 12 imidoesters, active carboxylic esters e.g. N-hydroxy succinimide 13 or ~-nitrophenyl, isocyanates, isothiocyanates, active 14 halide and the like.
La~elG~ Receptor 16 ` The receptor may be labelGd with either the quencher 17 or ~he chemiluminescent source component. Where the label 18 is a sm~-l molecule of about 125 to 1,000 molecular weight, 19 there wi~l usually be at least one label per receptor and not more than about one per 1,500 per molecular weight of 21 receptor more usually not mor~ than about one label per 22 2,500 molecular weight of receptor and preferably not more 23 than about one label per 5,000 molecular weight of receptor.
24 Where the receptor is an antibody, IgG, the number of labels will generally be from about 2 to 20, more usually from 26 about 2 to 12. Where the label is a macromolecule, that is ~7 of from about 10,000 to 300,000 molecular weight, there will 28 generally be from about 1 to 10 labels, more usually from 29 ~bout 1 to 6 labels.

~ ~ 7 1 ¦ The manner of conjugation to the receptor has been 2 ¦ indicated previously.
3 ¦ Kits 4 ¦ In carrying out the subject assays, in order to ~ ¦ obtain reproducible results, it is desirable that the critical 61 reag~nts be provided in predetermined ratiosl so as to 7 ¦ optimize the sensitivity of the assay. In the assay for ~¦ ligand, the critical reagents include labeled ligand, including ~¦ poly(ligand analog)-label and labeled receptor. In the 10¦ assay for receptor, ligand may also be a critical reagent.
11 ¦ Besides the desire to have the critical reayents in predetermined 12¦ proportions, it is frequently desirable that ancillary 13¦ materials, such as buffer, stabilizers and the like, be 14 ¦ included with critical reagents, so that dry powders or 15¦ concentrates may be diluLed to form assay solutions directly, 16¦ avoiding the necessi'~ eDighing the various materials.
17¦ In the kit, tl1e i-~gents will be provided in 18¦ relative proportions, so as to substantially optimize the 19¦ sensitivity of the assay tG the concentration range of 20¦ interest.i In addition, included with one or both of the -agents may be buffer, inert proteins, such albumins, abilizers, such as sodium azide and the like. Desirably, 23 ¦the reagents are provided as dry powders, particularly with 24 ¦labeled polyepitopic ligand and receptors, where these materials 25 ¦may be lyophilized.
26 ¦ The following examples are o~ered by way of 28 ~ ustxation and not by way o~ limitation.

29 l , 1 ¦ EXPERIMENTAL
2 ¦ All temperatures not otherwise indicated are 3 ¦ in centigrade. All percents or parts not otherwise indicated 4 ¦ are by weight except for mixtures of liquids which are by 5~ volume.

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6 ~ .

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; 28 :.'. ' :
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~ 17 : 1 E ~PLE 1. Conjugation of horseradish peroxidase to human gamma-ylohulin (hIaG) 4 The method employed is described in Nakane and ~ Kawaoi, J. Hist. Cyto. 22 (12), 1084-1091 (1974).

6 In lml of 0.3M sodium bicarbonate buffer, pH 8.1, 7 was introduced 6mg horseradish peroxidase (HRP) and 100~1 of 8 a 1% aqueous solution of 2,4-dinitro-1-fluorobenzene and the 9 mixture incubated for lhr. The mixture was then dialzyed against O.OlM sodium carbonate, pH 9.5 for 2hrs followed by 11 dialysis against 0.3M sodium bicarbonate buffer, pH 8.1.
12 An approxima~ely 1.5ml solution of the HRP material 13 prepared above was added to lml 0.4M sodium periodate and 14 the mixture allowed to stand for 45min at room temperature.
15 To the solution was then added 25~1 of 0.32M aqueous ethylene 16 glycol, the mixture allowed to stand for lhr, followed by 17 dialysis for 2hrs in a collodion bag apparatus against O.OlM
l8 sodium carbona~e, pH 9.5. The residue in the dialysis bag 19 was then combined with 5mg hIgG and the mixture allowed to stand for lhr. At this time, 15mg sodium borohydride was added, the mixture allowsd to stana for 1.25hrs at room ?~-:emperature and the product then dialyzed overnight in a 23 collodion bag apparatus against PBS, pH 7. The residue in 24 the dialysis bag was then chromatographed on Sephadex G-200 2B with O.OlM PBS, pH 7. Fractions were collected, with fraction 26 7 spectrophotometrically found to be 2.5 x 10 7M hIgG and 28 ~5 x 10- ~RP.

. .

8~3 87 1 il EXAMPLE 2. Conjugation of fluorescein to ~nti~hI~

-3 ¦ Into a vial fitted with stirring b~r wa5 irtr~d~
4 5mg lyophilized rabbit anti(hIgG) (Miles Laboratorie~,, Lot 5 18, Code 64-155) and the mixture dissolved in 0.5ml ~c~ueou~
6 sodium phosphate, pH 8.0 and the pH adjusted to 9 wi~h 7 aqueous sodium carbonate buffer. A solution of 0.3mg ~luo esceir.
8 isothiocyanate in 0.3ml D~F was added over about 40s~cs wi-h S vigorous stirring and the mixture stirred for 60min. At the 10 end of this time, the reaction mixture was chromotographed 11 on a Sephadex (G-25) column and the fractions collected. A
12 fraction was obtained having 2.4mg/ml of anti(hIgG~ with a t 13 fluorescein/anti(hIgG) ratio of about 5 to 1.
14 ¦ In order to demonstrate the subject invention, the 15¦ following assay was carried out at room temperature. The ,61 solu~ions employed were a 1.86mg/ml aqueous solution of 17¦ hIgG, a 0.023~g/ml solution of the fluorescein-anti(hIgG) lB¦ conjugate (prepared above) and a 2~5 x 10 8M solution of ~he 19¦ HRP-hIgG eonjugate. In addition~ 100~1 each of aqueous 20¦ solutions 10 3M luminol and 10 5M hydrogen peroxide were 21 also added. The following table indicates the amounts of 22 materials added and the results as read as counts per O.lmin 23 at different times ~xom the time of mixing.
24 * SEPHADEX is a trade mark.
2~

32 46 , . ' .

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3 Fluorescein- H~P-Counts per 5 sec from hIgG anti(hIgG) hIgGtime of mixing in min 4 ~ 1(in thousands) _ ~ 0 0 25 23.3 38.3 56.
7 1 5 25 27.2 41.1 60.
2.5 5 25 3~.9 50.3 71.
8 5 5 25 36.8 44.7 81.

4g.8 66.0 78.
11 The apparatus employed was a ~-Mate Scintillation Counte , non-coincidence mode.
13 It is evident from the above results, that a standard 14 curve can be prepared for determining the amount of an analyte in an assay ~edium. The method is quite simple in that the 16 reagents i~y ~ rapidly combined and a reading taken within 17 a very shoxt period of time. In addition, after about 0.5 hour 18 the readings stabilize, so that the timing of the reading 19 becomes les~ cl-itical.
2~ The subject assay provides a convenient means for etermining quantitatively a wide variety of analytes. In 22 addition, the method allows for multiplication of the signal, ~3 by employing a catalytic system, either enzymatic or non-~4 enzymatic, which provides a plurality of events for each molecule of analyte present in the medium. In addition, 26 the method avoids the problems of light scatter and protein 27 absorption and emission interfering with the results which 28 are determined.
29 Although the foregoing invention has been described in some detail by way of illustration and examplP for purposes 31 of clarity of understanding, it will be obvious that certain 1 changes and rnodifications rnay be practiced within the scope of e~le~cl~--~Zl 7'

Claims (31)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-1. A method for determining in an assay solution, the presence of an analyte in a sample sus-pected of containing said analyte, said analyte being a member of an immunological pair consisting of ligand and antiligand, said ligand having at least one epitopic site and said antiligand being capable of specifically binding to said epitopic site of said ligand; wherein a light emitting reciprocal pair are employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source having at least one component and a quencher capable of quenching the light emitted by said chemiluminescence source without collision, said labels being conjugated to members of said immunological pair to form a chemiluminescence label conjugate and a quencher label conjugate, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium;
said method comprising:
A. combining in an aqueous medium to form an assay solution:

1. said sample;
2. said chemiluminescence label conjugate;
3. said quencher label conjugate;
4. any additional components of said chemiluminescence source;

with the proviso that:
a. when said analyte is monoepitopic ligand and neither of said labels are conjugated to ligand, poly(ligand analog) is included in said assay solution;
b. when said analyte is polyvalent antiligand for monoepitopic ligand, poly(ligand analog)-label or poly(ligand analog) or the combination of quencher label ligand and chemiluminescer label ligand is included in said assay solution and when said analyte is monovalent antiligand, poly(ligand analog)-label is included in said assay solution;
c. when said analyte is antiligand for polyepitopic ligand and neither label is conjugated to ligand, ligand is included in said assay solution;
d. when said analyte is polyepitopic ligand and both the quencher label and the chemiluminescer label are bonded to ligand, antiligand is included in said assay solution;
wherein said ligand analog has at least one epitopic site common with said ligand and capable of com-peting with ligand for antiligand; and B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

2. A method according to Claim 1, wherein the assay solution is at a pH in the range of about 5 to 11 and is at a temperature in the range of about 10 to 50°C.

3. A method according to Claim 2, wherein said analyte is a haptenic ligand of from about 125 to 2,000 molecular weight.

4. A method according to Claim 2, wherein said analyte is an antigen of at least about 2,000 molecular weight.

5. A method according to Claim 2, wherein said analyte is antiligand.

6. A method for determining in an assay solution, the presence of an analyte in a sample sus-pected of containing said analyte said analyte being a member of an immunological pair consisting of monoepitopic ligand of from about 125 to 2,000 molecular weight and antiligand, said antiligand being capable of binding to said epitopic site of said ligand, wherein a light emitting reciprocal pair are employed as labels, said light emitting reciprocal pair consisting of a chemi-luminescence source having at least one component and a quencher dye capable of quenching the light emitted by said chemi-luminescence source without collision, said labels being conjugated to members of said immunological pair to form a chemiluminescence conjugate and a quencher label conjugate, at least one of said labels being conjugated to other than ligand, and wherein in said assay solution the amount of quencher brought within quenching distance of said chemi-luminescence source is related to the amount to analyte in said assay medium;
said method comprising:
A. combining in an aqueous medium at a pH in the range of 5 to 11 and a temperature in the range of 10 to 50°C
to form an assay solution:
1. said sample;
2. said chemiluminescence label conjugate;
3. said quencher label conjugate;
4. any additional components of said chemiluminescence source;
with the privso that, when said analyte is antiligand for monoepitopic ligand, and neither label is conjugated to ligand, poly(ligand analog) is included in said assay solution, wherein said ligand analog of said poly(ligand analog) has an epitopic site common to said ligand and is capable of competing with said ligand for antiligand; and B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

7. A method according to claim 6, wherein said analyte is antiligand.

8. A method according to claim 6, wherein said analyte is monoepitopic ligand.

9. A method according to claim 8, wherein said chemiluminescence source has at least two components, and one of said labels is conjugated to ligand and the other of said labels is conjugated to antiligand.

10. A method according to claim 9, wherein said ligand is an alkaloid.

11. A method according to claim 9, wherein said ligand is a steroid.

12. A method according to Claim 9, wherein said ligand is a lactam of from 5 to 6 annular members.

13. A method according to Claim 9, wherein said ligand is an aminoalkylbenzene, and said alkyl is of from 2 to 3 carbon atoms.

14. A method according to Claim 9, wherein said ligand is a benzheterocycle.

15. A method according to Claim 9, wherein said ligand is a purine.

16. A method according to Claim 9, wherein said ligand is an amino acid.

17. A method according to Claim 16, wherein said amino acid is a polyiodothyronine.

18. A method according to Claim 9, wherein one of said components of said chemiluminescence source is an enzyme and said chemiluminescence label conjugate is ligand conjugated to said enzyme.

19. A method according to Claim 18, wherein said enzyme is a peroxidase and another component of said chemiluminescence source is luminol.

20. A method for determining in an assay solu-tion, the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of an immunological pair consisting of polyepitopic ligand of at least about 2,000 molecular weight and antiligand, said antiligand being capable of binding to the epitopic sites of said ligand, wherein a light emitting reciprocal pair is employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source having at least one component and a quencher dye capable of quenching the light emitted by said chemilumnescence source, without collision, said labels being conjugated to members of said immunolo-gical pair to form a chemiluminescence label conjugate and a quencher label conjugate, at least one of said labels being conjugated to other than ligand, and wherein in said assy solution the amount of quencher brought within quenching distance of said chemiluminescence source is related to the amount of analyte in said assay medium;
said method comprising:
A. combining in an aqueous medium at a pH in the range of about 5 to 11 and at a temperature in the range of about 10 to 50°C:
1. said sample;
2. said chemiluminescence label conjugate;
3. said quencher label conjugate;
4. any additional components of said chemi-luminescence source;
with the proviso that when said analyte is anti-ligand and neither of said labels is conjugated to ligand, ligand is included in said assay solution; and B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount of light emitted from an assay solution having a known amount of analyte.

21. A method according to Claim 20, wherein said analyte is antiligand.

22. A method according to Claim 20, wherein said analyte is polyepitopic ligand.

23. a method according to Claim 22, wherein said chemiluminescence source has at least two components, and one of said labels is conjugated to ligand and the other of said labels is conjugated to antiligand.

24. A method according to Claim 23, wherein said ligand is a polypeptide.

25. A method according to Claim 24, wherein said polypeptide is a globulin.

26. A method according to Claim 25, wherein said globulin is an immunoglobulin.

27. A method according to Claim 24, wherein said polypeptide is a hormone.

28. A method according to Claim 23, wherein said component of said chemiluminescence source conjugated to a member of said immunological pair is an enzyme.

29. A method according to claim 28, wherein said enzyme is a peroxidase and luminol is another component of said chemiluminescence source.

30. A method for determining in an assay solu-tion, the presence of human globulin in a sample suspected of containing said human globulin, wherein said assay employs as reagents anti (human globulin) and a light emitting reciprocal pair employed as labels, said light emitting reciprocal pair consisting of a chemiluminescence source comprised of peroxidase and luminol and a quencher dye capable of quenching the light emitted by said chemi-luminescence source without collision, said peroxidase being conjugated to human globulin to form a human globulin-peroxidase conjugate and said quencher dye being conjugated to anti(human globulin) to form quencher label anti(human globulin), and wherein in said assay solution the amount of quencher is brought within quenching distance of said chemiluminescence source is related to the amount of human globulin;
said method comprising:
A. combining in an aqueous medium at a pH in the range of about 7 to 10 and at a temperature in the range of about 10 to 50°C to form an assay solution;
1. said sample;
2. said peroxidase-human globulin conjugate;
3. said quencher-anti(human globulin) conjugate;
4. any additional components of said chemilu-minescence source; and B. measuring the amount of light emitted from said assay solution at at least one wavelength as compared to the amount to light emitted from an assay solution having a known amount of human globulin.

31. A method according to claim 30, wherein said quencher dye is fluorescein.