CA2013214C - Catalyzed reporter deposition - Google Patents

Abstract

The present invention concerns a method to catalyze reporter deposition to improve detection or quantitation of an analyte in a sample by amplifying the detector signal which comprises immobilizing an analyte dependent enzyme activation system which catalyzes deposition of reporter by activating a conjugate consisting of a detectably labeled substrate specific for the enzyme system, said conjugate reacts with the analyte dependent enzyme activation system to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system. In another embodiment the invention concerns an assay for detecting or quantitating the presence or absence of an analyte in a sample using catalyzed reporter deposition to amplify the reporter signal. Also described are novel compounds which can be used as reagents to prepare HABA-type conjugates.

Description

TITLE
Catalyzed Reparter Deposition FIELD OF THE INVENTION
This invention relates to assays and, more particularly, to catalyzing reporter deposition via an activated conjugate to amplify the detector signal thereby improving detention and/or quantitation of an analyte in a sample.
BACKGROUND OF THE INVENTION
The introduction of immunodiagnostic assays in the 1960s and 1970s greatly increased the number of analytes amenable to precise and accurate measurement. Radioimmunoassays (RIAs) and immunoradiometric (IRMA) assays utilize radioisotopic labelling of either an antibody or a competing antigen to measure an analyte. Detention systems based on enzymes or fluorescent labels were developed as an alternative to isotopic detection systems.
Enzyme based assays proved to be more sensitive, faster, less dependent upon expensive, sophisticated instrumentation.
The need for diagnostic assays having simpler formats, increased sensitivity with less dependence upon sophisticated and expensive instrumentation prompted investigators to try to harness the catalytic power of enzymes to develop these newer assays.
D. L. Bates, Trends in Biotechnology, pages 204-209, Vol. 5 No. 7 (1987), describes diagnostics which use a method of enzyme amplification to develop more 0»2790 11:12 LUFONT LEGHL N0.010 005 sensitive and simple immunoassays. in this method a second ~nzyme system is coupled to the primary enzyme label, e.g., the primary enzyme can be linked catalyticaily to an additional system such as a substrate Cycle or an enzyme cascade. Thus, the essence of enzyme amplification according to Bates is the Coupling of catalytic processes wherein an enzyme is modulated by the action of a second enzyme, either by direct modification or by interaction with the product of the Controlling enzyme.
v.s. patent ~,6s~,62~, is'ued to ~oellga~t on May 26, 1987, describes application of an enzyme-linked coagulation as$ay (ELCA) to develop an amplified immunoassay using the clotting cascade to enhance sensitivity ~f detection of $.mmune complexes. The process involues clot formation due to thrombin activated fibrin formation from insolubilized fibrinogen and labeled solubilized fibrinogen. Amplification of th~ amount of reportable ligand attached to solid phase is obtained only by combinia~g use of clotting factor conjugates with subsequent coagulation cascade reactions, One of the disadvantages of this system is that it can only be used to measure the presence of materials which modulate the activity of one or more of the blood clotting factors. Another disadvantage is that the primary enzyme, thrombin, cannot be immobilized or coupled to a reporter or a member of a specific binding pair, U.S. Patent 4,463,090, issued to Harris on Juiy 31, 1984, describes a cascade amplification immunoassay requiring a combination of at least two sequential Catalyses wherein a first enzyme activates a second enzyme which in turn acts upon the substrate.
mother amplification system is described in U.S.
Patent 4,598,042, issued to Self on ,7uly 1, ~g86, and ' 2 ll:lc uuru~m ttUHt rvU.010 006 . Patent Application No. 2,059,x21 which was published on Apxi1 23, 1981, which disclose an immunoassay using an enzyme label to produce directly or indirectly a aubstanoe that is capable of influencing a oatalytic event without itself being consumed during the Catalytic ev~nt. Mor~ specifically, a primary enzyme system produces or remov~s a substance capable of modulating a secondary enzyme system which results in amplification. The enzyme systems use unconjuqated enzymes to avoid the tendency to inactivate certain enzymes on conjugation.
European Patent Application Publication I~o. 123,255 which was published on pctober 31, 1984, describes another cascade amplification immunoassay wherein a zymogen-derived°enzymd is coupled to a zymogen-to-enzyme cascade reaction sequence to obtain multiple stages of amplification in producing detectable masker material used to quantify analyte amount.
European Patent l~pplication Publication No.
199, 799, published ~Yune 19, 1985, describes a specific binding assay based on enzyme cascade amplification wherein the label component employed in the detectant reagent ie a participant in or a modulator of an enzym~
cascade reaction whenein a first enzyme acts on a first substrate to product a second enzyme. The production of the second enzym~ can be followed or the second enzyme can act on a second substrate to produce a third enzyme.
Similarly, U.S. Patent 9,318,980, issued to Boguslaski et al. on MasCh 9, 1982, de9Cribea a hetcrogenous specific binding assay using a conjugate formed of a specific binding substance coupled to the reactant, i.e., an enzymatic reactant. The ability of the reactant to participate in the monitoring reaction to detect the presence of analyt~ is altered by the presence of the ligand in the medium. Thus, the t~J~ c r ~ ?~~ m : 1 s 1W rUN I LtLar-rL (J0. 010 conjugate in its free state is more active in th~
monitoring reaction than in its bound state.
A heterogenous specific binding assay using enzyme amplification is described in Eritish Patent Application 'S No. 1,901,29? which was published on ,Tuly 30, 19'75 and L1.S. Pat~nt 4,376,825, issued to Rubenstein et al. on March 15, 1993. Amplification is achieved by bonding the compound to be assayed or a counterfeit of it to an enzyme. The re$ulting enzyms--bound-liqand competes with Eras ligand for specific receptor sites. When the ~nzyme-bound ligand is displaced by the free ligand the enzyme is than free to react with a large numb~r number of substrate molecules and the concentration of the remaining substrate or of the product oan be measured.
1S PCT International Publication No, t~~ 81/00'125 which was published on March 19, 1981 describes a method of det~rmining a substrata is a sample which comprises converting the substrata to a product in a first stage of a cyclic reaction sequence and converting the product back to the substrate in a second reaction stags of the CyCliC reaction sequence. At least one of the first and second reaction stages is enxym~ catalysed.
PCT Application having Tnt~rnational Publication Number PTO 84/02193, which was published on June 7, 1989, describes a chromgenic support immunoassay wherein the analyte is contacted with an enzyme--labeled antibody and in which the signal generated by the reaction of the enayme with its substrata is concentrated on an active support.
European Patent Application Publication No.
181,?62, published on May 21, 1986, describes a method to determine enzymatic activity in a liquid sample by particle agglutination or inhibition of particle agglutination. .

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Substrato/cofactor cycling is another exempla of amplification which is based on the cycling og a cofactor or substrate which is gen~rated by the primary 0nayme label. The primary enzyme converts the primary substrate to an active ~orm which can be cycled by two enzymes of the amplifier cycle. These two enzymes and provided in high concentration and ar~ poised to turn over high concentrations of substrata but era prevented from so doing until the cycling substrate is formed. The product of the primary enzym~ is a catalytic activator of the amplifier cycle which r~sponds in proportion to th~
concentration of substrate and hence the concentration of th~ ~nxym~ label.
in th~r early sixties, dowry et al., Journal of Biological Chemiat~ry, pages 2746-2755, Vol. 236, No. 10 (October 1962), dascsibad the measurement of pyridine nucleotides by enzymatic cycling in which the coenzyme to be d~termined was made to amplify an enzymatic dismutation between two substrates.
p, more complex substrate cycling system is described in U.S. Patent 4,75,054, issued to Rabin at al, on May 17, 1988. The Rabin system involves using a sme~li anzymically inactive peptide fragment of an enzyme as a label and conjugated with the complementary fragment to form an enzyme which catalyzes a pra.mary reaction whose product is, or leads to, an essential coenzyme or prosthetic group for a second enzyme which catalyzes a secondary reaction leading to a detectable result indicating the pres~nce of analyte.
vary et al., Clinical Chemistry, pages 1696-1701, Vol. 32 (1986) describes an amplification method suited to nucleic acids. This is the strand displacement assay which uses th~ unique ability of a polynucleotide to act as a substrata label which can be released by a 3S phosphosyla'e.

SUMMARY OF THE INVENTION
The present invention concerns a method to catalyze reporter deposition to improve detection or quantitation of an analyte in a sample by amplifying the detector signal which comprises immobilizing an analyte dependent enzyme activation system which catalyzes deposition of reporter by activating a conjugate consisting of a detectably labeled substrate specific for the enzyme system, said conjugate reacts with the analyte dependent enzyme activation system to produce an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system.
In another embodiment the invention concerns an assay for detecting or quantitating the presence or absence of an analyte in a sample using catalyzed reporter deposition to amplify the reporter signal.
This invention also concerns novel compounds which can be used to prepare novel HABA type conjugates.
Further aspects of the invention are as follows:
A conjugate comprising a detectably labeled phenol.
A method for the detection or quantitation of an analyte in an assay which comprises using an analyte dependent enzyme activation system comprising at least one enzyme to react with a conjugate consisting of a detectably labeled substrate specific for the enzyme system to form an activated conjugate which covalently deposits substantially wherever at least one receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system, wherein deposited detectable labels either directly or indirectly generate a signal which can be detected or quantitated.
A method for producing an activated conjugate comprising reacting a peroxidase enzyme with a detectably labeled phenol.

6a A method for the detection or quantitation of an analyte in an assay which comprises using an analyte dependent enzyme activation system comprising at east one enzyme to react with a conjugate consisting of a detectably labeled substrate specific for the enzyme system to form an activated conjugate which deposits substantially wherever at least one receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system, wherein deposited detectable labels either directly or indirectly generate a signal which can be detected or quantitated.
An assay for detecting or quantitating the presence or absence of an analyte in a sample which comprises a) immobilizing the analyte;
b) reacting the product of step (a) with an analyte-dependent enzyme activation system;
c) reacting the product of step (b) with a conjugate consisting of a detectabty labeled substrate to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system; and d) detecting or quantitating the presence or absence of the analyte in the sample.
An assay for detecting or quantitating the presence or absence of an analyte in a sample which comprises a) reacting an analyte dependent enzyme activation system with a conjugate consisting of a detectably labeled substrate to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system; and b) detecting or quantitating the presence or absence of the analyte in the sample.

6b A compound which is 6-(phenoxy-(4'-azo-2"-carboxyethylphenyl))-hexanoyl-alkaline phosphatase.
BRIEF DESCRIPTION OF FIGURES
Figure 1 is a graph comparing results of an HSV antigen assay run with and without catalyzed reporter deposition.
Figure 2 is a graph comparing results of an HIV p24 core antigen assay using conjugate concentrations of 0.2, 0.4, and 0.8 NI/ml (Amp 1, 2, and 3, respectively). "HRP" represents a non-amplified assay wherein the detector antibody was directly labeled with HRP. "Biotin" indicates another non-amplified assay wherein the detector antibody was conjugated to biotin and detected with HRP labeled streptavidin.
Figure 3 is a graph of a mouse IgG assay run using an HRP ADEAS to catalyze deposition of biotin-tyramine which was det~cted with streptavidin-HRP (HRP~l~mp Hgp) or with sir~ptavidin-AF (HRP-imp AP). The as'ay was also run using only 1~F~P lab~led dctectox antibody or AP
lab~lod deteotor antibody.
Figure ~ prosonts two graphs comparing results obtained from a Du Pont*8iV p24 antigen ELISA run with and without using catalyzed reporter d~position to amplify reporter signal.
~'igurs 5 is a graph comparing r~sults of a mouse IgG assay without catalyzed reporter deposition (HIBP) and with catalyzed reporter deposition (HRF~-~-Oal).
Figuxe 6 depicts a preparation of ethyl 2_(q._hydroxyphenylazo)ben~oat~-6valkaline phosphatase (HEE-6-AP), the synth~sia of which is described in Example 10.
~°igur~ '7 i~.lustrat~s the ~sterase catalyzed conversion of IiEE to 2-(d°-hydraxyphenylazo)benzoic acid (FiA9,~) /str~ptavidin complex.
~'he.term analyte dependent enzyme activation System (SEAS) refers to an ~nxyme system wher~in (i) at least ono enzymQ is coupled, in any manner known to those skilled in the art, to a m~mber of a specific binding pair, or (ii) the enzym~ need not be coupled to a member of a specific binding pair when it is the analyte. Th~
enzym8, 8ither by itselg or in connection with a second enzyme, catalyzes the formation of an activated conjugate which then is deposited wherever a receptor far the activated conjugate is immobilized, The term amplification as used herein means amplification of reporter signal due to d~position of a Conjugate activated by an F~DEAS.
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Th~ term conjugate means a datactabiy labeled substrate specific for th~ Id~EAS whether it ba a single enzyme ,~pEAg or mufti-~nzyma ADEaS. Tha substrate must have at least one component but is not limited to such.
For exempla, the substrat~ can consist of two components. One component contains th~ binding site for ths~ r~ceptor and is dat~ctably labeled. The oth:r component is a constituent which prevents or interferes with binding to the receptor until such tim~ as the AREAS prim~s the conjugate as is discussed below.
Another example of a Conjug~,ta is biotin-tyramine wherein tyramine is the substrate portion and biotin aonetitut~s the detectable label as described below, Conjugates are described in greater detail below as well.
The term datectably labeled means that th~
substrate can ba coup.led to either a reporter or to an unlabeled first member of a specific binding pair provided that the reporter introduces a dilf~rant moiety to th~ substrate as is discussed below. S9hen the substrate is coupled to an unlabeled member of a speoific binding pair, gollowing deposition, the aubstrate~apecifiC binding partner complex is reacted with the second member of the binding pair which is coupled to g reporter. Alternately, the substrata-specific binding partner complex can ba pre-reacted with the detectably labeled other member of the specific banding pair prior to deposition.
The term deposition means diraoted binding of an activated conjugate to the receptox which results from either the formation of a covalent bond or a specific binding pair interaction as described below.
~'he term receptor means a site which will bind to the activated conjugate either through the formation of t~~ i G r ~ ?C~ 11 : 1 r LrUt'UN I LtUwL NU. 010 012 i. ~ ~ ~. t.~

a covalent bond or a specific binding pair interaction as described b~low.
The term activated conjugate means that the conjugate has been primed by the ApEAS to bind with the r~ceptor.
One of the unique features of this invention is the analyte dependent enzyme activation system which catalyzes deposition of conjugate by converting th~
substrate portion of the eoajugate to an activated form which is deposited wherever e~ specific receptor for the activated conjugate is immobilized. ~ehe AREAS dose not utilize enzyme cascade reactions or enzyme cycling to eff~ct amplification. Rather, it uses either a single enzymes or combination of enzym~s to activate the 35 conjuc~at~. Deposition of conjugate occurs only if the analyta and analyte dependsnt enzyme activation system, which can be the same if the analyte is an enzyme, for exampl~ in the detection of an enzyme such as alkaline phosphatase, or different, have been immobilized and a r~ceptar, as described below, is immobiliz~d to bind the activated conjugate. Thug, the At~EAS, conjugate, and receptor are chosen to form an operational trio.
The following is one embodiment of a single enzyme AREAS system applied to a forward sandwich immunoassay format; the test sample containing the analyte is reacted with an immobilized capture reagent, such as an antibody, excess reagents are wash~d off; the immobilized capture antibody-analyte complex is reacted with an AD~AS, such as a second antibody specific ~or the analyte which has been coups~d to an enzyme, e,c~, horseradish peroxidase (HRpy, alkalia;e phosphatase (APy, etc. The AREAS will bind only if the analyte has been bound by the capture reagent. Otherwise the reagents w~.ll be washed off. Coupling of the enzyme to a specific binding partner does not affect the enzyme°s~

c~_,~~.~=m ~~~..~ Lmryvi LCUrt~ NU.bll1 F~ ~. e~3 t;, ~ ~~
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ability to react with the substrata portion of the conjugate. MTh~n conjugat~ such a's biotin-tyramine or HA~9A-tyramine e~nalog (~.g., N-(q~~o-hydroxyphenethyi)--6-(phenoxy-(q~~azo-2'~-benzoic acid~)hexamide) is added to the immobilized capture antibody-analyte-second antibody-enzyme compl~x, the enzymes r~acts with the substrate portion of the conjugate, e.g., with the tyramine portion of the conjugate, convestinq it to an active form which will bind to an immobilized receptor which is either endogenous or eacogenous to the assay syat~m. The amount of conjugate deposited will be a function of immobilized AREAS. t~eposited conjugate such as biotin-tyramine ar FIASA tyramine analog can then bs detected by reacting with streptavidin-F3A8 and orthoph8nylmnediamine. Th~ term FiABA-tyramine analog means, g~nerally, unsubstituted or substituted FIAr3A, coupled with or without a spacer, to a hydroxy-phenyl containing compound such as tyraming. If the conjugate la liuva~suelm-~.yramin~ cnen the peppglteCl Con~ugat~ can b~ det$Cted dirgotly, or following reaction with a labeled anti-fluoreacein antibody, Thus, the AREAS is used to catalyze the deposition of detectably labeled substrata (the Conjugate) to generate additional signal. The AREAS is detected directly as part of the overall signal when the enzyme component of the AREAS is the same as the enzyme used ~as the reporter. Figure 3 illustrates this situation as wdll as the situation where an AD~p,S enzyme component and reporter enzyme are different and thus, the AREAS
enzyme component is not detected directly as part of the overall signal.
A multi-enzyme AREAS immunoassay fermat would involve a similar approach. In the example above, the ApEAS can be an antibody coupled to an enzyme such as AP. In addition to the immobilized capture asntibody-F~J~tli7t~ A1:1C LUh'I.INI LtL~NL NO.C~10 0lLj ~~.~~~i~.
13.
analyte-second antibody-Ap complex, a second enzyme such as H~ could b~ immobilized on the support. Th~
conjugate can be a d~tectably labeled phenylphoaphate which cannot. seact with Fill until it is dephosphorylated. hP dephosphorylates the phenol which th~n is fees to react with the immobilized HRP to gosm an activated pheraolic con~u$ate which deposits wherever receptors aze immobilized. After removing excess reagent, deposited reporter is detected and c~uantitated.
Alternatively, HRP can be coupled to the second antibody and AP can be immobilized on th~ surface of the support.
Th~ instant invention is surprising and unexpected because amplification of reporter signal is obtained via d~sposited activated conjugate without using cascade 13 mechanisms os enzyme cycling. The ADEAS reacts with the conjugate to form an activated conjugate which will bind with immobilized receptor specific for the activated conjugate. The amounts of receptor and activated conjugate are in excerss of the amount of ADEAS
immobilized.
The choice of an ADEAS is governed by the ability of the enzyme or enzymes to convert a conjugate to an activated form which will bind to an immobilized receptor whether endogenous or exogenous. Accordingly, a detailed knowledge of catalytic properties of each specific enzyme is needed in order to properly design the substsaC~ and receptor. Other important factors include availability of the enzyme or enzymes, relative ease or difficulty to couple it to the member of a specific binding pair, stability of the enzyme or enzymes as well as the stability of the conjugate and the receptor. ~n some cases, an ADEAS can be purchased, depending on the assay format.
Enzym~s suitable for use in an ADEAS include hydrolases, lyases, oxidoreductases, transferases tJ» C ! i 71:~ 1 1 : 1 7 L~Ur' U[V I LGUHL N~. 81 isomeras~s and liga$~a, There can b~ mentioned peroxidase, glucose oxidase, phosphatase, esterase and glycosides~. Specific examples include alkaline phosphatase, lipase~s, beta-galactosidase, horseradish p~roxidase, ;and porcin~ liver este~ras~, Memb~ra of specific binding pairs suitable for uaa~
in practicing the invention can be of the immune or non-immune type. Immune specific binding pairs axe ex~ampiified by antigen/antibody systems or hapten/anti-hapten systems. The antibody member, whether polyclonal, monoclonal or an immunoreactive fragment thereof, of the binding pair can be produced by customary m~thods familiar to those e~cili~d in the art.
The terms immunoreactive antibody fragment or 13 immunoreactive fragment mean fragments which contain th~
binding region of the antibody. Such fragments may be Fab-type fragments which a.re defined as fragments devoid of the Fc portion, e.g., Fab, Fab' and ~'(ab')2 fragments, or may b~ ao-called "half-molecule" fragments obtained by reductive olaavage of the disulfide bonds connecting the heavy chain components of the inteect antibody. If the antigen member of the specific binding pair is not immunogenic, e.g,, a hapten. it can be covalently coupled to a carrier protein to render it immunogenic.
Non-immune binding pairs include systems wherein the two components shar8 a natural affinity for each other but are not antibodies. Exemplary non-immune binding pair3 are biotin-avidin or biotin-streptavidin, folic acid-folate binding protein, complementary prolbe nucleic acids, etc. Also included are non-immune binding pairs which form a covalent bond with each other but are not antibodies, Exemplary covalent binding pairs include sulfhydryl redctiv~ groups such as maleimides and haloacetyi derivatives and amine reactive k'1Ji G ( i 7th 1 1 : ck~ LUYkIlV I LtUHL r/G. 010 8 ~el ~ ~~.. C,a groups such as isothiocyanates, succinimidyl eaters and sulfonyl halides, etC.
Suitable supports us~d in assays include synthetic polymer supports, such as polystyrene, polypropyl~ne, substituted polystyren~, e.g,, aminated or carboxylated polystyren~; polyacrylamidesJ polyamides;
polyvinylchlorid~, etc.d glass beadsi agarose~
nitrocellulos~, etc.
l4nother important component of the invention is the conjugate, i.~,, a detestably labeled substrate which must be specitio for the ADEAS. As was stet~d above, when the conjugate reacts with the AREAS, the enzyme ar esnzymss catalyse formation of an activated conjugate which binds wherever a receptpr is immobilized whether exogonaus or endogenous, An immobilised exogenous receptor means a receptor which does not originate within the assay. Tt must be immobilized on the surface of the support prior to adding the oon~ugate to the reaction mixture. An endogenous rea~ptor means a receptor which originates within the assay and does not require immobilization prior to adding the conjugate because the receptor is immobilized Within the assay system, For example, when an HRP 1~DEAS (H~ coupled to a member of a specific binding pair) is reacted with conjugate containing a phenolic substrate, an activated phenolic substrate is produced, It is b~lieved thnt the activated phenolic substrate binds to electron rich moieties such as tyrosine and tryptophan present in the proteins on the solid support. However, if a cliff~rent conjugate is used, such as a labeled 3-methyl-2-benzothiazolinone hydrazine (MBTH) which is discussed below, a receptor, such as 3-(dimethylamino)benzoio acid (DMAB), must b~ immobilized prior to addition of oonjugato.

r~s~ c r ~ 7r~ 11: ~u LUPpNI LE(iAl.. N0. 010 01'7 Another embodiment involves reacting a conjugate which becomes pho~phorylated by an Ad9EAS. Th~ activated fphoaphorylatodD conjugated can then rgaet with sn antibody sp~cific for the~aCtivated Conjugate, yn moll another variation, an ~DHAS can be reacted with a conjugate Consisting of a component which when activated will bind to a receptor and which ie Coupled to a component having a thiol reactive group such as a maleimid~. The deposited mal~imide moiety can then be d~tected by reacting with a sulfhydryl-containing r~porter which Can be endogenous to the reporter, e.g., beta-galaCtoaidas~, or the sullhydryl groups can bo added to reporters such as H12F or AP using thiolating reagents such as N-'uGCinimidyl~S~acetylthioacetate 1~ (~~T~), g-aCetylmercaptosuccinic anhydride (SAMSA1, or eaCCinimidyl-3-(acetylthio)-propionate (9ATP), Alternatively, the ~eubetrato can b~ coupl~d to a protected sulfhydryl containing group and this can be used as the conjugate. After binding to the receptor, this can be deprotected using conventional techniques known to those skilled in th~a art. Detection can be ' effected using s reporter having a thiol reactive group such as maleimide-HRP or iodoacetyl-gIIZP.
Another alternative is to use a conjug$te wherein the substrate has two components as described above, a datectably labeled first component which will band to the receptor after the second compan~nt has been activated or removed by the AD>;AS. An example of this is a small organic molecule such as 2-(4'-hydroxy-phenylazo)-benzoic acid (HA13A) which binds specifically to avidity and streptavidin. The terms avidity and streptavidin are used herein int~rchangeably. ~I~~ oan ba detectabiy labeled using any of the reporters described below, e.g " radioisotopes, enzymes, etc. Per instance, alkaline phosphatase (AP) can be conjugated to t9.~~ G l i 710 11 : G1 LK.It'U~V I l.tl7HL ~U. 0S 0 016 ~3 '~ r.
ld .~ zc ~ a ~.3..
HABA, using techniques w~11 known to those skilled in th~ art, with or without a spacer, to a functional group on NASA. An exd~tpl~ of such a functional group is thd~
~°-hydroxyl moiety, Nloreovsr, NASA can ba modified to 5 possess a second component which prevents binding until it has been removed by the AREAS. F'or exempla, estsrification of HABA with ethanol produces a Hd~A
ethyl ester which doss not bind to straptavidin.
~etsctably label~d A ethyl asters will not bind to 10 strl~pt,avidin until the aster $soup has bean hydrolyzed to the corresponding carboxylic acid. ~3ydrolysia can bs off~cted using an enzyme such as an esterase, ~e.g., porcine liver esteraas. 3~hu8, detestably labeled HAHA
~stsrs such as 6-(phenoxy-(~'-azo-2°'-15 carboxyethylph~nyl)-hexanoyl-alkaline phosphatase can be depoeitsd using an AREAS having a suitable esterase which will hydrolyzo the ester to paranit binding of de~tsctnk~ly label~d HASA with streptavidin (i.~., exogenous receptor) which ha3 bean immobi~.ized on the surfac~ of a support.
Compounds of the formula II
a5 wher~~.n Rl through R~ arm the same or difi'erent and are selected from the group consisting of straight chain i~

h~~cr~7y ll:ec UUt'UN1 LEG~sL N0~010 019 as or branched alkyl groups having 1-~ carbon stoma, F, Cl, Rr Os 3 j 2 is phenyl or naphthyl~
Y1 is 1«iy x is P1, 0, ~; and R~ can b~e H ar straight chain or branch~d alkyl group having 1-9 carban atoms can be used to synthesize t3ARA-type conjugates as d~scribed in thg examples below. The term F3AEA-type conjugat~o m~ans A
derivatives: gi) which can b~ substituted or unsubetituted and are coupled with a spaoe~r t~ a reporter and (11) which contain an .SEAS activatabie moiety that prevents the conjugate from binding t~
Stseptavidin until it has been activated or removed by the ADEAS. The synthesis of such a compound is illustrat~d in Figure 6 as w~11 in Example 10 below.
fibs approach described below can be modified by those skilled in the art qen~rally to synthesise any of these compounds using procedureB well known to those sk111ad in the tart .
Other small organic molecule/receptor combinations which are suitable to praot~.ce th~ invention include haptens/antibodies, sugars and oligosaccharides/lectins, biotin and dyes/avidin and/or streptavidin.
A8 is shown in 'Table 1, a number o! receptors are available. The choice o~ a receptor will depend upon the aonjugat~ seleoted.
The optimal concentration of conjugate is determined according to the procedure explained in Example 1. Optimal concentrations will vary depending upon enzyme used in the ADEAS and substrate selected to produce conjugate.
Conjugate can be synthesized using conventional coupling and labeling techniques. substrate choice will depend upon the ADEAS selected. To reiterate, detailed ~. 6 t~_~~ ~ ~ ~ . c.. i i . cc uurl4~ i LtUh-1~ NU. 1~11'J 020 ~f'~9 r 6~ '[~?'~ '~
PJ~, ~ ~. C.

knowl~dg~ is required of the catalytic properties of ~ach specific en$yme in order to propegly d~sign a useful synthetic substrate and, if necessary, a receptor.
A wid~ variety of reporters .are available for coupling to the substrate to produce tha conjugate or to couple to a member of a specific binding pair. As ryas discussed above reporter should introduce a different moiety to th~ substrate. Reporters can be a radioactive ieotopt~, such as, 125g~ enzymes, fluorogenic, chemiluminescent, electrochemical or magnetic materials.
Interrit~lly labeled reporters (e. g., tritium ar other such radionuclides) which do not introduce a differ~nt moiety to the substrate are not contemplated for practicing the invention.
Examples of reporter enzymes which can be used to practice the indention include hydrolases, lyases, oxidoreductasea, transferases, isomerases and ligases some preferred examples are phosphatases, esterases, glycosidases and peroxidases. There can be mentioned peroxidase, glucose oxida,sa, phosphatase, esterase and glycosidase. Specific examples include alkaline phpsphvtase, lipases, beta-galactosidase, horseradish peroxidase and porcine liver esterase. As was noted above, if an enzyme is used as a reporter, it can be the same as ox different from the enzyme or enzymes used in the AREAS. ~'he instant invention can be used to catalyze deposition of a radioisotopicaily labeled conjugate or an enzyme-labeled conjugate, etc.
Another embodiment of the forward sandwich immunoassay descrimed akrove would involv~c reactzng a capture-antibody-analyte-second antibody complex with an AREAS consisting of an anti--antibody coupled to an enzyme such a9 HRP or AP. The anti-antibody would bind an epitope on the second antibody.

t~» G I ~ Jt' 1 1 . Gs murur~ i ~tuHL NU, 010 021 Thin invention is net limited to sandwich immunoassays. Tt is applicab~,~e to a widt~ v~ri~ty o!
assay lormata, !or example, nuc~.aic acid hybridisation assays for both RD1A and ~~a~.
To lurth~r illustrate the invention, ~xamplaa ~!
singi~ and mufti-enzyme ~E~S', con~u~atas, receptors, and race~ptor types ar~ present~d in Table 1 below.

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°' a~ ~ ~
a 'v ' ~4 ~N

11:24 PUPONT LEGHL N0.010 0 In th~ AP/~IRP mufti-enzyme ADEAS described above, the conjugate must be dephosphorylated befors it wfli react with HRpd and in the ~-gal/HRP mufti-enzyme ADE~1S, the con~ugat~ moat be deglycosylated before it will 5 react with HRP.
=t should b~e clear to those skill~d in the art that a larg8 numb~r of variations era possible and all these variations fall within the scope of the invention.
The following examples are intended to illustrate 10 the invention. Unless otherwise indicated, 100 dal of alI reagents were used. The one exception was that 200 ~,1 of blocking buffer was used, 15 prenar~tion of ~bni~ca~ and pare-hydroxypheny7.propionyl biocytin (IiPPB) was prepared by mixing a solution of p-hydroxyphenyl-propionic acid-N-hydroxysuccinimide ester (50 mg [0,2 2D mMoi)/2 ml dimethyi sulfaxide) with biocytin (70.75 mg [0.2 mMol,/2 ml 0.1 M NaHC03) overnight at room temperature (RT). Biotin-tyramine (BT) was prepared by mixing a solution of tyramine (40 mg (0.3 mMol~/1 ml dimethyl sulfoxide) with biotin-N-hydroxysuccinimide ester (100 mg [0.3 mMol)/1 ml dimethyl sulfoxide) overnight at RT. The solutions of FiPFB and BT were used as is. 'The Calculated conc~ntrntions were 26 mg/ml for HPPB and 55 mg/ml for HT, polystyrene EIA strips (N(JflG) were coe~ted witty polyclonal anti-Herpes Simplex virus (HSV) antibody (nako, Carpenteria, C~) in 0.1 M carbonate buffer pH 9,6 overnight at 4°C, and them block~d with Z~ bovine sexum albumin (BSA) in carbonate buffer and then washed with 10 mM phosphate buffered saline, 0.05% Tween 20, pH 7.4 (pBST) , !~ dilution of HSV antigen in 1% BS,~, 10 mM

e3~~1~~~ 11:5 DLIPONT LEGHL N0.010 024 phosphate buffered salineo 0.05 Twe~n 20 pH 7.4 (BSA-PBST), or buffer without antigen, was incubated for 1 hour at 37~C. The dilution waa sufficient to obtain the optical densiti~s in the range reported in Tabie 1. It was washed with PBST. The analyta dependent enzyme activation syr~tem consisted of HRP coupled to anti-HSv ~HRP AOBAS) which was purchased from Dako. The HRP
ADBAS was added and incubated for 30 min, at RT and was washed with PBST. Various conoentrations of HPPB or Bx as set forth is Table 1 below, ware added in 50 mM tria-HCl, 0.019 Hg~2, pH 5.0, for 15 min. at RT. After washing with PBST, streptavidin-HRP was added and incubated for 13 min, at RT to react with deposited biotins. The plate was then washed with PBST. l~n HF3P
13 substrate, a-phenylenediamine (OPD), was added, incubated for 30 min. at RT, and atopp~d with 4 N HZS04, Optical d~naiti~s at 990 nm were recorded on a miorotiter plate read~r.
ao Result' are presented in Table 2. Column 1 presents the various concentrations in ul/ml of HPPB or 8T. Columns 2 and 3 present the optical densities r~corded as a function of HPPS concentration. Columns A
25 and 5 present the results obtained using BT.
HPpB and BT were oonverted to activated forms by HRP AbEAS. Catalyzed reporter deposition was achieved without immobilizing a receptor.
In choo'ing the optimal concentration, one must 30 look at both the magnitude of signal amplification as wall as the signal to noise ratio. With this in mind, the optimal concentration of HPP$ was 20 H1/ml (approximately 0.5 mg/ml), and that of $m, was about 0.3 ~1/ml (approximately 16 ~g/ml).

~~y, ~ ": U 1 ~, : ~e UUF'UtV 1 LEGAL N0. 010 canc . IiF~P~

3 ar sa imps CorravG~TE PT cot~,~tl~~T~

c~l/mi~ xsv ~ut~~r xsV xu~f~r cw/a Ag' cw/~ ~g>
*

p 0.079 0.031 0.079 0.031 20 1.155 0.181 0.700 0.165 10 0.904 0.140 __ _~

5 0.99 0.120 2,060 0.430 2.5 0.177 0.063 __- ~-' 1.25 0.113 0.062 2.230 0.502 0.625 0.103 0.048 -~ _-0.313 -- --_- 1.850 0.169 0.078 ~- -- . 0.263 0.051 0.020 -- - 0.090 0.040 * w/o ~,g ~ without tigen an AntioHS'V coated EIA etrips were prapar~d as desCrib~d in ~xampia 1. ~ 1.:100 dilution of ~1SV anti~~n wa~~ prepared and serially lour-fo7.d diluted. These dilutians of HSV were incubated for 2 hours at 37'C with the antl-xSV coated ET~1 stripe. Excess r~agant was washed ofg with PBST. Th~ SEAS was the same as that described in ~xampla 1 aboee. It was added to the anti-xSV coated EIA strips Containing the anti~HaV_F?SV
complex and incubated !or 30 min. at ~tT and th~n w~.shed with PSST. 20 ~1/ml o~ HPPB conjugate as determined in Exempla 1 wao added in 50 mM tris-HC1, 0.0~.~ Ha02, p&I
6.0, and was incubated !ar 15 min. at RT and than washed u~: ~ ~ : ~u ~ ~ : co uurutv i LtIaHL NU. 010 026 with PB9T. Dctposited biotin~9 were re$cted wi~Gh 9treptavidineHRP iSA-HRP) for 15 min. at R'T. It was wash~d with PBST. The substrata, OPD, was added and incubated 30 min, at AT, stopped with 4 N FizSO~, and the absorbance at 490 nm was record~d on a microtiter plats read~r.
I4on-amplified ~~~~ys were run in which (a) no H~P~H
and no SA-HIaP were used; (b) HPPE was used without SA-HRP= ta) S.~r-HRP was used without HPPH.
The results shown in Figure 1 demoa~strate that (a) catalyzed depositioa~ of reporter was obtained and (b) both the conjugate and SA-HRP were needed for detection b~cause the conjugate contained an uniabel~d member of a ~pecifiC binding pair.
Results for the non-amplified assay (no I~PpB, no SA-HRP) were plotted. The results for the other assays were not plotted because the additional plots would overlap with the non-amplified results already plotted.
Polystyrene EIA stripe (NUtdC) were coated with rabbit anti-HZV p24 antibodie~ in 0.1 M carbonat~
buif8r, pH 9.6, overnight at 4°C, send th~n blocked with 2% BSA in carbonate buffer gollowed by washing with PEST. HIV antigen was incubated for 2 hours at 37°C
(concentrations are indicated in E'igure 2). The plate was then washed with PHST. A rabbit anti-H=V p24-HRP
analyte dependent enzyme activation system was then incubat~d for 2 hours at 37°C, and washed with PBST.
various concentrations of BT conjugtate, (0.2, 0.9, and b~~ ~ r~ ~u 11: a r DUFONT LEGr~L N0. 010 027 !a ~~ . _~,. -e~ %v .~. ~~~

0.9 ttl/ml) in 0.1 M borate buffer, 0.01 Fi2~~, pH 9.5, were incubated for 15 min, at RT faliowed by washing with FPS'. Then streptavidin-F3&tP was incubated for 15 min a ~t RT .
S ~g a comparison, a biotinylated anti-x=V p24 antit~ody was used, and detected with str~ptavidin-xRP.
OPD was added and incubated for 30 minutes, stopp~d with 9 N H~S~O~, and optical densities at 490 nm were record~d on a microtit~r plate reader.
The results are shown in Figure 2 where Amp 1, Amp 2, and Amp 3 refer to RT at concentrations of 0.2, 0.4, and 0.9 ~llmi respectively. Different lev~ls of 25 amplification w~sre achi~ved using catalyzed report~r deposition depending on the concentration of conjugate:.
pigure 2 also presents results far a non-amplified assay using a biotinylated antibody/SA-xRP detection system (biotin) and a non-amplified assay wherein anti-2o x~v p24 deteotor was directly labeled with aR~. ~h~
results obtained using the anti-xTV p24-xRP detector were inferior compared to the significant increase in detector signal obtained using catalyzed reporter deposition.
25 Depending upon the concentration of conjugate, signals a8 good, and greater, as those obtained with the biotinylated antibody were obtained using th~ cataly$ed reporter deposition method of the instant invention.
Best results were obtained using conjugate concentration 30 near the optimal amount s:s was determined in Rxa.mple 1.
g,~eDarat on ~n Pharao~!,;r~t .o Biotin. Tyr~gine preparation of biotin-tyramin~: a solution of 35 biotin-N-hydroxysuccinimide, 170 mg (0.5 mMoles), and 2a d~,~I,~L~ 11:2g DUFUNT LEG~iL N0.010 028 ~~~.~-~~~"~~
tyramine (recrystallized i'rom water), 5~.5 mg (0.5 mMol~s), in 25 ml dim~thylformamide was treated with 10 ml of 1 M triethylammonium bicarbonate, pH 7.5, and then heated at 50°C for 3 hours.
5 isolation: the solution was concentrated to dryness on a rotary ~~raporator, and th~ residue was recryatailixed from water, with a yield of 72~.
Ch,sraaterizationt the melting point was determined to b~ 192193°C .
FXA'MPl.fl! S
~t i ~ aid ~.~~5~~,~ e~ g,'~~A Ma~9 Asia ~ ~aa~ r~at~,ly~~Pd Rer~or .er ~eposition Polystyrene CIA stripe tNt7NC) were Coated with goat anti-mouse zgG (Fa fragment specific) antibody (TCt~) in 0.1 M carbonate buffer pH 9.5, overnight at R'I. They were then biock8d with 2~ eS~1 in carbonate buffer and washed with PHST. Dilutions of mouse Ig~ in BSA-~P8ST
were incubat~d in the wells for 1 hour at 37°C followed by washing with pBST. Conc~sntrations are set forth in Figur~ 3. Goat anti-mouse Ig~-HIP (HRp A~~~1S) and goat anti-mouse IgG-alkaline Pho9phatase d~ ~8~) (8oehringer Mannheim) were diluted as recommended by the manufacturer and incubated for 1 hour at 37°C. Assays were run with and without oat~alyzed r~porter deposition.
The AP ADEPuS was not used to catalyze reporter depo$ition in this experiment.
For catalyzed reporter deposition using the 1~RP
ADEAS, a 1 mg/ml stock solution of biotin tyramine (as described in Example 9) in dimethyl sulfoxide was prepar8d, and then added to a 0.1 M borate buffer, pH g.5, 0.01% H202, at 10 ~t7./ml (10 ug/ml biotin tyramine) and incubated for 15 min. at RT. "Ths plate was then washed with PHST. Streptavidin-filtP (for HFtP-Amp HItP), os streptawidin-Alkaline Phosphatase (for HFtP-e, ,~ ~ r ~ ~n 1 i : ~a L~uruN i i_EGH~ IdO. 010 029 ;,u ~. a~ ~ .> >.3 zs Amp Alk Phos) were incubated for 1S mia, at RT and the plate was washed with P9ST. Spectrophotometric detection was achieved after the addition of oPD (for HRF~), or p~nitrophenyl phosphat;r (for AP) for 15 min. at R~'. Reactions wer~ stopped by the addition of A N HaSfJ~
(xRF~/OPD), or 1 N NaOH (Alk Phoa/pNph). Optical densities, at 490 nm for ~iRF~/OHD and 405 nm for Alk Phos/pidPP, were recorded on a microtiter plate reader.
The results are shown in figure 3. As is apparent, one Can achieve signal amplification with a concomitant lower detection limit by allowing the HRP t~ERS to catalyze deposition of an activated ET conjugate followed by detection with atreptavidir~ coupled to HRp or AP. Thie exempla shows that if the reporter is an enayme, it can be the same as, or different from, the ~nzyme used in the ADEA.9.
E, , 6 ~~thhict'1 y~~lizPa, A H~o~~~irl~ ~ t~~r~n~r,.
Antibod~ /r Str~o ,vide n- RF p,steet t on~S a p~
The Du 8ont HIV p29 l~ntigen ELISA (catalog number 23 NEK OSO) was modified for catalyzed reporter deposition as follows: SA-HRP was used at 1/4 the concentration indicated in the directions. This was followed by a 15 min. RT incubation with biotin-tyramine, 10 ~tg/ml, in 0.1 M borate, 0.01% HaOZ px 8.5 buffer (as in 90 Example 5). Following washing with pBST, SR1-HRf at 1/16 the Concentration was incubated for 15 min. at RT.
Finally, OPD was added as per kit directions. Except for e~ttending the standard concentrations down to 0.39 pg/ml, no other changes wer~ mad~.

ro ,~ : j a a i.r ~ .~ c~ a ~. ~Z

The results ar~ shown in ~°igure 4. This experiment d~monatrated that one can amplify the signal generat~d by a biotinylatad ~antibody/SA-H~ system using catalyz~d reporter d~poaition. Because the concentration of SA-HRP for both incubations was much lass than that for the non-amplified away, it was clear that th~ incr~asad ai~nal was attributabl~ to reporter depo,~ition and not to a double 9A-HRP incubation.
~e~dt~rt s~ D~~~~ 1 ~ ~ en_an M~mhran~ 9 Nitrocellulose tSchlaichar & Schuall, 8A S5~ was spott~d with HSV antigen, and than blocked with 1~ SSA, 1~ non-fat dry milk, in PBS buffer, overnight. xh~
membranes were incubated for 1 hour at R~ with the analyta dependent enzyme activation sygtam described in Example 1 above. mha membranes were than incubated with biotin tyramina (from Exempla 1) at 2 y~lll0 ml 50 mM
tris-HC1, 0.01 H~02, pH ~.0 buffer for 15 min. at R°~, which was followed by incubation with atreptavidin-alkaline phosphatass !or 15 min. at R~°. Controls ware sun where biotin tyramine was incubated without streptavidin-alkaline phosphataaa, and straptavidin-alkaline phosphatasa was incubated without biotin-tyraminc. visualization of deposited alkaline phosphatasa was lacilitat~d by the addition of SCIp/N~'r (Kirkegaard & parry). BCTP is S-bromo-4-chloro-ir~doxyl phosphate and NBT is 2,2'-di-ip-nitrophenyl)-5,5'-diphenyl-3,3'-43,3'-dimethoxy-4,4'-diphenylene)-ditetrazolium chloride, visualization of th~ bound anti-HSv-HRP conjugate was facilitated by th~ addition of diaminob~nzidine (D
trade mark 032?~90 11:30 DUPONT LE6r~L N0.010 031 as Addition of DAB produced observable brown spotx where HSV antigen wag spotted on the nitrocellulose membrane. Rddition of BCIP/1VBT produced observable blue spots wh~r~ HSV antig~n was spott~d when biotin tyramine and streptavidin-alkaline phosphatas~ were incubated with the membrane. This showed that alkalin~
phosphatase waa deposited due to HRP activation of the biotin tyramine conjugate, followed by streptavidin-alkaline phosphatase detection.
~~rQM~.~~~~8s and A~~f y '~on b~l ~''ILfSY'Bg~°~Tnm Polystyrene EIA strips (~1t11~C) ware Coated with goat anti-mouse ggG (fc lrag~rnent specific) antibody (3Cid) in 0.1 M carbonate bufger, pH 9.6, overnight at RT. They w~ra than block~d with 2% BSa~ in carbonate buffer and washed with PBST. Concentrations of mouse igG in sSA-2p PBST, as set forth in ~°igure ~, were incubated for 1 hour at 37°C followed by washing with BST. Goat anti-mouse IgG-HRP (AREAS) purchased from Boehringer Mannheim was diluted as recommended by the manufacturer and incubated fCr 1 hour at 37°C. The plate was then washed 23 with PBST. A 1 mg/ml stock solution of HT conjugate (as describ~d in Hxample 4) in dimethyl sulfoxide was prepared, and then added to a B.1 M borate, 0.01 Hz02, pH 8.5 buffer at 10 ~tl/ml (10 ~tg/ml biotin tyramin~) , The mixture was added to the plate and incubmted for 15 30 min, at RT, and then washed with PBST. 8treptavidin-beta galactosidase (B~thesda Research Labs) was added and incubated for 15 min. at Rfi. The assay was also run without catalyzed reporter deposition, i.e., without adding HT. Colorimetric detection of thg non-amplified 33 assay was achieved after incubation with OPD (for HRp), 03i27i90 11:30 DUPONT LEGAL I~1Q.010 032 ~' ~
2 9 !~ ~'. l.~
!or 15 min. at RT. Fluorescent detection of the amplified assay was achieved after the addition of 4-methylumbelliferyi beta-D-galaotoside (MUO) (for HRF-beta Gal). Optical densities at 490 nm w~re recorded far HRP/~PD on a microtitar plat~ reader. ~'luorescence for HRP-b~ta Gal/MU~ was recorded on a fluoregc~nce microtiter pieta reader (Dynatech Laboratories).
The results era shown in Figure 5. The fluorescent signal was due to the quantitative deposition of biotintyramine by the HRP ADHAS (allowed by incubation with streptavidin beta-galactosidas~e.
Am~~al~~iCg~~ori ef a M~m~ran~ 10.a~~~
Fluoresceia~°tyramine (FT) was prepared as followsa Solutions of 96.6 mg of 5-(and 6)-carboxyfiuorescein succinimidyl ester in 0.3 ml dimethyl sulfoxide and 14.6 mg tyramirae in 0.3 ml dimethyl sulfoxide were pxepared.
Conjugation was achieved by mixing 0.25 mi of each solution overnight at FtT. The solution was used as is.
Three nitrocellulose (Schl~icher & Sshuell, 19A S3) strips were spotted wfth 1 ~tl of mouse gg0 at 10 wg/ml, and serial two fold dilutons in PBS. The membranes were blocked with 5% non-fat dry milk in PBST for 30 min, amd then wash~d three times its ASST, A goat anti-mou~e IgG-H~r conjugate (Hoehringer Mannehim) diluted 1/2000 in 1%
BSA-PBST was incubated for 30 min. at RT, and the membrane were washed thre~ times with PBST. The third membrane was incubated with FT at 20 ~g/m~. in 0.1 M
borate, 0.01% H20~, pEE 8.5 buffer for 15 min. at RT, and washed three times in PBST, Then, the secoxid and third membranes were incubated with an anti-fluorescein 3!f antibody (Chemicon) which was conjugated to HitP (by the NJ~G~i7b 11:J1 LUh-'UN1 L~GHL N0.010 033 '' -;
a 3o r~.
SMCC method of gshibcawa, ~., et al., ~T. gmmunoa9aay, .~, 209-327, 19~3) diluted in 1~ HSA-PSST for 15 min at RT, and washed three times in P~3T. Vieualixatian of all three strips was facilitated by the addition of diaminobenzidin~ for 5 mi.n.
Three spots could be assn on the ~isgt two strips indicating a detection limit o~ 2.5 ~.g/ml, and that the anti-fluoresCein-HRP con~ugat~e did not contribute to additional signal. Six spots could been seen on the third strip indicating a detection limit of 313 ng/ml.
Th~ us~ of the catalyzed reporter deposition amplification method of the invention impsoved the detection limit of the assay eight fold over that of the non-amplified assay, ~,yntl2esis ef the C~;~~~ ~~~ ~~
~uccin~ mi dY.l) -6- tnh~nox~,l~ ~ -axo-z °~-~ar$Wxy~thyloh_~nYt'~xanoah.a" H~~-S-I~7i3~ and A13cm74ne~
h~yha~ase_..IAP
The following reaction scheme is illustrated in Figure 6: Ethyl 2-(~~-hydroxyphenylazo)benzoate (HEE), 23 i.s prepared from 2-(4°-hydroxyphenylazo)-benzoic soil (HABA, (~)), anhydrous ethanol and a catalytic amount of acetyl chloride. The ethyl ester (HEE) is reacted with t°butyl 6-iodohexanoate and sodium hydride using the general procedure reposted by Castellanos et sl., Tetrahedron, pages 1691-1696, Vol. 37, (191), to produce t-butyl-6-(phenoxy-(4~-azo-2"_ carboxyethylphenyl)hexsnoate (HEE-6-t--BU, (~)). The tart-butyl ester is hydrolyzed by tr~atment with txifluoroaceti.c acid using the general procedure 3S reported by Bryat: et al., ~?ournal of the American t~_~ c r ~ ?t~ 11 ~ Jc UUf ~I[~~ I LtUHL ~V~J. 010 i Chemical Society, pages 23x3-2355, Vol. 99, (1977), to produce 6-(phenoxy-(4'-azo-2"-carboxyethylphcnyl)-hexanoic acid (HH~-6-~I). (N-succinimidyl)-6-(phenoxy-t4'-azo-2°-carboxyethylphenyl)hexanoate (HSE-6-N13S, (~)) is prepar~d =rom HEE-6-H, N-hydroxy9uccinimide and dicyclohexylcarbodiimida in THF using the g~ngral procedur~ reported by Aryan et al., Makromoleouiar Chami~, pages 2375-2382, Vol. 186, (1985). The uHS
ester (~) is dissolved is a minimum volum~ of DMSO and adda~d tc~ a buffered aqueous solution of alkaline phosphatase (e. g. calf intestine) using the g~neral procedure reported by p'Sullivan et al., Methods in Enzymology, page9 147-166, Vol. 73, 11981) to 4ive the con jugate gHEE~6-~, E
SVnf~,~"~ at Of HABA-'f'vrami nr t~nn_imesrs~øw Iet W
from IN-Qnr.n~g1'~ m~ dy1 j -6- ( hi~~4 (~ ~-aZy a ~
~rboxvathyynY' ) hsYanoat~ (HEJ~~~,S r~~ ~
~ , The NHS ester (5) (1 mmol) described in Hxample 10 abov~, and tyramina (1 mmol) are dissolved in DNtF (5 ml,) and stirred at RT for 48 hrs. The solution is evaporated to dryness in vaGUO. The residue is suspended in I~ZO (pFd 8.0, 50 mL) and porcin~ liver esteras~ (100 mq) ie added. The pH of the solution is maintained by adding 0.1 N Na~H as required. The solution is evaporated to dryness after 24 hours, The resulting H-T conjugat$ is isolated by chromatography (silica gal, chloroform/methanol).

t~3~~7i~0 11:32 UUFONT LEGAL N0.010 935 L~ 12 c~m~zx~c~r,ion oz uer~~s,~~-q~'~ g M;p~9~ ~pC Aam~v .LCaawmn rvav.sma u~~~y~.~,~~~~1 LnLaw~~a ~Iicrotiter plate strips (Nuno) are coated wi~.h a miactura of goat anti-mouse igG (~'c fragment speoific) antibody (ZCN) and atreptavidin (Scripps ~aboratori~s) in 0.1M carbonate buffer pH 9.6 overnight at FtT. They area then block~d With 2% ESA in PBS and wa3hed with PEST. nilutions of mouse ggG (0-100 ng/mL) in sSA-p~ST
are incubated in the wells for 1 hr, at 37°C followed by washing with BEST. A preparation of goat anti-mouse YgG°PLE (PhE.:~DEAS) (0.75 mg/mh) is prepared by the general method deecrib~sd by Hashida et al,, journal of Appli~d Eiochemiatry vol. 6, pages 56-63, 1984 and diluted 1:100-102000 with phosphat~ buffer (0.1 ~R, pH
8.0, 0.24 ESA) and is incubated for i hr. at 37°C and washed with DEBT. ~tEE°6-AP (1 mg/mL) (prepar~d as described in Example 10) is added to the microtiter plate woll and incubated for at least 15 min. at 37°C.
. The plate is then wa9hed with PBST. SpeGtrophotometric detection is achieved after the addition og p-nitrophenyl phosphate. Heactiona are stopped by the addition of 1 N NaOH. Optioal densitie3 at 405 nm are r~corded on the miorotiter plate reader. hmplification of detector signal in this example results from catalyzed reporter-enzyme deposition, i.e., PLE
Catalyzes deposition of HEE-6-AFB whesc the receptor, streptavidin, has been immobilised on the microtiter plate surface.

03~27i90 X1:33 DUPONT LEGI~I. N0.01~ 036 1~~~.P~~ a t~

D~mon~ rat icon o~ ~h~ ~n~a~m~ l~od~1 ~t.~d g~ ndina o~ ~
locked finder R suspension of porcin~ liv~s esterase was added t10 uL, 2850 t1/mL, Cat . I~o. F3128, Sigma Chemical, 5t .
Louis, M~) to a solution of ethyl 2-t4'-hydroxy-phenylazo)benzoate tHEE) 10.25 mM) and atreptanvidin t0.2 m~/mL) in phoaphat~ buffer t0.1 M, pH 8.0, 2m~). A
second solution identioal to the gisat was prepared which contained no streptavidin. fhe absorbanc~ of the two solutions w~a measured at 500 nm sa a function of time. Figure 7 shows that th~ absorbents of the solution which contained no atreptavidin decreased over time indicatingr hydroly'is of HEM to 2-t~9°-hydxoxy-phenylazo)benzoic acid iFIAB~). The absorbents of the solution whieh contained streptavidin inor~ased over time indicating the formation o~ the HAS~:streptavidira complex, which is known to have a strong absorbents at 50A nm.

Claims (43)

1. A conjugate comprising a detectably labeled phenol.

2. A conjugate according to Claim 1 wherein the detectable label is selected from the group consisting of a fluorescent label or a member of a specific binding pair.

3. A conjugate according to Claim 2 wherein the detectable label is fluorescein, tetramethyl rhodamine, coumarin or biotin.

4. A method for the detection or quantitation of an analyte in an assay which comprises using an analyte dependent enzyme activation system comprising at least one enzyme to react with a conjugate consisting of a detectably labeled substrate specific for the enzyme system to form an activated conjugate which covalently deposits substantially wherever at least one receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system, wherein deposited detectable labels either directly or indirectly generate a signal which can be detected or quantitated.

5. A method according to Claim 4 wherein at least one enzyme of the analyte dependent enzyme activation system is selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases, and ligases.

6. A method according to Claim 5 wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatase, esterases and glycosidases.

7. A method according to Claim 6 wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and betagalactosidase.

8. A method according to Claim 7 wherein the enzyme is horseradish peroxidase.

9. A method according to Claim 8 wherein the conjugate is selected from the group consisting of biotin tyramine, p-hydroxyphenylpropionylbiocytin, or fluorescein tyramine.

10. A method according to Claim 4 wherein the conjugate is reacted with detectably labeled antibody.

11. A method according to Claim 4 wherein the conjugate is reacted with a detectably labeled member of a specific binding pair.

12. A method according to Claim 4 wherein the conjugate is reacted with detectably labeled streptavidin.

13. A method according to Claim 4 wherein the detectable label is selected from the group consisting of enzymes, radioactive isotopes, fluorogenic, chemiluminescent, or electrochemical materials or a member of a specific binding pair.

14. A method for producing an activated conjugate comprising reacting a peroxidase enzyme with a detectably labeled phenol.

15. A method according to Claim 14 wherein the detectable label is selected from the group consisting of a fluorescent label and a member of a specific binding pair.

16. A method according to Claim 15 wherein the detectable label is fluorescein, tetramethyl rhodamine, coumarin or biotin.

17. A method according to Claim 4 wherein the conjugate is 6-(phenoxy-(4'-azo-2"-carboxyethylphenyl))-hexanoyl-alkaline phosphatase.

18. An assay for detecting or quantitating the presence or absence of an analyte in a sample which comprises a) immobilizing the analyte;
b) reacting the product of step (a) with an analyte-dependent enzyme activation system;
c) reacting the product of step (b) with a conjugate consisting of a detectably labeled substrate to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system; and d) detecting or quantitating the presence or absence of the analyte in the sample.

19. An assay according to Claim 18 wherein the analyte dependent enzyme activation system has at least one enzyme selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases, and ligases.

20. An assay according to Claim 19 wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

21. An assay according to Claim 20 wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase, and beta-galactosidase.

22. An assay according to Claim 21 wherein the enzyme is horseradish peroxidase.

23. An assay according to Claim 18 wherein the detectable label is selected from the group consisting of enzymes, radioactive isotopes, fluorogenic, chemiluminescent, or electrochemical materials or a member of a specific binding pair.

24. An assay according to Claim 21 wherein the enzyme is horseradish peroxidase and the conjugate is selected from the group consisting of biotin tyramine, p-hydroxyphenylpropionyl-biocytin or fluoresceintyramine.

25. An assay according to Claim 18 wherein the conjugate is 6-(phenoxy-(4'-azo-2"-carboxyethylphenyl))-hexanoyl-alkaline phosphatase.

26. An assay according to Claim 18 wherein the conjugate is reacted with a detectably labeled member of a specific binding pair.

27. An assay according to Claim 18 wherein the conjugate is reacted with detectably labeled antibody.

28. An assay according to Claim 18 wherein the conjugate is reacted with detectably labeled streptavidin.

29. An assay according to Claim 18 wherein said assay is an immunoassay.

30. An assay for detecting or quantitating the presence or absence of an analyte in a sample which comprises a) reacting an analyte dependent enzyme activation system with a conjugate consisting of a detectably labeled substrate to form an activated conjugate which deposits substantially wherever receptor for the activated conjugate is immobilized, said receptor not being reactive with the analyte dependent enzyme activation system; and b) detecting or quantitating the presence or absence of the analyte in the sample.

31. An assay according to Claim 30 wherein the analyte dependent enzyme activation system has at least one enzyme selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases, and ligases.

32. An assay according to Claim 31 wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

33. An assay according to Claim 32 wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase, and beta-galactosidase.

34. An assay according to Claim 30 wherein the detectable label is selected from the group consisting of enzymes, radioactive isotopes.
fluorogenic, chemiluminescent, or electrochemical materials or a member of a specific binding pair.

35. An assay according to Claim 30 wherein the conjugate is reacted with a detestably labeled member of a specific binding pair.

36. An assay according to Claim 30 wherein the conjugate is reacted with detestably labeled streptavidin.

37. An assay according to Claim 30 wherein the conjugate is reacted with detestably labeled antibody.

38. An assay according to Claim 30 wherein said assay is an immunoassay.

39 39. A compound which is 6-(phenoxy-(4'-azo-2"-carboxyethylphenyl))-hexanoyl-alkaline phosphatase.

40. An assay according to Claim 29 wherein the conjugate is a detectably labelled phenol.

41. An assay according to Claim 33 wherein the enzyme is horseradish peroxidase.

42. An assay according to Claim 41 wherein the conjugate is a detectably labelled phenol.

43. An assay according to Claim 42 wherein the detectably labelled phenol is selected from the group consisting of biotin tyramine, p-hydroxyphenylpropionyl-biocytin or fluorescein-tryamine.