CA2301818C - Bifunctional compounds useful in catalyst 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

Bifunctional Compounds Useful in Catalyst Reporter Deposition FIELD OF THE INVENTION
This invention relates to assays and, more particularly, to catalyzing reporter deposition via an activated conjugate to amplify the defector signal thereby improving detection 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. Detection 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 sensitive and simple immunoassays. In this method a second enzyme system is coupled to the primary enzyme label, e.q., the primary enzyme can be linked catalyticaliy 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 proCesseB wherein an enzyme is modulated by the action of a second enzyme, either by direct modificat~.on or by interaction with the product of the controlling enzyme.
U.B. Patent 4,668,621, issued to Doellgast on May 26, 1987, describes application of an enzyme-linked coagulation assay (ELCA) to develop an amplified immunoassay using the clotting cascade to enhance is sensitivity of detection of immune complexes. The process involves clot formation due to thrombin activated fibrin formation from insolubilized fibrinogen and labeled solubilized fibrinogen. Amplification of the amount of reportable ligand attached to solid phase is obtained only by combining use of clotting factor eonjugatts 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 July 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.
Another amplification system is described in U.S.
Patent 9,598,042, issuCd to Self on July 1, 1986, and ' 2 ~.K. Patent Application No. 2,059,421 which was published oa April 23, 1981, which disclose an immunoaeaay using an $nzyme label to produce directly or indirectly a eubstanee that is capable of influencing a catalytic event without itself being consumed during the vatalytic event. More specifically, a primary enzyme system produces or xemoves a substance capable of modulating a secondary enzyme system which results in amplification. The enzyme systems use unconjugated enzymes to avoid the tendency to inactivate certain enzymee on Conjugation.
European Patent Application Publication No. 123,2b5 which was published on October 31, 1989, describes another cascade amplification immunoassay wherein a zymogenrderived-enzyme is coupled to a zymogen-to-enzyme cascade reaction sequence to obtain multiple stages of amplification in producing detectablm marker material used to quantify analyte amount.
European Patent Application Publication No.
144,744, published June 19, 1985, describes a specific binding assay based on enzyme cascade amplification wherein the label component employed in the dttectant reagent is a participant in or a modulator of an enzyme cascade reaction wherein a first enzyme acts an a first substrate to product a second enzyme. The production of the second enzyme can be followed or the seCOnd enzyme can act on a second substrate to produce a third enzyme.
Similarly, U.S. Patent 4,318,980, issued to Bogusiaski et al. on March 9, 1982, describes a hetervgenous specific binding assay using a conjugate formed of a specific binding substance coupi~d to the reactant, i.e., an enzymatic reactant. The ability of the reactant to participate in the monitoring reaction tv detect the presence of analyte is altered by the presence of the ligand in the medium. Thus, the conjugate in its free state is more active in the monitoring rQaction than in its bound state.
A heterogenous specific binding assay using enzyme amplification is described in British Patent Application No. 1,901,297 which was published on July 30, 1975 and U.S. Patsnt 4,376,825, issued to Rubenstein et ai, on March 15, 1983. Amplification is achieved by bonding the compound to be assayed or a counterfeit of it to an enzyme. The resulting enzyme~bOund-ligand competes with free ligand for specific receptor sites. when the enzyme-bound ligand is displaced by the free ligand the enzyme is then tree to react with a large number number of substrate molecules and the concentration of the remaining substrate or of the product can be measured.
PCT International Publication No. Wp 81/00725 which was published on March 19, 1981 describes a method of determining a substrate in a sample which comprises converting the substrate to a product in a first stage of a cyclic reaction sequence and converting the product back to the substrate in a second reaction stage of the cyclic reaction sequence. At least one of the first and second reaction stages is enzyme catalyzed.
PCT Application having International Publication Number WO 84/02193, which was published on June 7, 1984, 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 enzyme with its substrate is concentrated on an active support.
European Patent Application Publicatipn No.
181,762, published on May 21, 1986, describes a method to determine enzymatic activity in a liquid sample by particle agglutination or inhibition of particle agglutination.

Substrate/cofactor cycling is another example of amplification which is based on the cycling of a cofactor or substrate which is generated by the primary enzyme label. The primary enzyme converts the primary substrate 5 to an active form which can be cycled by two enzymes of the amplifier cycle. These two enzymes are provided in high concentration and are poised to turn over high concentrations of substrata but are prevented from so doing until the cycling substrate is formed. The product of the primary enzyme is a catalytic activator of the amplifier cycle which responds in proportion to the concentration of substrate and hence the concentration of the enzyme label, In the early sixties, Lowry et al., Journal of eiologicai Chemistry, pages 2746-2755, Vol. 236, No. 10 (October 1961), described the measurement of pyridine nucleotides by enzymatic cycling in which the coenzyme to be determined was made to amplify an enzymatic dismutation between two substrates.
A more complex substrate cycling system is described in U.S. Patent 4,745,054, issued to Rabin et al, on May 17, 1988. The Rabin system involves using a small enzymieally inactive peptide fragment of an enzyme . as a label and conjugated with the complementary fragment to form an enzyme which catalyzes a primary 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 presence of analyte.
vary et al., Clinical Chemistry, pages 1696-1701, Vol. 32 (1986) describe an amplification method suited to nucleic acids. This is the strand displacement assay which uses the unique ability of a polynucleotide to act as a substrate label which can be released by a phosphorylase.

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 compound of the formula:
O
R-L-IC-(H2C)n-X R6 R5 \ C OR9 R'/R$ 'N=N R' z2 ~
Ra ~ R3~ R2 wherein R' through R$ are the same or different and are selected from the 6a group consisting of H, OH, OCH3, straight chain or branched alkyl groups having 1-4 carbon atoms, F, CI and Br;
Z' and Z2 are independently phenyl or napthyl;
n is 1-19;
X is N, O or S;
R9 is selected from the group consisting of glycosides and straight chain or branched alkyl groups having 1-4 carbon atoms;
L is (NH(CH2)SCO)~ where s is 1-5 and r is 0-5; and R is a reporter selected from the group consisting of radioisotopes, fluorogenic materials, light scattering materials, chemiluminescent materials, electrochemical materials, magnetic materials and enzymes provided that said enzymes do not react with OR9.
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 pNl/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 detected with atreptavidin-HRP (HRP-Amp HRp) or with streptavidin-AP (IiRP-Amp AP). The assay was also run using only HltF labeled detector antibody or AP
labeled detector antibody.
Figure 4 presents two graphs comparing results obtained from a Du Pont* HIV p24 antigen ELISA run with and without using catalyzed reporter deposition to amplify reporter signal.
Figure 5 is a graph comparing results of a mouse IgG assay without catalyzed reporter deposition (HRP) and with catalyzed reparter deposition (NRP-~-Gal).
Figure 6 depicts a preparation of ethyl 2-(4'-hydroxyphenylazo)benzoate-6--alkaline phosphatase (HEE-6-AP), the synthesis of which is described in Example 10.
Figure 7 illustrates the esterase catalyzed conversion of HEE to 2-(4'-hydroxyphenylazo)benzoic acid (IiAHA) /streptavidin complex.
DETAILED DESGRIPTTnN OF THE I .NTTON
The term analyte dependent enzyme activation system (AREAS) refers to an enzyme system Wherein (i) at least one enzyme is coupled, in any manner known to those skilled in the art, to a member of a specific binding pair, or (ii) the enzyme need not be coupled to a member of a specific binding pair when it is the analytE. The enzyme, either by itael! or in connection with a second enzyme, catalyzes the formation of an activated conjugate which then is deposited wherever a receptor for the activated conjugate is immobilized.
The term amplification as used herein means amplification of reporter signal due to deposition of a conjugate activated by an AREAS.
* trade mark The term conjugate means a detestably labeled substrate specific for thm ADEAS whether it be a single enzyme ADEA9 or mufti-enzyme ADEAS. The substrate must have at least one component but is not limited to such.
For example, the substrate can consist of two components. One component contains the binding site for the receptor and is detestably labeled. The other component is a constituent which prevents or interferes with binding to the receptor until such time as the ADEAS primes the conjugate as is discussed below.
Another example of a conjugate is biotin-tyramine wherein tyramine is the substrate portian and biotin constitutes the detectable label as described below.
Conjugates are described in greater detail below as well.
The term detestably labeled means that the substrate can be coupled to either a reporter or to an unlabeled first member of a specific binding pair provided that the reporter introduces a different moiety to the substrate as is discussed below. When the substrate is coupled to an unlabeled member of a specific binding pair, following deposition, the r substrate-specific binding partner complex is reacted with the second member of the binding pair which is coupled to~a reporter. Alternately, the substrate-specific binding partner complex can be pre-reacted with the detestably labeled other member of the specific binding pair prior to deposition.
The term deposition means directed binding of an activated conjugate to the receptor which results from either the formation of a covalent bond or a specific binding pair interaction as described below.
The term receptor means a site which will bind to the activated conjugate either through the formation of a a covalent bond or a specific binding pair interaction ae described below.
The term activated conjugate means that the conjugate has been primed by the AREAS to bind with the receptor.
One of the unique features c! this invention is the analyte dap~ndent enzyme activation system which catalyzes deposition of conjugate by converting the substrate portion of the conjugate to an activated form which is deposited wherever a specific receptor for the activated conjugate is immobilized. The AREAS does not utilize enzyme cascade reactions or enzyme cycling to effect amplification. Rather, it uses either a single enzyme or combination of enzym:s to activate the eonjugat~. Deposition o! conjugate occurs only if the analyte and analyte dependent enzyme activation system, which can be the same if the analyte is an enzyme, for example in the detection of an enzyme such as alkaline phosphatase, or different, have been immobilized and a receptor, as described below, is immobilized to bind the activated conjugate. Thus, the AREAS, 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: th. te$t sample containing the analyte is reacted with an immobilized capture reagent, such as an antibody excess reagtnts are washed off; the immobilized capture antibody-analyte complex is reacted with an AREAS, aueh as a second antibody specific for the analyte which has been coupled to an enzyme, e.g.
horseradish peroxidase (HRp), alkaline phosphatase (AP), etc. The AREAS will bind only if the analytc has been bound by the capture reagent. Otherwise the reagents will be washed off. Coupling of the enzyme to a specific binding partner does not affect the enzyme's ability to react with the substrate portion of the conjugate. When conjugate such aye biotin-tyramine or RAHA-tyramine analog (s. g., N-(4 " ~-hydroxyphenethyi)~6-(phenoxy-(4'-azo-2 "-benzoic acid))hexamide) is added to 5 the immobilized capture antibody-analyte-second antibody-enzyme complex, the enzyme reacts with the substrate portion of the conjugate, e.g., with the tyramine portion of the conjugate, converting it to an active form which will bind to an immobilized receptor 10 which is either endogenous or exogenous to the assay system. The amount of conjugate deposited will be a function of immobilized AREAS. Deposited conjugate such as biotin-tyramine or HAHA tyramine analog can then bs detected by reacting with streptavidin-HRP and orthophenylenediamine. Ths term HAHA~tyramine analog means, generally, unsubstituted or substituted I3A9A, coupled with or without a spacer, to a hydroxy-phenyl containing compound such as tyramine. If the conjugate 1~ liuvrd~~~lu-~yramin~ znen Lne aeposiLea con~ugats Can bs detected directly, or following reaction with a labeled anti-fluore$cein antibody.
Thus, the AREAS is used to catalyze the deposition of detectably labeled substrate (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 well as the situation where an AREAS 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 format would involve a similar approach. In the example above, the AREAS can be an antibody coupled to an enzyme such as AP. In addition to the immobilized capture antibody-analyte-second antibody-Ap complex, a second enzyme such as HRP could be immobilized on the support. The conjugate can be a detectably labeled phenylphosphate which cannot react with HRP until it is dephosphorylated. AP dephosphorylatee the phenol which than is free to react with the immobilized HRP to form an activated phenolic conjugate which deposits wherever receptors are immobilized. After removing excess reagent, deposited reporter is detected and quantitated.
Alternatively, HRP can be coupled to the second antibody and AP can be immobilized on the surface of the support.
The instant invention is surprising and unexpected because amplification of reporter signal is obtained via deposited activated conjugate without using cascade mechanisms or enzyme cycling. The AREAS reacts with the conjugate to farm an activated conjugate which will bind with immobilized receptor specific for the activated conjugate. The amounts of receptor and activated conjugate are in excess of the amount of AREAS
immobilized.
The choice of an AREAS is governed by the ability of the enzyme or enzymes to convert a conjugate to an activated form whioh 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 substrate and receptor. Other important factors include availE~bility of the enzyme or enzymes, relative ease or difficulty to couple it to the member of a specific binding pair, stability of the enzyme ox enzymes as well as the stability of the conjugate and the receptor. ~n some cases, an AREAS can be purchased, depending on the assay format.
Enzymes suitable for use in an AREAS include hydrolases, lyases, oxidoreductases, transferases isvmerases and ligases. There can be mentioned peroxidase, glucose oxidase, phosphatase, esterase and glycosidase. Specific examples include alkaline phosphatase, lipases, beta-galactosidase, horseradish peroxidase, and porcine liver esterase.
Members of specilic binding pr~ira suitable for use in practicing the invention can be of the immune or non-immune type. Immune specific binding pairs are exemplified by antigen/antibody systems or hapten/anti--:0 hapten eyatema. The antibody member, whether polyclonal, monoclonal or an immunoreactive fragment thereof, of the binding pair can be produced by customary methods familiar to those skilled in the art.
The terms immunoreactive antibody fragment or immunoreactive fragment mean fragments which contain the binding region of the antibody. Such fragments may be Fab-type fragments which are defined as fragments devoid of tha Fc portion, e.g., Fab, Fab~ and F(ab')2 fragments, or may be so-called ~~half-molecule~~ fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the intact 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 share a natural arfiriity for each other but are not antibodies. Exemplary non-immune binding pairs are biotin-avidin or biotin-streptavidin, folic acid-folate binding protein, complementary probe 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 reactive groups such as maleimides and haloaCetyl derivatives and amine reactive groups such as isothiocyanatea, succinimidyi esters and sulfonyl halides, etc.
Suitable supports used in assays include synthetic polymer supports, such as polystyrene, polypropylene, substituted polystyrene, e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides;
polyvinylchioride, etc.; glass beads; agaroaet nitrocellulose, etc.
Another important component of the invention is the conjugate, i.e., a detestably labeled substrate which must be specific for the ADEAS. As was stated above, when the conjugate reacts with the ADEAS, the enzyme or enzymes catalyze formation of an activated conjugate which binds wherever a receptor is immobilized whether exogenous or endogenous. An immobilized exogenous receptor mean8 a receptor which does not originate within the assay. ~t must be immobilized on the surface of the support prior to adding the conjugate to the reaction mixture. An endogenous receptor 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 ADEAS (HRF coupled to a member of a specific binding pair) is reacted with conjugate containing a phenolic substrate, an activated pheaolic substrate is produced. It is believed that the activated phenolie substrate binds to electron rich moieties such as tyrosine and tryptophan present in the proteins on the solid support. However, if a different conjugate is used, such as a labeled 3-methyl-2-benzvthiazolinone hydrazine (METH) which is discussed below, a receptor, such as 3-(dimethylamino)benzoic acid (DMAH), must be immobilized prior to addition of conjugate.

Another embodiment involves reacting a conjugate which becomes phosphorylated by an AREAS. The activated (phosphorylated) conjugated can then react with an antibody specific for the~activated conjugate.
In still another variation, an AREAS can be reacted with a conjugate consisting of a component which when activated will bind to a receptor and which is coupled to a component having a thiol reactive group such as a maleimide. The deposited maleimide moiety can then be detected by reacting with a sulfhydryl-containing r~porter which can be endogenous to the reporter, e.Q., beta-qalactoeidase, or the sulfhydryl groups can be added to reporters such as HRP or AP using thiolating reagents such as N-sueeinimidyi-S-acetylthioacetate (SATA), 8-acetyimercaptosuccinic anhydride (SAMSA), or succinimidyi-3-(acetylthio)-propionate (SATp).
Alternatively, the substrata can be coupled 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 the art. Detection can be effected using a reporter having a thiol reactive group such as maleimide-HRP or iodoacetvl-HRp.
Another alternative is to use a conjugate wherein the substrate has two components as described above, a detectably labeled first component which will bind to the receptor after the second component has been activated or removed by the AREAS. An example of this is a small organic molecule such as 2-(4'-hydroxy-phenylazo)-benzoic acid (HAHA) which binds specifically to avidin and st reptavidin. The terms avidin and streptavidin are used herCin interchangeably. HAHA can be detectably labeled using any of the reporters described below, e.g., radioisotopes, enzymes, ete. For instance, alkaline phosphatase (AP) Can be conjugated to HAHA, using techniques well known to those skilled in the art, with or without a spacer, to a functional group on HABA. An example of such a functional group is the 9'-hydroxyl moiety. Moreover, HABA can be modified to 5 possess a second component which prevents binding until it has been removed by the AREAS. For example, esterification of HABA with ethanol produces a HABA
ethyl eater which does not bind to etreptavidin.
DeteCtabiy labeled NASA ethyl esters will not bind to 10 streptavidin until thQ ester group has been hydrolyzed to the corresponding carboxylic acid. Hydrolysis can be affected using an enzyme such as an esterase, e.g., porcine liver esterase. Thus, detectably labeled HAHA
esters ouch as 6- (phenoxy- (4'-azo-2 "-15 carboxyethylphenyl)-hexanoyl-alkaline phosphatase can be deposited w ing an ADEAS having a suitable eatera~e which will hydrolyze the ester to permit binding o!
detectably labeled xASA with streptavidin (i.e., exogenous receptor) which has been immobilized on the surface of a bupport.
Compound9 of the formula (~~n ~X
/~
H
~N ~
wherein R1 through RB are the same or different and are selected from the group consisting of straight chain or branched alkyl groups having 1-4 carbon atoms, F, Cl, Br or It Z is phenyl or naphthyis n is 1~19J
X is N, 0, S; and R9 can be H or atraisht chain or branched alkyl group having 1-4 carbon atoms can be used to synthesize t3AHA-type conjugates as described in the examples below. The term BABA-type conjugate means fiAHA
derivatives; (i) which can be substituted or unsubstituted and are coupled with a spacer to a reporter and iii) which contain as AREAS activatable moiety that prevents the conjugate from binding to streptavidin until it has been activated or removed by the AREAS. The synthesis of suoh a compound is illustrated in Figure 6 as well in Example 10 below.
The approach described below caa be modified by those skilled in the art generally to synthesize any of these compounds using procedures well known to those skilled in the art.
Other small organic molecule/xeceptor combinations which are suitable to practice the invention include haptens/antibodies, sugars and oligosaccharides/lectins, biotin and dyes/avidin and/or atreptavidin.
As is shown in Table 1, a number of receptors are available. The choice of a receptor will depend upon the conjugate selected.
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 AREAS and substrate selected to produce conjugate.
Conjugate can be synthesized using conventional coupling and labeling techniques. Substrate choice will depend upon the AREAS selected. To reiterate, detailed lfi 1~
knowledge is required of the catalytic properties of ~ach specific enzyme in order to properly design a useful synthetic substrate and, if necessary, a receptor.
A wide variety of reporters are available for coupling to the substrate to produce the conjugate or to couple to a member of a specific binding pair. As was discussed above reporter should introduce a different moiety to the substrate. Reporters can be a radioactive isotope, $uch aa, l2sI, enzymes, fluorogenic, chemiluminescent, electrochemical or magnetic materials.
Inter~aily labeled reporters (e. g., tritium or other such radionuclides) which do not introduce a different moiety to the substrate are not contemplated for practicing the invention.
Examples of reporter enzymes which can be used to practice the invention include hydrolaaea, lyaaes, oxidoreductases, transferasea, isomerasea and ligases soma preferred examples are phosphatases, esterases, glycosidases and peroxidases. There can be mentioned peroxidase, glucose oxidase, phosphatase, esterase and glyeosidase. Specific examples include alkaline phosphvtase, 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 or different fxom the enzyme or enzymes uatd in the AREAS. The instant invention can be used to catalyze deposition of a radioisvtopically labeled conjugate or an enzyme-labeled conjugate, Ctc.
Another embodiment of the forward sandwich immunoassay described above would irivolvt reacting a capture-antibody-analyte-second antibody complex with an AREAS consisting of an anti-antibody coupled to an enzyme such as HRP or AP. The anti-antibody would bind an epitope on the second antibody.

This invention is not limited to sandwich immunoassays, It is applicable to a wide variety of assay formats, for example, nucleic acid hybridization assays for both RNA and DNA, To further illustrate the invention, examples of single and mufti-enzyme ADEAS', conjugates, receptors, and receptor types are presented in Table 1 below.

'8 . ~ ~ ;
o ~, °.' v ~ mw o ., H ~ .ar ~i ar ,~ ai ~' ','~., w ~ o, ~~'a~ ~ ~ ~ w A n w , ~ .~ . ~;~ .
~~ ~g r,, ~ ~ m 0t , A . ~ ~ a ~ w a d a .~ .~ ~ o ~ o '" .~ p, O w .~ N . a .a ~r ~' ~ m .,,, Q o ~ ~ o ~ ~ w ~r ~m ~ ~p~w ~ ~ai~ ~~4 ~~~ a~a~~"~ ~ a 0.a O
n w N
W
'i No N
o ~ .e ar ~ ~ ~ m as i ~ a .~
~ N f~f In the AP/HRP multi-enzyme ADEAS described above, the conjugate must be dephosphorylated before it will react with HRP~ and in the ~-gal/HRP multi-enzyme ADEAS, the conjugate must be deglycosylated before it will react with IiRP .
It should be clear to those skilled in the art that a large number of variations are possible and ail these variations fall Within the scope or the invention.
The following examples are intended to illustrate 10 the invention. Dnless otherwise indicated, 100 girl of all reagents were used. The one exception was that 200 ui of blocking buffer was used.
15 preparation of on;j~~r~toa and para-hydroxyphenyipropionyl biocytin (HPPB) was prepared by mixing a solution of p-hydroxyphenyl-propionic acid-N-hydroxysuccinimide ester (50 mg [0,2 20 mMoi]/2 ml dimethyl sulfoxide) with biocytin (70.75 mg [0.2 mMol]/2 ml 0.1 M NaHCpg) overnight at room temperature (RT). Hiotin-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 HPPB and HT were used as is. The calculated concentrations were 26 mg/ml for HPPB and 55 mg/ml for 8T.
Polystyrene EIA strips (NUNC) were coated with polyclonal anti-Herpes Simplex virus (HSV) antibody (Dako, Carpenteria, CA) in 0.1 M carbonate buffer pH 9,6 overnight at 4°C, and then blocked with 2% bovine serum albumin (BSA) in carbonate buffer and then washed with 10 mM phosphate buffered saline, 0.05$ Tween ZO, pH 7.4 (PEST). A dilution of HSV antigen in 1% BSA, 10 mM

phosphate buffered saline, 0.054 Tween 20 pH 7.9 (BSA-PBST), or buffer without antigen, was incubated for 1 hour at 37°C. The dilution wa3 sufficient to obtain the optical densities in the range reported in Table 1. It was washed with PBST. The analyte dependent enzyme activation system consisted of HRP coupled to anti-HSv (HRP ADEAS) which was purchased from Dako. The HRP
ADEAS was added and incubated for 30 min, at RT and was washed with P88T. Various concentrations of HPPB or HT
as set forth in Table 1 below, were added in 50 mM tria-HC1, 0.014 HZOz, pH 8.0, for 15 min. at RT. After washing with PEST, streptavidin-HRP was added and incubated for is min, at RT to react with deposited biotins. The plate wad then washed with PEST. An HRP
substrate, o-phenylenediamine (OPD), was added, incubated for 30 min. at RT, and stopped With 4 N HZS04, Optical densities at 990 nm were recorded on a mierotiter plate reader.
Results are presented in Table 2. Column 1 present: the various concentrations in ul/ml of HppB or 8T. Columns 2 and 3 present the optical densities recorded as a function of HPPB concentration. Columns 9 and 5 present the results obtained using HT, HPpB and 8T were converted to activated forms by HRP Ab~AS, Catalyzed reporter deposition was achieved without immobilizing a receptor.
In choosing the optimal concentration, one must look at both the magnitude of signal amplification as well as the signal to noise ratio. With this in mind, the optimal concentration of HPPB was 20 ui/mi (approximately 0.5 mg/ml), and that of BT, was about 0.3 ~1/ml (approximately 16 ~g/ml).

Conc. HPpB

or BT HPPB CONJUGATE BT CONJUGATE

(~l/ml) HSV Buffer HSV Buffer (w/o AQ) * ~w/o Aq) 0 0.079 0.031 0.079 0.031 20 1.155 0.181 0.700 0.165 10 0.904 0.140 --- ---5 0.499 0.120 2.060 0.430 2.5 0.177 o.os3 _-_ ___ ' 1.25 0.113 0.062 2.230 0.502 0.525 0.103 0.04$ --- ---0.313 --- --- 1.880 0.169 0.078 "- --- 0.263 0.051 0.020 '-- --- 0.090 0.040 * w/o Aq a without antigen A ili f ti ca mQ
.
on cs D~teetnr Sicrna~l T,n,, Anti-HSV coated EIA Strips were prepared as described in Example 1. A 1:100 dilution of HSV antigen was prepared and serially four-fold diluted. These dilutions of HSV were incubated for 2 hours at 37C with the anti-HSV Coated EIA strips. Excess reagent was washed off th PBST. The AREAS was the same as that wi described in Example 1 above, It was added tv the anti-HSV coated A strips containing the anti-HSV-HSV
EI

complex and ncubated for 30 min, at RT and then washed i with PEST. 0 ul/ml of HPPe conjugate as determined 2 in Example 1 was added in 50 mM tris-HC1, 0.01 H202, pH

$.0, and was incubated for 15 min. at RT and then washed with FHBT. DepO~ited biotinn were reacted with streptavidin-HRP (SA-HRP) for 15 min. at RT. It was Washed with PEST. The substrate, OPD, was added and incubated 30 min, at RT, stopped with 4 N HZS04, and the absorbance at 490 nm was recorded on a microtiter plate reader.
Non-amplified as8ays were run in which (a) no HPPB
and no SA-HRP were used; (b) HPPB was used without SA-HRP; (c) SA-HRP was used without HPPB.
Besulta The results shown in Figure 1 dsmonstrate that (e) Catalyzed deposition of reporter was obtained and (b) both the conjugate and SA-HRP were needed for detection because the conjugate contained an unlabeled member of a apecifio binding pair.
Results for the non-amplified assay (no HPpB, no SA-HRP) were plotted. The results for the other assays were not plotted becauee the additional plots would overlap with the non-amplified results already plotted.
o, i na Catalyrzed ~~porter b ne° ii, r-Etfeet Of on j,~rat - _on pn+~ rwt ~ nn Polystyrene EIA strips (NUNC) were coated with rabbit anti-HIV p24 antibodies in 0.1 M carbonate buffer, pH 9.6, overnight at 4°C, and~then blocked with 2% BSA in carbonate buffer followed by washing with PEST. HIV antigen was incubated for 2 hours at 37°C
(concentrations are indicated in Figure 2). The plate was then washed with PEST. A rabbit anti-H=V p24-HRP
analyte dependent enzyme activation system was then incubated for 2 hours at 37°C, and wt~shed with PBST.
various concentrations of BT conjugate, (0.2, 0.4, and 0.8 ul/ml) in 0.1 M borate buffer, 0.01 HZO2, pH 8.5, were incubated for 15 min, at RT followed by wa$hing with FBST. Then atreptavidin-HRP was incubated for 15 min. at RT.
As a comparison, a biotinylated anti-HIV p24 antibody was used, and detected with streptavidin-HRP.
OPD was added and incubated for 30 minutes, stopped with 4 N H2S0~, and optical densities at 490 nm were recorded on a microtiter plate reader.
The results are shown in Figure 2 where Amp 1, Amp 2, and Amp 3 refer to BT at concentrations of 0.2, 0.4, and 0.8 ul/ml respectively. Different levels of amplification ware achieved u3ing catalyzed reporter deposition depending on the concentration of conjugate.
Figure 2 also presents results for a non-amplified assay using a biotinylated antibody/SA-HRP detection system (biotin) and a non-amplified assay wherein anti-HIV p24 detector was directly labeled with HRP. The results obtained using the anti-HIV p24-HRP detector were inferior compared to the significant increase in detector signal obtained using catalyzed reporter deposition.
Depending upon the concentration of conjugate, signals as good, and greater, as those obtained with the biotinylated antibody were obtained using the catalyzed reporter deposition method of the instant invention.
Beat results were obtained using conjugate concentration near the optimal amount as was determined i.n Example 1.
Preparation of biotin-tyramine: a solution of biotin-N-hydroxysuccinimide, 170 mg (0.5 mMOles), and tyramine (recrystallized from water), 68.5 mg (0.5 mMoles), in 25 ml dimethylformamide was treated with 10 ml of 1 M triethylammonium bicarbonate, pH 7.5, and then heated at 50°C for 3 hours.
Isolation: the solution was concentrated to dryness on a rotary evaporator, and the residue was recrystailized from water, with a yield of 72$.
Characterization: the melting point was determined to b~ 192--193°C .
A~liiicati_t~n of Detwntnr Caanal Tr, ~ wt""ee Polystyrene EIA stripe (NUNC) were coated with goat anti-mouse IgG (Fe fragment specific) antibody (ICN) in 0.1 M carbonate buffer pH 9.6, overnight at RT. They were then blocked with 2% 8SA in carbonate buffer and washed with PEST. Dilutions of mouse IgG in BSA-PEST
were incubated in the wells for 1 hour at 37°C followed by washing with pBST. Concentrations are set forth in Figure 3. Goat anti-mouse IgG-HRP (HRp AREAS) and goat anti-mouse IQG-Alkaline Phoaphatase (AP AREAS) (BOehringer Mannheim) were diluted as recommended by the manufacturer and incubated for 1 hour at 37°C. Assays were run with and without catalyzed reporter deposition.
The iorp AREAS waa not used to catalyze reporter deposition in this experiment.
For catalyzed reporter deposition using the HRP
AREAS, a 1 mg/ml stock solution of biotin tyramine (as described in Example 9) in dimethyl sulfoxide was prepared, and then added to a 0.1 M borate buffer, pH 8.5, 0.01% H202, at 10 ~tl/tnl (10 ~g/ml biotin tyramine) and incubated for 15 min. at RT. The plate was then washed with PBST. Streptavidin-HRP (for HRP-Amp HRP), ar streptavidin-Alkaline Phosphatase (for I~RP-Amp Aik Phos) were incubated for 15 min. at RT and the plate was washed with P$ST. Spectrophotometric detection was achieved after the addition of OPD (for HRP), or p-nitrophenyl phosphate (for AP) for 15 min, at RT. Reactions were stopped by the addition of 4 N H2Sp4 (HRP/OPD), or 1 N NaOH (Alk Phoe/pNPP). Optical densities, at 490 nm for HRP/OPD and 405 nm for Aik Phos/pNBP, were recorded on a microtiter plate reader.
gesulte The results are shown in Figure 3. As is apparent, one can achieve eiqnal amplification with a concomitant lower detection limit by allowing the HRp AREAS to catalyze deposition of an activated BT conjugate followed by detection With streptavidin coupled to HRp or AP. This example shows that if the reporter is an enzyme, it can be the name as, or different from, the enzyme used in the AREAS.
Ths Du Pont HIV p29 Antigen ELISA (catalog number NEK 060) 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 ug/ml, in 0.1 M borate, 0.01% Hz02 pH 8.5 buffer (as in Example 5). Following washing with PBST, SA-HRP at 1/16 the concentration was incubated for 15 min. at RT.
Finally, OPD was added as per kit directions. Excapt for extending the standard concentrations down to 0.39 pg/ml, no other changes wire made.

$gg~
the results are shown in fiigure 4. This experiment demonstrated that one can amplify the signal generated by a biotinylated antibody/SA-HRP system w ing catalyzed reporter deposition. Because the concentration of SA-HRP for both incubations was much less than that for the non-amplified assay, it was clear that the increased signal was attributable to reporter deposition and not to a double SA-HRP incubation.
Aepnrt~~o~ i_t i en on Memhran~
Nitrocellulose (Schleicher & Schuell, BA 85~ was spotted with HSV antigen, and then blocked with 1% HSA, 1% non-fat dry milk, in P8S buffer, overnight. The membranes were incubated for 1 hour at RT with the analyte dependent enzyme activation system described in Example Z above. The membranes were then incubated with biotin tyramine (from Example 1) at 2 ~tl/10 ml 50 mM
tris-HC1, 0.019 HZ02, pH 8.0 buffer for 15 min. at RT, which was followed by incubation with atreptavidin-alkaline phosphatase for 15 min. at RT. Controls were run where biotin tyramine was incubated without streptavidin-alkaline phosphatase, and streptavidin-alkaline phosphatase was incubated without biotin-tyramine. Visualization of deposited alkaline phosphatase was facilitated by the addition of eCIB/NHT
(Kirkegaard & perry). BCIP is 5-bromo-4-chloro-indoxyl phosphate and NBT is 2,2'-di-(p-nitrophenyl)-5,5'-Biphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene)-ditetrazolium chloride. visualization of the bound anti-HSV-HRP conjugate was facilitated by the addition of diaminobenzidine (DAB).
* trade mark Addition of DA8 produced observable brown spots where HSV antigen was spotted on the nitrocellulose membrane. Addition of BCIP/NBT produced observable blue spots where HSV antigen was Spotted when biotin tyramine and etreptavidin-alkaline phosphatase were incubated with the membrane. This showed that alkaline phosphatase was deposited due t0 HRP activation of the biotin tyramine conjugate, followed by streptavidin-alkaline phosphatase detection.
S
~~il L b D
Dsaosition e~ Hsta-Gala _ .n~ad~4~ ~,x Horserad~,~,o Peroxidase, and Detpoi~ i on b~,r glLOrea~pnre Polystyrene EIA strips (NUNC) were Coated with goat anti-mouse IqG (Fc fragment specific) antibody (ICN) in 0.1 M carbonate buffer, pH 9.$, overnight at RT. They were then blocked with 2~ BSA in carbonate buffer and washed with PEST. Concentrations of mouse IgG in HSA-PBST, as set forth in Figure 5, were incubated for 1 hour at 37°C followed by washing with PBST. Goat anti-mouse IgG-HRP (AREAS) purchased from Boehringer Mannheim Was diluted as recommended by the manufacturer and incubated for 1 hour at 37°C. The plate was then washed with PBST, A 1 mg/ml stock solution of BT conjugate (as de8cribed in Example 4) in dimethyl sulfoxide was prepared, and then added to a 0.1 M borate, 0.01% HZO2, pH 8.5 buffer at 10 ul/ml (10 ~tg/ml biotin tyramine) .
The mixture was added to the plate and incubated for 15 min, at RT, and then washed with PBST. Streptavidin-beta galactosidase (Bethesda Research Labs) was addCd and incubated for 15 min. at R1", The assay was also run without catalyzed reporter deposition, i.e., without adding BT. Colorimetric detection of the non-amplified assay was achieved alter incubation with OPD (for HRp), for 15 min. at RT. Fluorescent detection of the amplified assay was achieved after the addition of 4-methylumbelliferyl beta-D-galactoside (MUG) (for HRP-beta Gal). Optical densities at 490 nm were recorded for HRP/OPD on a microtiter plate reader. Fluorescence for HRP-beta Gal/MUG was recorded on a fluorescence microtiter plate reader (Dynatech Laboratories).
Re s Lip, The results are shown in Figure 5. The fluorescent signal was due to the quantitative deposition of biotintyramine by the HRP ADEAS followed by incubation with atreptavidin beta-galactoaidaae.
~7,~,~~ Get ~ t~ri Of a ~~~ml~rswa Aq,gy Fiuorescein-tyramine (FT) was prepared as follows:
Solutions of 46.6 mg of 5-(and 6)-carboxyfluorescein succinimidyl ester in 0.3 ml dimethyl sulfoxide and 14.6 mg tyramine in 0.3 ml dimethyl sulfoxide were prepared.
Conjugation was achieved by mixing 0.25 mi of each solut~.on overnight at RT. The solution was used as i9.
Three nitrocellulose (Schieicher & Schuell, BA 83) strips were spotted with 1 ~1 of mouse IgG at 10 ug/ml, and serial two fold dilutons in PBS. The membranes wire blocked with 5% non-fat dry milk in pBST for 30 min, and then washed three tima$ in 88ST. A goat anti-mouse IgG-HRP conjugate (Boehringer Mannehim) diluted 1/2000 in 1%
BSA-PBST was incubated for 30 min. at RT, and the membrane were washed three times with PEST. The third membrane was incubated with FT at 20 ~tg/ml in 0.1 M
borate, 0.01% H202, pH 8.5 buffer for 15 min. at RT, and washed three times in PBST. Then, the second and third membranes were incubated with an anti-fiuorescein antibody (Chemicon) which was conjugated to HRP (by the SMCC method of Ishikawa, E., et al., J. Immunoassay, q, 209-327, 1983) diluted in 1% 8SA-PEST for 15 min at RT, and washed three times in PEST. Visualization of all three strips was facilitated by the addition of 5 diaminobenzidin~ for 5 mi.n.
Three spots could be seen on the first two strips indicating a detection limit or 2.5 ~,g/ml, and that the 10 anti-fluorescein-HRP conjugate did not contribute to additional signal. Six spots could been seen on the third strip indicating a detection limit of 313 ng/ml, The use of the catalyzed reporter deposition amplification method of the invention improved the 15 detection limit of the ae~ay eight fold over that of the non-amplifi~d as:ay, EX~yE Z O
20 $ ~ _ _roam,.i~5r11,-6-(fix-(4'-aso-2~'-j'-arboxVethy7 ohe_n_V1) hexarida ~,, HEE-6-NHS arid A1 llr~ 1 ~ n-phoanha a4 _ (,~
I
The following reaction scheme is illustrated in Figure 6: Ethyl 2-(4'-hydroxyphenylazo)benzoate (HEE), 25 is pr~pared from 2-(4'-hydroxyphenylazo)-benzoic acid (SBA, (1)), 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 Qeneral procedure reported by Caatells,no$ et al., 30 Tetrahedron, pages 1691-1596, Vol. 37, (1991), to produce t-butyl-6-(phenoxy-(4'-azo-2"-carboxyethyiphenyl)hexanoate (HEE-5-t-Bu, (~l). The tent-butyl ester is hydrolyzed by treatment with txifluoroacetic acid using the general procedure reported by Bryan et al., Journal of the American Chemical Society, pages 2353-2355, Voi. 99, (1977), to produce 6-(phenoxy-(4'-azo-2"-carboxyethylphenyl)-hexanoic acid (HEE-6-H). (N-euccinimidyl)-6-(phenoxy-(4'-azo-2~-carboxyethylphenyl)hexanoate (FiEE-6-NHS, (~)) ie prepared from HEE-6-H, N-hydroxyauccinimide and dicyclohexylcarbodiimide in TFiF using the general procedure reported by Bryan et al., Makromolecular Chemie, pages 2375-2382, Voi. 186, (1985). The NHS
ester (~,) is dissolved in a minimum volume of DMSO and added to a buffered aqueous solution of alkaline phosphatase (e. q, calf intestine) using the general procedure reported by 0'Suilivan et al., Methods in Enzymology, pages 147-166, Vol. 73, (1981) to give the con jugate (HEE-6-AF, (~,) ) .
EXB~LE~
- ~~-~arboxyethyoeheTn~p ? hexanoate th~;~-6-NHS (5)1, 2~ 8,~,_ Tyrramine The NHS ester (5) (1 mmol) described in Example 10 above, and tyramine (1 mmoi) are dissolved in DMF (5 mL) and stirred at RT for 48 hra. The solution is evaporated to dryness in vacuo. The residue is suspended in H20 (pH 8.0, 50 mi,) and porcine liver esterase (100 mg) is added. The pH of the solution is maintained by adding 0.1 N NaOFi a9 required. The solution is evaporated to dryness after 24 hours. The resulting H-T conjugate is isolated by chromatography (silica gel, chioroform/methanol).

Amnlifi~a .idr of D .tPntnr Signal T~ a Mous~e~~~ na~s~v Usinc Porcine Liver Fatera~. (pLE) atrl~r~
A~norter-En iym~ D~~,os~,r Microtiter plate strips (Nuns) are coated with a mixture of goat anti-mouse IgG (Fc fragment specific) antibody (ICN) and streptavidin (Scripps Laboratories) in 0.1M carbonate buffer p8 9.6 overnight at RT. They are then blocked with 2~ HSA in P8S and mashed with PeST. Dilutiona of mouse iqG (0-100 ng/mL) in sSA-PEST
are incubated in the wells for 1 hr, at 37°C followed by washing with PeST. A preparation of goat anti-mouse IqG-PLE (PLE AREAS) (0.75 mg/mL) is prepared by the general method described by Hashida et al., Journal of Applied Biochemistry vol. 6, pages 56-63, 1984 and diluted 1:100-1:2000 with phosphate buffer (0.1 M, pH
8.0, 0.2~ eSA) and is incubated for.i hr. at 37°C and washed with FBST. HEE-6-AP (1 mg/mL) (prepared as described in Example 10) is added to the micrQtiter plate well and incubated for at least 15 min. at 37°C.
The plate is then washed with PsST. Spectrophotometric detection i9 achieved after the addition of p-nitrophenyl phosphate. Reactions are stopped by the addition of 1 N NaOH. Optical densities at 405 nm are recorded on the miorotiter plate reader. Amplification of detector signal in this example results from catalyzed reporter-enzyme deposition, i.e., PLE
catalyzes deposition of HEE-6-AP where the receptor, streptavidin, has been immobilized on the microtiter plate surface.

Demonstration of the Enzyme Modulated Binding of a Blocked Binder A suspension of porcine liver esterase was added (10 uL, 2860 U/mL, Cat. No. E3128, Sigma Chemical, St. Louis, MO) to a solution of ethyl 2-(4'-hydroxy-phenylazo) benzoate (HEE) (0.25 mM) and streptavidin (0.2 mg/mL) in phosphate buffer (0.1 M, pH 8.0, 2 mL). A second solution identical to the first was prepared which contained no streptavidin. The absorbance of the two solutions was measured at 500 nm as a function of time. Figure 7 shows that the absorbance of the solution which contained no streptavidin decreased over time indicating hydrolysis of HEE to 2-(4'-hydroxy-phenylazo)benzoic acid (HABA). The absorbance of the solution which contained streptavidin increased over time indicating the formation of the HABAatreptavidin complex, which is known to have a strong absorbance at 500 nm.
This application is a divisional application of Canadian Patent Application No. 2013214, filed March 28, 1990.

Claims (2)

CLAIM

1. A compound of the formula:
wherein R1 through R8 are the same or different and are selected from the group consisting of H, OH, OCH3, straight chain or branched alkyl groups having 1-4 carbon atoms, F, CI and Br;
Z1 and Z2 are independently phenyl or napthyl;
n is 1-19;
X is N, O or S;
R9 is selected from the group consisting of glycosides and straight chain or branched alkyl groups having 1-4 carbon atoms;
L is (NH(CH2)5CO)r where s is 1-5 and r is 0-5; and R is a reporter selected from the group consisting of radioisotopes, fluorogenic materials, light scattering materials, chemiluminescent materials, electrochemical materials, magnetic materials and enzymes provided that said enzymes do not react with OR9.

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