CA1230289A

CA1230289A - Enhanced sensitivity in enzymatic poly reactant channeling binding assay

Info

Publication number: CA1230289A
Application number: CA000449458A
Authority: CA
Inventors: Robert Dinello; Ian Gibbons; Edwin F. Ullman
Original assignee: Syva Co
Current assignee: Syva Co
Priority date: 1983-03-14
Filing date: 1984-03-13
Publication date: 1987-12-15
Also published as: EP0122059A2; JPH0643997B2; JPS59178362A; US4687735A; AU2556184A; DE3476834D1; EP0122059A3; EP0122059B1; ATE40926T1; AU575315B2

Abstract

ABSTRACT OF THE DISCLOSURE

Improved sensitive immunoassays are provided involving channeling involving one, usually two enzymes, where the enzymes are related by the product of one enzyme being the substrate of the other enzyme. A
dispersed aggregation is formed in the assay medium of (1) the analyte, (2) one of the enzymes bound to a second binding member ("SBM") (enzyme - SBM conjugate) which conjugate is non-covalently bound to a first binding member ("FBM"), and (3) a multiplied amount of the other enzyme bound in the complex. The large amount of enzyme or reactant in the complex is achieved by having a multiplicity of linkages binding the enzyme or reactant directly or indirectly to FBMs. The enzyme channeling provides for a detectable signal which can be related to the amount of analyte in the medium.

Description

ENHANCED SENSITIVITY IN ENZYMATIC POLY-REACTA~T
SHEA SLING BINrIN~ ASSAY

An important component in the diagnosis and treatment of disease is the ability to determine the nature of the pathogen, the strain of the pathogen, the affected cells, the presence of abnormal proteins, and 20 the live, as well as the anility to determine the level of bound or unbound drug in a physiological fluid, during the treatment of a disease. Determination of drugs is also of interest in cases of drugs of abuse, the ingestion of toxins, and the like. Also, in many instances, one is concerned with the presence of specific receptors, particularly antibodies, in relation to disease diagnosis or determining the health of the individual. Assays also find use in flood typing, ALA
typing and the determination of other phenotypic products.
In many situations, one is concerned with detecting an extremely small amount of material in a complex composition containing a variety of other materials, which may be of similar or different structure. In order to detect the presence of a specific material, antibodies or other receptors have been employed which bind 0633K ~1~30-FF

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specifically to a determinant site. this complex formation between a loosened an receptor has fount extensive use for a qualitative or quantitative determination of the presence of a defined determinant 5 site in a sample.
More recently, there is increasing interest in toe ability to detect pathogens, mutations an genetic related diseases by polynucleoti~e hybridization. wince these will frequently be concern in detecting unique 10 sequences which are only a minute part of the total DNA
and/or RNA, sensitive methods will ye necessary to ensure accuracy of results.
As the need has grown to detect an ever increasing variety of analyzes, there has been increased efforts to 15 develop new techniques with enhanced sensitivity, ease of operation, opportunity for automation, reproducibility arid low incidence of error.
In the prior art US. Patent No. 4,233,402 describes a homogeneous channeling assay. US. Patent No. 4,299,916 20 describes a heterogeneous channeling assay. Stuart and Porter, Exp. Cell. Rest (1978) 113:219-222, describe moo-atonal antibodies to DNA-RNA hybrid complexes.
According to the invention novel assays are provided which involve enzymatic production of a product which 25 interacts with a second component of a signal producing system. The enzyme is conjugated to a member of a specific binding pair. The second component is polymerized under conditions where the enzyme is included in the polymer only as the conjugate bound to its reciprocal binding member.
30 The high mole ratio second component to enzyme in the polymer provides for an enhanced signal for each binding event. The observed signal is related to the amount of analyze in a sample by comparison with standards.

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In accordance with the subject invention, a novel, sensitive assay technique employing channeling is provided for the determination of annihilate, particularly 5 at low analyze concentrations. The method involves at least one enzyme, normally two enzymes, where the two enzymes are related by the product of one being the substrate of the other.
The subject invention is predicate on the 10 employment of a signal producing system involving at least three members. The three members include an enzyme, its substrate, and a material which interacts with a product of the enzyme. The signal producing system component which interacts with the product of the 15 enzyme can itself be a different enzyme which interacts with the product of the first enzyme in a manner which provides a change in a measurable signal or may be a reactant, which reacts with the product of the first enzyme, to result in a change in a measurable signal.
The subject invention provides a number of advantages in that the molecules involved in the signal producing system are, for the most part, relatively small molecules, as compared to most particles. Thus, the molecules enjoy a rapid rate of diffusion in the assay 25 medium. Employing en. enzyme, provides for a large amplification, which in the present invention will be associate with a winding event. By polymerizing a member of the signal producing system, a large amount of such member will be brought into close proximity. By 30 further providing that the first enzyme will be incorporated in the polymer by virtue o-f a binding event between specific Bering members, one brings the first enzyme into close proximity to a high multiple and localize concentration of the subsequent component Of 35 the signal producing system, which interacts with the 0633K ~l930-FF

~3(~12~9 product of the first enzyme, or, if it is an enzyme, may produce a product which is acted upon by the first enzyme. In effect, one can enjoy the high concentration of a label or reactant which is achieved by employing 5 particles, while avoiding the problem of the slow difrcusion of the particles. In this manner, the sensitivity of the assay is greatly enhanced and a large signal can result from a single binding event.
One may divide the signal producing system into two 10 major categories: enzyme channeling; and enzyme product reaction. The preferred method would be the enzyme channeling system.
The winding event involves members of a specific binding pair which will be ligands and receptors, which 15 will be arbitrarily referred to without indicating which members intended as a first binding member ("IBM") and a second binding member ("SUM"). eye first enzyme will be conjugated to an SUM to provide an enzyme-SBM conjugate.
The enzyme of the enzyme-SBM conjugate will be in limited.
20 amount based on the limited number of available binding sites of the FEM.
The other enzyme will be part of a system that involves polymerization of the other enzyme used, that includes the enzyme-SBM conjugate in the polymer to an 25 extent related to the extent of complex action between the enzyme-SBM conjugate and IBM. In effect, a substantial proportion of the total other enzyme in the assay medium becomes involved in colloidal copolymeric aggregates.
By the appropriate choice of enzymes, one can provide for a high localized concentration of substrate of the second enzyme in the catalytic sequence or one can ensure that a substantial fraction of the product produced by the first enzyme in the catalytic sequence is 35 consumed by the second enzyme in the aggregate. In this 0633K ~1~30-FF

way, great sense tivi try is produced by having the oboe rued signal related to the amount of analyze present in the assay medium greatly enhancec7 as compared to the background signal resulting from production of product 5 unrelated to the amount of analyze in the medium.
Optionally, one may use a scavenger in the bulk solution to further reduce background signal. Furthermore, since the binding of SUM to IBM occur us in a homogeneous solution prior to formation of particles, binding occurs 10 more rapidly than when the binding rate is limited by diffusion to a surface, such as a wall or partial.
The assay can measure both first and second binding members. Where SUM is the analyze, one can have a predetermined amount of IBM included in the reagents 15 employed, where the amount of SUM reduces the amount of enzyme-SBM in the copolymeric aggregate. Usually FBI`5 will be the analyze, so that the amount of enzyme-SBM in the cop lyre n c egg no go lo w i 11 be do no e if y no 1 a lo d to the amount of analyze in the assay medium. Thus, where IBM
20 is the analyze, the observed change in signal will usually increase with increasing amounts of IBM.
A wide variety of interactions can be provided for by varying the nature of the enzymes and their substrates. One can provide for activation my the 25 production of a co-effector, deactivation by p~ocluction of an inhibitor, or deactivation, where the two enzymes compete for the same substrate, but only the enzyme of the enzyme-SBM eon gigawatt results in a product producing a de lea table signal .
The enzyme channeling reaction is predicate' upon two enzymes communicating, so that the product of a first enzyme is the substrate of a second enzyme. The second enzyme produces a product which results in a change in a detectable signal as comparec7 to a standard. By virtue 35 of having the two enzymes of the channeling system in 0633K ~1930-FF

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close proximity, an enhanced rate will be observed for formation of the product that provides for the change in the observed signal Furthermore, by having a high concentration of one of the enzymes in proximity to the 5 other enzyme, the production of the signal producing product in the aggregation will be much greater than the production of such product in the bulk solution.
In many instances, enzymes will have substantially different turnover rates, so that it is desirable to have 10 a plurality of one enzyme in relation to the otter enzyme. In other situations, ore wishes to maximize the rate of turnover of an enzyme by having a high localized concentration of the substrate. Another consideration is whether one can have one enzyme substantially surrounded 15 by the other enzyme, so that one minimizes the amount of the intermediate substrate which diffuses into the solution. Furthermore, by substantially sweeping the assay medium, so that a substantial proportion, preferably a major proportion, of one of the enzymes is 20 involved in the aggregations, the background can be substantially reduced. Also, one may further reduce the background by employing a scavenger in the bulk medium.
One of the enzymes will be bound to a SUM, normally covalently bound, so that for each binding site to which 25 the SUM binds, one or a few enzymes will become nonequivalently bound to the IBM. I've other enzyme will be part of a system which provides for the copolymerization of the other enzyme with the complex of the IBM and enzyme-SBM conjugate. In effect, one 30 provides a linking system which provides for hock polymerization or copolymerization of one of the enzymes with intermittent incorporation of the complex in the polymeric aggregation. The IBM, will be polyvalent, that it have a plurality of birding sites, or be provided in a 0633K ~1930-FF

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polyvalent form, that is, having a plurality of FBM's joined together.
Various cor.figur2tions and reagents may be employed to provide the desired polymeric aggregation. Once 5 aggregates have begun to form or have been formed, by adding the appropriate substrate and other reagents, the formation or destruction of a product providing a delectable signal can be achieved. The signal may then be determined in relation to standards, and the presence 10 or amount of particular analyze may be determined.
Definitions Analyze - The compound or composition to be measured, which may be a first binding member or second binding member, where the first and second binding 15 members are a specific binding pair. One of the binding members will ye a ligand or polynucleotic,e. The other binding member will be a receptor or polynueleoti~e which is homologous or complementary to the first binding member.
First Binding Members and Second Binding Members ("IBM" and "SUM", respectively) - The first and second binding members are organic compounds which are members of a specific binding pair and are reciprocal to each other. The compounds are ligands and receptors, where 25 ligands are any organic compound for which a receptor is available or can be made, which ligand may have one or a plurality of sites to which the receptor binds. The sites may be referred to as determinant sites or epitopie sites, which define a particular spatial and polar 30 organization to which the reciprocal or homologous receptor binds. Depending upon the nature of the receptor, the receptor may be a macro molecular palpated or a polynucleotide, either DNA or PEA, usually having at least about ten bases. The receptor 35 will bind to a specific polar and spatial organization of 0633K 91~30-FF

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the ligand, having a surface complementary to a number of features of the ligand surface. The polynucleotide receptor will have a sequence of bases where all, or substantially all, of the bases are complementary to the 5 ligand polynucleotide.
For the purposes of the subject invention, the focus of the FBMs and Sums is their ability to specifically bind to one another. Therefore, a variety of receptors are treated as equivalent in having the same binding 10 specificity. For example, antibodies, enzymes, natural receptors, or the like may be useful for binding to the same ligand. In addition, fragments of a receptor, such as a Fob, F(~B')2 or TV will be equivalent to the intact antibody for the purpose of this invention. In 15 addition, the various types of immunoglokulins may be interchangeable in various situations.
Similarly, with large ligands, it may be feasible to use only the portion of the ligand associated with the determinant site or epitome, rather than the entire 20 ligand in some instances.
Modified Second Binding Member ("modified SUB
The modified SUB will have covalently bonded to the SUM a label which is part of a linking system which involves the inclusion of the modified SUM in the polymeric 25 aggregate. The modification may be as a result of covalent or non-covalent binding to the SUM to provide for the multiplicity of the member of the signal producing system in the copolymer aggregate.
Signal Producing System - The signal producing 30 system involves at least three members, the essential members being an enzyme, a substrate for the enzyme which produces a product, and an interacting member which interacts with the enzyme product to provide for a change in a measurable signal in the assay medium. The 35 interacting member may ye a second enzyme which employs the product of the first enzyme as a substrate or may be a chemical compound which reacts with the product of the enzyme to produce a product which provides for a change in a detectable signal of the three essential members 5 of the signal producing system, two will be involved in the polymeric aggregate with one of the two at a high molar multiple to the other. For the most part, the two members in the polymeric aggregate will be enzymes, involved in an enzyme channeling reaction, although one 10 of the enzymes may be substituted with a compound which can react with the product of the other enzyme.
Linking System - The linking system provides a method for polymerizing a member of the signal producing system, which is either an enzyme or a compound which 15 reacts with a product of an enzyme to produce a charge in a detectable signal. The polymeric aggregate will involve the polymerized member of the signal producing system and the enzyme bound to the SUM through the intermediacy of the IBM, providing for a high multiple of 20 the polymerized signal producing system member to the enzyme bound to the SUM. In order for enzyme-SBM to be involved in the polymeric aggregate, it will be necessary that the IBM be polyvalent or provided in polyvalent form. For a hasten acting as the IBM, the hasten could 25 be provided as a plurality of hastens joined together or the hasten joined to another molecule as a conjugate which molecule serves as the linking member to the copolymeric aggregate.
Ligand Analog - A modified ligand which can compete 30 with the analogous ligand for a receptor, the modification providing Myers to join a plurality of ligand analogs in a single entity or to provide a means for binding to a label. The ligand analog will differ from the ligand by more than replacement of a hydrogen 0633K 91~30-FF

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with a bond which links the ligand analog to a hub nucleus or label.
Poly(ligand analog) - A plurality of ligand analogs joined together covalently, normally to a hub nucleus.
5 The hub nucleus is a polyfunctional material, normally polymeric, usually having a plurality of functional groups e.g. hydroxyl, amino, Marquette, ethylenic, etc. as sites for linking. The posy ligand analog well may be water soluble and will normally be at least about 30,000 10 molecular weight and may be lo million or more molecular weight. Illustrative hub nuclei include polysaccharides, polypeptides (including proteins), nucleic acids, and the like.
Receptor - Any macro molecular organic compound or 15 composition capable of recognizing a particular spatial and polar organization of a molecule e.g. epitopic or determinant site. The receptor will normally be at least as large as, usually much larger than, the specific organization to which the receptor binds, e.g.
20 macro molecular. Illustrative receptors include naturally occurring receptors, antibodies, enzymes, Fob fragments, pectins, and the like. For any specific ligand, the receptor will be referred to as antiligand. The receptor-antiligand- and its reciprocal ligand form a 25 specific binding pair.
Aggregate - A colloidal particle composed of a polymer of a member of the signal producing system, in which such member is incorporated independent of the occurrence of FBM-SBM binding and an enzyme is 30 incorporated which is dependent upon such kinking, resulting in a high multiple of the signal producing system member of the polymer to the enzyme.
Label - A compound which is either directly or indirectly involved with the production of a detectable 35 signal and is bonded, either directly or indirectly, to 0633K 1~30-FF

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ligand, ligand analog or receptor. In the subject invention, the labels will ye components involved in the signal producing system, e.g. enzymes and reactants, and compounds employed in the linking system for forming the 5 polymeric aggregation. The labels will therefore be distinguished between signal producing member labels and the linking labels.
Intermediate Substrate - The intermediate substrate is the product of one enzyme which acts as the substrate 10 of the other enzyme.
Final Product - The product of the final reaction of the signal producing systems, which will be distinguishable from the substrate of the first enzyme, so that the formation of the Final Product will result in 15 a change in a detectable signal in the assay. Usually, the final product will provide for a signal involving electromagnetic radiation, particularly involving ultraviolet or visible light, as a result of absorption or emission, as a result of fluorescence, 20 chemiluminescence or phosphorescence.
Final Reactant - A compound capable of being polymerized and reacting with an enzyme product to result in a product which provides for a change in a detectable signal.
ASSAY
The subject assay is carried out at least in part in an aqueous zone at a moderate phi generally close to optimum assay sensitivity. The aqueous assay zone in which the complexes are formed for the determination of 30 analyze is prepared by employing an appropriate aqueous solution, normally buffered, the unknown sample which may have been subject to prior treatment, enzyme-SBM
conjugate, the components of the linking system for the other enzyme and the remaining members necessary for the 35 change in observable signal in the assay medium.

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~12-(Hereafter, when the two enzymes of the channeling system are referred to together, the two enzymes will be referred to as "enzyme+" and "enzyme ," so as to avoid any indication of which enzyme is the first enzyme 5 in the series of enzyme reactions.) For the most part, the analyzes of interest will be polyepitopic. However, it is feasible to use the subject system for monoepitopic ligands, namely hastens. This can be achieved by preparing a poly(ligand analog), where 10 the haptenic ligand and poly(ligand analog) will compete for available receptors, so that the degree of channeling which occurs will be dependent upon the amount of hasten present in the medium. Or, one could have a labeled hasten where the label provides for polyvalency.
In carrying out the assay an aqueous medium will normally be employed. Other polar solvents may also he employed, usually oxygenated organic solvents of from 1-6, more usually from 1-4 carbon atoms, including alcohols, ethers, asides and the like. Usually these 20 cosolvents will be present in less than about 40 weight percent, more usually in less than about 20 weight percent. With polynucleotides, salts may be added, e.g.
Nail, generally at concentrations in the range of 0.1 to 1.5 M.
The pi for the medium will usually be in the range of about 4-11, more usually in the range of about 5-lQ, and preferably in the range of about 6.5-9.5. The pi is chosen so as to maintain a significant level of specific binding by the receptor while optimizing signal producing 30 proficiency. In some instances, a compromise will be made between these two considerations. Various buffers may be used to achieve the desired pi and maintain the pi during the determination. Illustrative buffers include borate, phosphate, carbonate, Trip, barbital and the 35 like. The particular buffer employed is not critical to I

this invention but in individual assays, one buffer may be preferred over another.
Moderate temperatures are normally employed for carrying ox t the assay and usually cons lent temperatures 5 during the period of the assay. The temperatures for the determination will generally range from about 10-60C., more usually f rum about 15-45C.
The concentration of analyze which may be assayed will generally vary from about 10 4 to 10 15 M, more 10 usually from about 10 to 10 14 M. Considerations as to whether the assay is qualitative, semi-quantitative or quantitative, the particular detection technique and the concentration of the analyze of interest will normally determine the concentration of other reagents.
While the concentrations of the various reagents will generally be determined by the concentration range of interest of the analyze, the final concentration of each of the reagents will normally be determined empirically to optimize the sensitivity of the assay over 20 the range of interest. Based on binding so toes of ligands and receptors, the ratio will usually not be greater than about 104, usually not greater than about 102. (I
polynucleotide complementary sequence will be one binding site. ) In referring to binding sites, this will usually, 25 by t no t always, intend, who no the ligand i s pulp topic, that one is concerned with all of the receptors which bind to a ligand. However, where there are two populations of receptors which do not compete for the same epitopic site, the ratios will be considered 30 independently. The concentration of the members of the linking system will be varied widely, being determined empirically to provide for polymerization of the signal producing system member. Therefore, where the polymer is a copolymer, usually the ratio of comonomers will be 35 relatively close to 1.

-~Z3~ 3 As indicated above, the concentration for each of the reagents will for the most part be determined empirically. The amount of each of the components involved in the signal change in the assay, namely 5 substrates and cofactors for the enzymes, and Final Reactants, will be in sufficient amount so as not to be rate determining. Thus, the change in concentration of such components durincJ the assay will not observably affect the rate of formation of final product.
The order of the addition of reagents may be varied, but will usually involve adding the enzyme~SBM conjugate and any other Sums specific for the IBM analyze (or the complementary IBM to an SUM analyze), followed by the remaining members of the linking system and the 15 components necessary for producing the Final Product.
For receptor analyze the labeled ligand may be added initially to the sample followed by the addition of the remaining binding members and signal producing system member linking system members.
after sufficient time for aggregates to form, optionally a portion of the assay mixture may be transferred to a solid support, desirably a bibulous support or fibrous support. To the aggregates, either in solution or on a support, is added the developer, 25 comprising the necessary reagents such as substrates and cofactors, to provide for a change in the observed signal.
In carrying out the assay, a sample containing the analyze will be obtained. The sample may be derived from a wide variety of sources and may be subject to prior 30 treatment. Illustrative sources include physiological fluids, such as blood, serum, urine, lymph, spinal fluid, pus, mucous, etc. Other samples of cellular materials may be tissue cultures, lusts, DNA, PA, membranes, etc. The sample need not be a physiological fluid or 35 cellular but may be involved with monitoring contaminants owe in water, chemical processing, or the like. Depending upon the nature of the sample, as well as the nature of the analyze, the sample may ye subjected to a wide variety of prior treatments which would be conventional 5 as to the particular analyze.
Other than various conventional analyzes in competitive protein binding assays or immunoassay, such as, lower molecular weight organic compounds e.g. drugs and synthetic chemicals, and macromolecules, such as 10 polysaccharides, polypeptides and proteins, oligo~ and polynucleotides may also serve as binding members. The polynucleotides may be derived from a wide variety of 60U fees, including genomic, episomal, plasm id mitochondrial, synthetic, or the like. Where 15 polynucleotides are involved, various prior treatments may be performed which involve cell louses, gradient centrifugation, electrophoresis, etc.
For a listing of exemplary ligands, see the description in US. Patent No. 4,233,402, beginning at column 9, line 20 65, and ending at column 17, lint 20.
Similarly, the ligand analog is described in US.
Patent No. 4,233,402 beginning at column 17, line 21 and ending at column 18, line 12.
The reagents which are employed and can be provided 25 in kits will comprise the enzyme~-SBM conjugate, the linking system for linking the enzyme of the enzyme pair to the polymeric aggregate or for linking the Final reactant, and the various components involved with the production of the detectable signal.
The first reagent to be discussed is the enzyme-SBM
conjugate The enzyme will usually be covalently bonded to the SUM. As already indicated the SUM will be a ligand, a receptor, usually a protein, e.g. antibody, or v~9 a polynucleotide, either polyribonucleotide or polydeoxyribonucleotide, being either the same or different sugar from the analyze. The polynucleotide SUM
will have a sequence complementary to a sequence of the 5 analyze and a linking chain, desirably an extended chain, covalently hounded to such sequence, which will allow for binding of enzymes to the SUM. The binding may be covalent between the enzyme and the SUM linking chain or non-covalent, for example, by employing antibody or one 10 or a few sequences, complementary to a polynucleotide extended sequence covalently bonded to the enzyme or other receptors complementary to ligands bound to the extended chain.
In this way, one or a plurality of enzymes may be 15 bound to the linking chain. Any complementary sequence will usually be at least about 6 bases, often being lo bases or more. The extended sequence will be at least about 15 bases, usually 20 bases or more, and is conveniently a homopolymer, conveniently posy G or posy C.
Depending upon the nature of the SUM and the enzyme, there may be more than one enzyme per SUM, or more than one SUM per enzyme. Factors to be considered are the relative molecular weights, the nature of the enzyme, the effect of having a plurality of one or the other 25 components on the functioning of either of the components, etc. Usually, the ratio of enzyme to SUM may vary from about Lowe or more. Various methods of linking are well known in the art. Any method may be employed which is satisfactory for providing the desired 30 ratio and retaining the desired activity of the SUM and the enzyme.
In choosing an enzyme where two enzymes are employed, one must consider the fact that the enzyme must be a member of a pair, where the substrate of one enzyme 35 is the product of the other enzyme and that the final :~30~

result is to destroy or produce a product which allows for a differential signal in the assay medium. A number of enzyme systems are known which provide for this result and are descried in US. Patent No. 4,233,~02.
The first type of enzymes to be considered is the oxidoreductases. These enzymes under the TUB
classification are Class 1. Of particular interest in this class are the groups of enzymes in 1.1.1 and 1.6, where nicotinamide adenine dinucleotide or its phosphate 10 (NOD and NAP) are involved. These enzymes can be used to produce the reduced form of the consumes AUDI and NAP or vice versa. Specific enzymes include the dehydrogenases, such as alcohol dehydrogenase, glycerol dehydrogenase, lactate dehycrogenase, palate 15 dehydrogenase, glucose-6-phosphate dehydrogenase, mannitol~l-phosphate clehydrogenase, glyceraldehyde-3-phosphate and isocitrate dehydrogenase.
Another group of enzymes in the oxidoreductase class are those that produce or destroy hydrogen peroxide.
20 Among these enzymes are those of group 1.11.1, such as kettles and peroxiclase, amino acid oxidize, glucose oxidize, galactose oxidize, unease, polyphenol oxidize and ascorbate oxidize. Another oxicdoreductase enzyme of interest is doffers.
another group of enzymes of interest is the transferases, Class 2 of TUB classification, particularly subclass 2.7, where phosphate is transferred to an alcohol, Class 2.7.1, e.g. hexokinase.
Another group of enzymes of interest is the 30 hydrolyses which are Class 3 in the TUB
classification. Of particular interest are the glycoside hydrolyses (glycosidase), which are in Class 3.2.1 and phosphatases in Class 3.1.3. Of particular interest are -aimless, cellulose, -glucosidase, 35 amyloglucosidase, `-galactosidase, amyloglucosidase, d ~-glucuronidase, acid phosphates and alkaline phosphates.
Two additional groups of enzymes of interest are the leases, in Class 4 and the isomerases in Class 5, 5 particularly subclasses 5.3 and I which include enzymes such as phosphoglucose isomers, trios phosphate isomers and phosphoglucose mutate.
As illustrative of the manner of action of the various enzymes, the following examples are given. The 10 first examples are concerned with the oxidoreductases, particularly those reducing NOD to NASH. These enzymes are for the most part dehydrogenases, where an hydroxylic group is taken to an ox group. NASH then becomes an intermediate substrate which can be combined with a 15 number of different enzymes to produce a product which may be detected. For example, the second enzyme can be doffers, which can react with a synthetic substrate, such as 2,6-dichlorophenolindophenol, ethylene blue or potassium ferricyanide. The NASH can be employer with a 20 flavoprotein, which includes such enzymes as glucose oxidize, amino acid oxidizes and dihydroorotate dehydrogenase, where the product of the flavoprotein and NASH and oxygen, namely hydrogen peroxide, may then be detected.
Alternatively, one can use an oxidoreductase which produces hydrogen peroxide. Such enzymes include glucose oxidize, cytochrome reeducates, unease, and the like.
These enzymes can be coupled with an enzyme which reacts with hydrogen peroxide, such as peroxides, with the 30 hydrogen peroxide reacting as the intermediate substrate. The hydrogen peroxide, plus the peroxides, plus a luminescent material e.g. luminol, can be employed for producing a chemiluminescent reaction.

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Hydrolyses can be effectively used employing compounds, which require the hydrolytic removal of two substituents in two separate steps.
For example, l-umbelliferyl-~-galactoside-6-5 phosphate must be converted to umbelliferone in order Tibetan a fluorescent signal. By employing alkaline phosphates as the first enzyme, l-umbelliferyl `-galactoside as the intermediate substrate, and `-galactosidase as the second enzyme, 10 one can obtain a detectable signal-fluorescence-which will be dependent upon the proximity of the two labels in a complex. Both enzymes are essential to the formation of umbelliferone which provides the detectable signal.
Alternatively, the hydrolyze may produce a product 15 which may then be used in a subsequent enzymatic reaction. For example, a consume may be functionalized so as to inhibit its activity and the functionality be removable by a hydrolyze enzyme and the free consume then able to interact with the second enzyme to produce a 20 detectable signal.
In addition, isomerases can be used to produce a substrate for a subsequent enzymatic reaction, particularly with saccharides, isomerizing Al doses and coituses by transferring a phosphate from one position to 25 another.
While the enzyme channeling system is by far preferred, it is feasible to have a reactant which can react with the product of the first enzyme to provide for a change in a detectable signal. For example, one could 30 employ derivatives of Ellman's reagent, where the disulfide is linked to a compound which allows for polymerization, e.g. button, which can be polymerized with avid in by employing an enzyme such as cholinesterase, one could release theistic acid, which 35 would react with Ellman's reagent to produce :~3(~2i~1~

carboxynitrophenylthiophenoxide, which would provide an observable signal. Alternatively, one could employ an enzyme which produces hydrogen peroxide and employ as the final reactant, a chelated metal catalyst which destroys 5 the hydrogen peroxide. One can then measure the hydrogen peroxide which is present in the assay medium. Other combinations could include employing TAD and a dehydrogenase, where the resulting AUDI would react with a compound to form or destroy a color, e.g. Mildly 10 blue. Other combinations are also available.
The next group of reagents are the linking system.
The linking system involves combinations of covalent or non-covalent linking members having specificity for each other, that is, having specific reactivity or being 15 homologous or cognate ligands and receptors. The linking system permits the binding of a large plurality of enzymes to the complex between the IBM and the enzyme-SBM
conjugate by polymerizing the enzyme and associating the complex with the enzyme polymer. Various linking 20 techniques can be employed, both covalent and non-covalent, preferably non-covalent.
The concept is to polymerize most of one of the enzymes into large aggregates and to associate the other enzyme with such aggregate through the complex of the IBM
25 and the e~zyme-SBM conjugate, particularly by binding to the IBM.
The members of the linking system are polyvalent, being at least diva lent. The polyvalency may be as a result of a plurality of reactive functionalities, which 30 under the conditions of the assay will form bonds which substantially preclude the binding of the enzyme-SBM
conjugate to the polymeric enzyme aggregate through other than the binding to the EM
Alternatively, non-covalent binding can be employed, 35 where homologous ligands and receptors form copolymers by having a plurality of epitopic or determinant sites on the enzyme, either naturally present or synthetically introduced, and a polyvalent receptor having a plurality of binding sites specific for the sites present on the 5 enzyme.
In the course of the polymerization rapid diffusion ox the enzymes to the growing polymer results to produce large aggregations of the enzyme, while substantially depleting the bulk solution of the enzyme. In addition, 10 as the aggregation of the enzyme is formed, enzyme-SBM
bound to IBM is bound to the aggregation so as to bring the two enzymes involved in the channeling in close proximity to each other, with one of the enzymes preferably in substantial mole excess to the enzyme of 15 the enzyme-SBM conjugate.
Among non-covalent techniques for linking, various receptors may be employed. One system can involve common determinant sites of a label on a receptor for the ligand ("antiligand") and a multivalent receptor which 20 recognizes these common determinant sites. The common determinant sites may be the determinant sites of an enzyme label, where the enzyme is bound to receptor and optionally additional enzyme is added which is not covalently bonded to receptor. Or determinant sites may 25 be introduced by covalently bonding various compounds, particularly low molecular weight organic compounds, to the antiligand and to the enzyme. Illustrative of such compounds are button, which may be used with its naturally occurring receptor, avid in; thyroxine, which 30 may be used with thyroxine binding globulin;
methotrexate, which may find use with dihydrofolate reeducates, etc.
Alternatively, one could use antibodies from various host sources, e.g. rabbit, sheep, or the like, and then 35 use antibodies against such host immunoglobulin to act as :~23(3~

the polymerizing system. For example, one could have an enzyme-sheep anti-ligand conjugate to which is added rabbit anti sheep immunoglobulin), wherein the enzyme-SBM conjugate incorporates a receptor other than 5 sheep immunoglobulin for the SUM.
Where polynucleotides are involved, an enzyme-antibody conjugate can be employed, where the antibody is directed to an epitome of the analyze that is absent in the SUM, for example an antibody directed to 10 single stranded polynucleotide having the sugar of the analyze i.e. DNA or PEA In this way the sequences other than the sequence of interest, in conjunction with the sequence of interest, will form large three dimensional aggregates of one of the enzymes about the complex 15 between the analyze nucleated sequence and the enzyme-SEM conjugate. For example, with a DNA sequence of interest, the SUM will be NOAH complementary to the DNA
analyze and will be linked to an extended polyribonucleotide to which one or more of the same 20 enzyme is bound, either covalently or through PUN
coupling or through anti-RNA-binding to PA. By adding enzyme*-antiDNA conjugate, the DNA present in the assay medium can copolymerize with the enzyme - antiDNA to form a large aggregate which will include the DNA analyze 25 -(RNA-enzyme ) hybridization product, so as to have a large amount of enzyme in close proximity to enzyme .
For covalent bonding, various reactive combinations can be employed, which while individually stable in the assay medium will specifically and preferentially form 30 covalent bonds under predetermined conditions. One reactant will be on ore member and the other reactant on the other member. Illustrative combinations include active halogen e.g. bromoacetyl, and they'll groups, activated ethylenes, e.g. maleimidyl and they'll groups, 0633K ~l930-FF

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thiols and Ellman's reagent derivatives, thiols and bis-arylmercury halides etc. One or a plurality of the groups may be covalently bonded to a reciprocal binding member for the IBM. For example, the enzymes may be 5 functionalized with maleimide groups and antibodies functionalized with mercaptans. Where the enzyme-SBM
conjugate has groups which may interact e.g. twill groups, these may be capped so as to prevent any reaction during the assay. For forming disulfides mercaptans may 10 be used and a mild oxidant, e.g. hydrogen peroxide, added or may be present as a product of an enzyme reaction.
By employing the linking system, one can sweep large amounts of enzyme or final reactant prom the assay medium and bring the these signal producing system members into 15 proximity to the enzyme-SBM conjugate and ligand complex. Thus, one can obtain high amplification of signal, while enjoying the benefits of rates of diffusion in an aqueous medium. In this manner, one achieves a uniformly dispersed aggregate while maintaining a stable 20 dispersion, which allows for signal determination in an aqueous medium without separation of enzyme-SBM conjugate bound to IBM and unbound enzyme-SBM conjugate.
Alternatively, one can separate the aggregates on a porous pad or filter before adding the reagents necessary 25 for signal production.
The remaining third group of substances employed in the assay are the members of the signal producing system, namely the substrates and cofactors which provide for the change in signal in the assay medium. The change in 30 signal may be as a result of a change in light absorption, a change in fluorescence, chemiluminescence, or other change in the effect of the system on electromagnetic radiation or changes in other properties such as electro-chemical. For the most part, the 35 detectable signal will be a light signal, either 0633K ~1~30-FF

ox I

ultraviolet or visible light. Because the substrates will vary with the particular enzymes employed, and for most, if not all, commercially available enzymes, substrates have been described or are generally available 5 which provide for the production of product which can be detected spectrophotometrically or fluorometrically, many of these substances or derivatives thereof may be used by providing for an appropriate combination of enzymes.
In some situations, it will be desirable to provide 10 for a scavenger of the product of the first enzyme. For example, where hydrogen peroxide is the product, kettles may be introduced into the medium, so as to ensure a very low or insignificant concentration of hydrogen peroxide in the bulk medium.
Various ancillary materials will be employed, particularly buffers, stabilizers, proteins, surfactants, more particularly non-ionic surfactants, or the like.
These may function in providing specific properties to the assay medium, such as maintaining a desired phi 20 inhibiting non-specific binding, imparting stability to the reagents during storage either as a lyophilized product or in an aqueous medium, or the like.
Desirably, the reagents can be provided as kits so that the specific ratios are optimized to provide for 25 optimum sensitivity over the concentration range of interest of the analyze. The kits will include either in combination in the same container or separately, in different containers, the enzyme-SBM conjugate, the members of the linking system, the enzyme substrates and 30 cofactors, the Final reactant, as appropriate, the ancillary members described above, bulking factors, etc.
The following examples are offered by way of illustration and not by way of limitation.

0633K 9lg30-FF

EXPERIMENTAL

Coupling of RIP to Antibody to 150 my HOP (Sigma Lot No. 31F9605) in 20 ml distilled water, pi 5~3, was added 4 ml of 0~1 M sodium peridot in distilled water and the mixture stirred for 20 min. at room temperature. After adding 2.4 ml 1 M
glycerol, the solution was stirred for 30 min. at room I temperature and then dialyzed against 2 my sodium acetate, pi 4.5, 3 x 500 ml, 2 hr., overnight, 2 hr.
A sample of antibody to polyribosephosphate (3 ml, Sample N-2, Lot AYE) was diluted to 9 ml and dialyzed against 3 x 10 my sodium carbonate, pi 9.5, 2 hr., 15 overnight, 2 ho-The oxidized HO sample was diluted 1-20 in 0.1 M
sodium phosphate, pi 6.0 (HOP - 1.072 x 10 4 M =
4.3 mg/ml, based on absorption at 403 no) to provide a final solution at a concentration of 3.13 mg/ml. A
20 reaction mixture was prepared of 21.64 ml oxidized HOP
and 4.67 ml of the above antibody, the solution diluted to 29.7 ml with 10 my sodium carbonate, and the pi then adjusted to 9.6 with 0.1 N sodium hydroxide. The reaction mix no was then stirred for 2 his. at room 25 temperature, followed by cooling to 0 on ice and then 1.5 ml (4 mg/ml) sodium bordered added. The reaction was protected from light by aluminum foil. After stirring for 2 his. at 0, the reaction mixture was dialyzed overnight against 1 L PBS (10 my sodium 30 phosphate, pi 7.2, 0.15 M awl The connate was concentrated to 3.5-4.5 ml using a collodion apparatus. The concentrate was then chromatographed on a Biogel*A5M column, 2.5 x 90 cm and 5 ml fractions collected. The effluent was monitored for 35 transmission at 280 no and the protein eluded with PBS

*Trade Mark I

containing 0.01% sodium aside. The column was run at 20 ml/hr. pooling fractions 39-48, 49-57, 58-61 and 62-66 to provide pools 1-4 respectively.

Conjugation of glucose oxldase to antiPRP
Glucose oxidize (40 ml Lot. 61F 9007) was dialyzed against 1 L each 3 x 0.3 M sodium bicarbonate (3 hr., overnight, 3 hr.). (Initial concentration of the glucose 10 oxidize was 5.3 mg/ml.) After completion of the dialysis, a 0.2 ml Alcott was removed and the remained r treated with l-fluoro-2,4-dinitrobenzene. The remaining volume, 35 ml, pi 8.23, was combined with 3.5 ml 1% (w/v) of 1-fluoro-2,4-dinitrobenzene in as. ethanol. After 15 stirring for 1 hr. at room temperature, the reaction mixture was dialyzed overnight against 1 L 0.3 M sodium bicarbonate and the next day the dialysis repeated with 1 L of fresh bicarbonate for 3 his. The sample was then concentrated to about t 20 ml using a collodion apparatus.
To the concentrated capped glucose oxidize was added 0.4 M sodium peridot in distilled water and the solution stirred for 20 min. at room temperature. To the reaction mixture was then added 1.1 ml of 1 M glycerol in water and the solution was s tiffed for an additional hour 25 at room temperature. The solution was then dialyzed 3 x 10 my sodium carbonate, pi 9.6 (3 hr., overnight, 3 hr., 600 ml each change), the residue having a volume of 27.5 ml.
AntiPRP (1.2 ml New York State Department of EIealth 30 Lot AYE) was diluted to 3.6 ml with 10 my sodium carbonate, pi 9.6, and the diluted sample dialyzed against 3 x 10 my sodium carbonate, pi 9.6, 3 hr., overnight, 3 hr., 300 ml each change), leaving a final volume of 3.2 ml. The concentration determined by 35 ultraviolet absorption at 280 no was 28.7 mg/ml.

To 27.5 ml of the capped glucose oxidize was added the antibody sample prepared above and the solution stirred for 14 hr., at room temperature, followed by cooling to 0 on ice. To the reaction mixture was then 5 added 1.55 ml of 4 mg/ml sodium bordered in distilled water, while the solution was protected from light and stirred for 2 hr. at room temperature. At the end of this time, the reaction mixture was dialyzed against 1 L
PBS for 3 his. at room temperature.
The PBS solution was concentrated to 5 ml using a collodion apparatus and then loaded onto a 2.5 x 90 cm Bejewel ARM column chromatographing as described previously and collecting fractions which were pooled as follows 40-45, 46-50, 51-56; 57-60: and 61-63, monitoring 15 the fractions at 280 no and 450 no, to provide pools 1-5 respectively.
To demonstrate the sensitivity and effectiveness of the subject technique, the following study was carried out. The enzyme reagents were prepared by diluting 1:40 20 pool 2 of the glucose oxidize conjugate (Example 2) and 1:32 of pool 1 of the HOP conjugate (Example 1). The buffer employed was PBS-Tween*(10 my sodium phosphate, pi 7.2, 150 my Nail, 0.05~ w/v Tweet 20). To 11 micro titer wells was added 10 I each of each of the enzyme 25 con gates and 10 1 of serially diluted PUP, using two-fold dilutions and ranging from 500 ng/ml to 0.5 ng/ml plus a blank for the Thea well. The plate was then covered and incubated for 10 miss. at 37~. To the mixture was then added 10 1 of 1:50 diluted sheep 30 antibody to amino modified glucose oxidize in Piston buffer. The mixture was then incubated for 60 miss. at 37 with shaking, followed by the addition of 220 1 of a developer solution (250 my glucose, 10 my ARTS
(2,2'-azino-di-3-ethylkenzthiazoline sulfonic acid), 0.8 35 mg/ml ovalbumin and 185 gel kettles). The mixture 0~33K ~lg30-FF

*Trade Mark was shaken at 37 for 11 miss. and read in the Artek vertical beam micro titer plate reader at 415 no. The results of the readings are set forth in the following table.

PUP Mean Artek Artek reading gone. reading SD corrected for ng~ml (avg. of 6) blank 500 1.4285~0.0818 0.7053 250 1.2893~0.0366 0.5661 100 1.1243+0.0~99 0.~011 0.9680+0.0358 0.2448 I 0.8772+0.0246 0.1540 0.8013+0.0160 0.07~0 0.7610~0.0156 0.0378 1 0.7600+0.009 0.0368 0.5 0.7113+0.0132 Blank 0.7232+0.0175 0 The above procedure was modified in a second assay.
Using the same reagents and materials as described previously, the assay of the two conjugates and PUP were placed in 10 x 75 mm glass tubes, the mixture incubated at 37 for 10 min. with shaking, followed by the addition 25 of the sheep antimony modified glucose oxidize) and the tubes incubated for an additional 30 min. at 37 with shaving.
At the end of this time, 270 I of PBS-Tween buffer was added to each tube, the tube vortexes and 30 100 I of the tube contents was then spotted out on an 8 x 8 mm pad of glass fiber paper placed on a second larger glass fiber pad. Onto the spot was then added 3 ml of the developer solution described previously to wash away unbound conjugates and to initiate development I
Trade Mark , ,. :

off I

of the colored product. Each pad was developed for

2.5 min. and then washed with 270 Al pBS-Tween buffer and pressed dry. A concentration of 0.5 ng/ml, the lowest concentration employed, was detectable. However, 5 development continued for some time after washing and pressing dry of the pad, so that eventually color appeared on the blank. Even after standing for an extended period, one could readily distinguish 25 ng/ml PUP from no PUP.
The above described experiment was repeated except that incubation with antimony modified glucose oxidize) was for 60 min. and development was for 440 sec. Similar results were obtained.
It is evident from the above results, that the subject method provides for a rapid, sensitive assay which can be carried out in a variety of ways. By fairly simple manipulations, extremely low concentrations of analyzes may be obtained. By employing a linking means which provides for high localized concentration of one 20 enzyme as compared to another enzyme, particularly where the highly concentrated enzyme provides the substrate for the second enzyme, the rate of production of a product which can be detected can be greatly enhanced. Thus t extremely low concentrations of analyze can be detected 25 by virtue of producing a strong signal in relation to a background signal. Quantitative results can be obtained by using incitements such as a spectrophometer or reflectometer, while semi-quantitative or qualitative results can be obtained visibly, particularly where 30 control is employed so as to provide a comparison.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced 35 within the scope of the appended claims.
* Trade Mark

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method for detecting the presence of an analyte in a sample suspected of containing said analyte, when said analyte is a member of a specific binding pair consisting of first and second binding members ("FBM" and "SBM", respectively), said method employing a channeling signal producing system comprising an enzyme, enzyme+, conjugated to an SBM to provide enzyme+ -SBM conjugate, an enzyme substrate and a Final Reactant, wherein said Final Reactant is (1) another enzyme, enzyme*, where the two enzymes are related by the substrate of one enzyme being the product of the other enzyme, or (2) a compound which reacts with the product of enzyme+, wherein the reactions of said enzyme+ in conjunction with enzyme*
or said compound results in a change in an observable signal in relation to the amount of analyte in said sample; and linking system providing for the polymerization of said Final Reactant and the incorporation of enzyme+
within the polymer as a function of the binding of enzyme+ -SBM conjugate to FBM so as to form a polymeric channeling aggregate;
said method comprising:
combining in an aqueous assay medium;
(a) said sample;
(b) said enzyme+ -SBM conjugate, and FBM when said analyte is a SBM, so as to form a complex between said FBM and said enzyme+ -SBM conjugate in relation to the amount of analyte present;
(c) any members of said linking system, whereby is formed a polymeric channeling aggregate of said Final Reactant and a complex of enzyme+ -SBM conjugate with FBM; and (d) remaining members of a signal producing system, whereby a Final Product is produced as a result of channeling of said members of said signal producing system in said polymeric channeling aggregate, which results in a change in a detectable signal; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

2. A method according to Claim 1, said method employing (1) enzyme channeling where two enzymes, enzyme+ and enzyme*, which are members of a signal producing system are employed, where the product of one enzyme is the substrate of the other enzyme, and enzyme+ is conjugated to a SBM to provide enzyme+-SBM
conjugate; (2) a linking system providing for the polymerization of enzyme* and the incorporation of enzyme+ through the binding of a complex of enzyme+-SBM conjugate to FBM to form a polymeric channeling aggregate; and (3) the remaining members of the signal producing system comprising substrates, including cofactors, of said enzymes, which results in a change in a detectable signal as a result of the formation of said polymeric channeling aggregate in relation to the amount of analyte present;
said method comprising:
combining in an aqueous assay medium;
(a) said sample;
(b) said enzyme+-SBM conjugate, and FBM wherein said analyte is a SBM, so as to form a complex between said FBM and said enzyme+-SBM
conjugate in relation to the amount of analyte present;

(c) members of said linking system, whereby is formed a polymeric channeling aggregate of enzyme* and the complex of enzyme+-SBM
conjugate with FBM; and (d) members of the signal producing system, whereby a final product is produced as a result of enzyme channeling of said members of said signal producing system in said polymeric channeling aggregate, which results in a change in a detectable signal; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

3. A method according to Claim 2, wherein said FBM is a ligand and said SBM is a receptor.

4. A method according to Claim 3, wherein said SBM is an antibody.

5. A method according to any of Claims 1, 2, or 3, wherein said linking system comprises enzyme*-SBM
conjugate and anti-enzyme*.

6. A method according to any of Claims 2, 3, or 4, wherein said linking system comprises modified enzyme*-SBM conjugate, where said modification is the bonding of ligands to said conjugate, and polyvalent receptor for said ligand.

7. A method according to Claim 2, wherein said enzymes are oxidoreductases.

8. A method according to Claim 2, wherein said assay medium is transferred to a porous support prior to the addition of said signal producing system to said polymeric channeling aggregates.

9. A method according to Claim 2, wherein said sample and said enzyme+-SBM conjugate are incubated together in said assay medium before the addition of a non-enzymatic member of said linking system.

10. A method according to Claim 1, wherein said analyte is a member of a specific binding pair consisting of ligand and receptor;
said method employing (1) enzyme channeling where two oxidoreductase enzymes, enzyme+ and enzyme*, are employed, which are members of a signal producing system;
where the product of one enzyme is the substrate of the other enzyme; and where enzyme+ is present conjugated to a first receptor, enzyme+-first receptor conjugate wherein first receptors bind to ligand; (2) a linking system providing for the polymerization of enzyme*, comprising enzyme* conjugated to first receptor, enzyme*-first receptor conjugate, wherein said enzyme*-first receptor conjugate has naturally occurring determinant sites or such sites are synthetically introduced, and a polyvalent second receptor for said determinant sites, whereby polymerization of enzyme* and the incorporation of enzyme+ through the binding of a complex of enzyme+-first receptor conjugate to ligand occurs to form a polymeric channeling aggregate; and (3) a signal producing system comprising substrates, including cofactors, of said enzymes, which results in the production of a final product which provides a change in a detectable signal as a result of the formation of said polymeric channeling aggregate in relation to the amount of analyte present;
said method comprising:
combining in an aqueous medium;
(a) said sample;
(b) said enzyme+-first receptor conjugate, and ligand when said analyte is a receptor, to form an enzyme+-first receptor and ligand complex;
(c) enzyme*-first receptor and second receptor for polymerizing enzyme*, whereby enzyme*
is polymerized and incorporates enzyme+ as part of said complex;
(d) members of the signal producing system, whereby said members in conjunction with the channeling of said enzymes produce a final product providing a detectable signal in an amount related to the amount of analyte present; and comparing said detectable signal to the detectable signal observed in an assay medium having a known amount of analyte.

11. A method according to Claim 10, wherein additional enzyme* is added as a member of said linking system.

12. A method according to any of Claims 10 or 11, wherein one of said enzymes is glucose oxidase and the other of said enzymes is horseradish peroxidase.

13. A kit comprising enzyme+-SBM conjugate and a linking system including as one component, enzyme*, where enzyme+ and enzyme* are related by the substrate of one being the product of the other, and means for polymerizing enzyme.

14. A kit according to Claim 13, wherein said linking system comprises enzyme -receptor conjugate and receptor for enzyme or a ligand bound to enzyme.