US3852157A - Compounds for enzyme amplification assay - Google Patents

Abstract

Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as ''''ligands'''') and binding an enzyme to the ligand or ligand counterfeit to provide an ''''enzyme-bound-ligand,'''' an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-boundligand and enzyme-bound-ligand combined with receptor. The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

Description

United States Patent [191 Rubenstein et al.

[ Dec. 3, 1974 1 COMPOUNDS FOR ENZYME AMPLIFICATION ASSAY [75] Inventors: Kenneth E. Rubenstein, Palo Alto;

Edwin F. Ullman, Atherton, both of Calif.

[73] Assignee: Syva Corporation, Palo Alto, Calif.

[22] Filed: Nov. 6, 1972 [21] Appl. No.: 304,157

Related US. Application Data [63] Continuation-impart of Ser No 143,609, May 14,

1971, abandoned.

[56] References Cited UNITED STATES PATENTS 2/1972 Csizmas et a1. 195/63 4/1972 Schuurs et a1. 195/1035 R Primary ExaminerAlvin E. Tannenholtz Attorney, Agent, or FirmTownsend and Townsend 5 7 ABSTRACT Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as ligands) and binding an enzyme to the ligand or ligand counterfeit to provide an enzyme-bound-ligand, an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-bound-ligand and enzyme-bound-ligand combined with receptor. The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

11 Claims, N0 Drawings COMPOUNDS FOR ENZYME AMPLIFICATION ASSAY CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of Application Ser. No. 143,609, filed May 14, 1971 and now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention There is a continually pressing need for rapid, accurate qualitative and quantitative determinations of biologically active substances at extremely low concentrations. The purpose of the determination can be extremely varied. Today, there is a wide need for determining the presence of drugs or narcotics in body fluids, such as saliva, bloord or urine. In addition, in medical diagnosis, it is frequently important to know the presence of various substances which are synthesized naturally by the body or ingested. These include hormones, both steroidal and polypeptides, prostaglandins, toxines, as well as other materials which may be involved in body functions. Frequently, one is concerned with extremely small amounts and occassionally, with very small differences in concentrations.

To meet these needs, a number of ways have been devised for analyzing for trace amounts of materials. A common method is to use thin layer chromatography (TLC). By determining the flow factors and using specific reagents, the presence of certain materials can be detected; in many instances the particular material can be isolated and identified quantitatively, for example, by mass spectroscopy or gas phase chromatography However, thin layer chromatography has a number of deficiencies in being slow, requiring 'a high degree of proficiency in its being carried out, being subject to a wide range of interfering materials, and suffering from severe fluctuations in reliability. Therefore, the absence of satisfactory alternatives has resulted in intensive research efforts to determine improve methods of separation and identification.

An alternative to thin layer chromatography has been radioimmunoassay. Here, antibodies are employed for specific haptens or antigens. A radioactive analog employing a radioactive atom of high flux is used and bound to the antigen. By mixing an antibody with solutions of the hapten or antigen and the radioactive hapten or antigen analog, the radioactive analog will be prevented from binding to the antibody in an amount directly related to the concentration of the hapten or antigen in the solution. By then separating the free radioactive analog from the antibody bound radioactive analog and determining the radioactivity of the separate components, one can determine the amount of hapten or antigen in the original solution.

The use of radioactive materials is not desirable for a variety of reasons. First, radioactivity creates handling problems and undesirable hazards. Secondly, the preparation of such compounds involves similar hazards, greatly enhanced by the much larger amounts of radioactive materials which are present. Because of their instability, the radioactive materials have only a short life. In addition, the use of radioactive materials requires a license from the Atomic Energy Commission, subjecting the licensee to review by the Commission as to the maintenance of minimum operating standards. These standards may change from time to time, so as to involve added expense and inconvenience to the licensee. Finally, the separation of the bound and unbound radioactive analog is difficult and subject to error. See, for example, Abraham, Prelim. Comm., 29, 866 (1969).

Besides the aforementioned materials, assays at extremely low concentrations would be desirable for a variety of pesticides, such as insecticides, bactericides, fungicides, etc., as well as other organic pollutants, both in the air and water. Organic pollutants may be assayed whenever a receptor can be devised and the pollutant is inert to the reagents employed.

2. Description of the Prior Art Use of radioimmunoassay is described in two articles by Murphy, 1. Clin. Endocr. 27, 973 (I967); ibid., 28, 343 (I968). The use of peroxidase as a marker in an immunochemical determination of anitgens and antibodies is found in Stanislawski et al., C. R. Acad. Sci. Ser. D. 1970, 271 (16), 1442-5. (CA. 74 1144 B). See also, Nakane, et al., J. of Histochem. and Cytochem. 14, 929 (1967) and Avrameas, Int. Rev. of Cytology, 27, 349 (1970). A general description of thin layer chromatography for assay may be found in Stahl, Thin Layer Chromatography, Springer Verlag, New York, 1969. See also, Peron, et al., Immunologic Methods in Steroid Determination, Appleton, Century'Crofts, New York, 1970.

Also of interest are publications by Van Weemen, et al., FEBS Letters 14, 232 (1971), and Engvall, et al., Immunochemistry, 8, 871 (I971) concerned with immunoassays employing enzymes. See also US. Pat. No. 3,654,090. See also, Cinader, Proceedings of the Second Meeting of the Foundation of European Biochemical Societies, Pergamon, Oxford, 1967, vol. II, chapter four.

SUMMARY OF THE INVENTION Detection of ligands is obtained at extremely low concentrations by using specific receptor sites for the ligand and enzyme applification of ligand displacement. By bonding a ligand or a ligand counterfeit to an enzyme while retaining enzymatic activity and then combining the enzyme-bound-ligand to a receptor for the ligand, the presence and amount of ligand in an unknown solution may be readily determined. By competition for receptor sites between the enzyme-boundligand and the free ligand, the two ligand moieties being added to the receptor simultaneously or sequentially, the difference in enzymatic activity resulting from the presence or absence of ligand may be determined in accordance with a particular analytical scheme. This difference will be related to the amount of ligand present in the unknown solution. Enzymatic activity is easily determined in known ways by following the change in concentration of an enzyme substrate or product of the substrate by standard techniques.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS This invention provides a method for detecting or assaying extremely low concentrations of a wide range of organic materials by relating the presence of a particular unknown to enzymatic activity. An amplification is obtained by having a large number of molecules formed or transformed as a result of the presence of one molecule. This amplification is achieved by bonding the compound to be assayed or a counterfiet of the compound to an enzyme. This assemblage is referred to as an enzyme-bound-ligand. The particular molecule to be assayed is referred to as a ligand. The ligand analog will include either a ligand which is modified by replacing a proton with a linking group to bond to the enzyme or a ligand counterfeit which is a ligand modified by other than simple replacement of a proton to provide a linking site the enzyme. The ligand and the enzymebound-ligand are both capable of binding in a competitive fashion to specific receptor sites. It should also be noted that other compounds of very similar structure may serve as ligands capable of competing for these sites, e.g., morphine glucuronide and codeine will compete with enzyme-bound-morphine for binding to certain types of morphine antibodies. In most instances, this is advantageous in permitting one to assay for a class of physiologically closely related compounds.

Various methods'or protocols may be employed in assaying for a wide variety of ligands. Normally, the ligand, enzyme-bound-ligand and receptor will be soluble in the medium employed. The substrate(s) for the enzyme may or may not be soluble in the medium. In some situations it may be desirable to provide a synthetic substrate which is not soluble or employ an insoluble natural substrate.

in carrying out the assay,-the enzyme-bound-ligand is combined with a high molecular weight receptor which results in inhibition of enzymaticactivity, When a ligand and enzyme-bound-ligand are introduced into a solution containing ligand receptor, the enzymatic activity of the solution after the three substances are com.- bined will be affected by the concentration of the ligand present in the solution. That is, the enzymebound-ligand and the ligand will compete for the receptor sites. The number of enzymebound-ligand molecules not inhibited by the receptor will be directly related to the number of ligand molecules present in the solution. One can achieve this in two ways: (I) either by competition, whereby the enzyme-bound-ligand and ligand are introduced to the receptor substantially simultaneously; or (2) the enzyme-bound-ligand or ligand may be first added to the receptor, and the system allowed to come to equilibrium, and then the ligand added or enzyme-bound-ligand added respectively, in effect, to displace the material originally added from the receptor. Since the enzymatic activity will be diminished or inhibited when the enzyme-bound-ligand is bound to the receptor, the enzymatic activity of the solution will be directly related to the amount of ligand present in the solution.

The assay can be carried out, either by considering the effect of ligand on the rate at which enzyme-boundligand binds to receptor or the effect of ligand on the equilibrium between the reagents: enzyme-boundligand and receptor. Where enzyme-bound-ligand and ligand are present with'receptor, one need not wait until equilibrium is achieved between the three species. If one measures the enzymatic acitivity at a specific time or interval of time from the time of combination of the three species, the enzymatic activity of the assay mixture will be a function of the effect of the ligand on the rate of binding of the enzyme-bound-ligand to the receptor. By determining standards under the same conditions, including the same time interval, employing different concentrations of ligand, a smooth standard curve is obtained.

By measuring the effect of the ligand on rate of binding, rather than the effect of equilibrium, a shorter time interval between the time of combining the reagents and unknown suspected of containing the ligand and the time for the determination will be involved, compared with waiting until equilibrium is achieved. it is frequently found that reproducible values can be obtained in from 0.1 to 5 minutes after combining the reagents and unknown. The rate of enzymatic activity is usually determined over a short time interval, e.g.. l minute. The time interval can be the second, third, etc. minute from the time when the reagents and unknown were combined.

The concentrations of the reagents: the enzymebound-ligand and the receptor, may be varied widely. Normally, the concentration of receptor (based on active sites) and enzyme-bound-ligand will be from about 10 to IO M, more usually from 10* to 10 M. The lower limit for the concentration of enzymebound-ligand is predicated onthe minimum amount which can be detected. This will vary with different enzymes as well as different detection systems.

The amount of receptor employed is normally calculated based on receptor sites and will vary with the concentration of enzyme-bound-ligand, the ratio of ligand to enzyme in the enzyme-bound-ligand, and the affinity of the receptor for the ligand. Usually, there will be at least 1 active receptor site per molecule of enzymebound-ligand and less than about 20 active sites per molecule of ligand as enzyme-bound-ligand, but siteligand molecule ratios may be as high as l,000 to 1, depending on the type of assay and the affinity of the receptor. Preferably, the ratio of receptor active sites to molecules of enzyme-bound-ligand will be at least one, usually at least two, and the ratio of active sites to molecules of ligand as enzyme-bound-ligand will be less than about 5 to l. The ratio will vary to a great degree depending on binding constants and the amount of ligand suspected of being present. The method of determining binding sites for the receptor will be discussed subsequently in the experimental section.

The en'zyme-bound-ligand will usually have mole cules of ligand to enzyme subunit ratios on the average over the entire composition in the range of 0.01 :1, frequently 0.0250:l, and more frequently about 0.04 25:1, wherein the number of ligands when the ligand is a protein is expressed as the number of ligand molecules times the number of its component polypeptide chains. For small ligands (less than about 10,000 molecular weight), there will generally be at least one ligand, more usually at least two ligands per enzyme, while with large ligands (greater than about 5,000 molecular weight) there will generally be at least one enzyme per ligand. In the area of overlap, the ratio will depend on the nature of the ligand, among other factors to be discussed.

, The number of small ligands per enzyme will be affected to some degree by the molecular weight of the enzyme. However, normally, the fewer molecules ofgand bound to an enzyme to achieve the desired degree of inhibitability with receptor, the more sensitive the assay. Therefore, the number of small ligands per enzyme will usually not exceed 40, more usually not exceed 30, and will not exceed 1 ligand per 2,000 molecular weight of enzyme on the average over the entire composition. Usually, the range of ligands will be 1 to 40, more usually I to 24, and with random substitution 2 to 20.

With large ligands, there will be on the average not more than one enzyme per 2,000 molecular weight, usually not more than one enzyme per 4,000 molecular weight, and more usually not more than one enzyme per 6,000 molecular weight.

In some instances, a number of enzymes bind together in a stable arrangement to form a multienzyme complex. Because of the juxtaposition of the enzymes, a number of reactions may be carried out sequentially in an efficient manner, providing localized high concentrations of reactions. Therefore, the ligand may be bound to a combination of enzymes, whereby there will be a plurality of enzymes per ligand. If a number of ligands were bound to the multienzyme complex, one could have 1:1 mole ratio of enzymes to ligand, although, in fact, there would be a plurality of enzymes and ligands involved in a single aggregation. The numberof enzymes bound together, either as a multienzyme complex or by another mechanism will rarely exceed 20, usually not exceed 10, and commonly be in the range of 2 to 5 enzymes.

All other things being equal, the greater the number of enzymes per large ligand, the greater the sensitivity of the assay. However, the enzymes may interfere with receptor recognition, affect solubility and be deleterious in other ways. Therefore, usually, the number of enzymes bonded to a large ligand will be such that there will be no more than one enzyme polypeptide chain for every 2,000 molecular weight of the ligand.

The concentration of receptor and enzyme will be related to the range of concentration of the ligand to be assayed. The solution to be assayed will be used directly, unless a relatively high concentration of ligand is present. If a high concentration is present, the unknown solution will be diluted so as to provide a convenient concentration. However, in many biological systems of interest, the amount of material being assayed will be relatively small and dilution of the unknown substrate will usually not be required.

To illustrate the subject method, a soluble receptor is employed for a particular ligand. For illustrative purposes, the ligand will be considered the hapten, morphine, and the receptor will be an antibody specific for morphine. It should be notedparenthetically, that antibodies generally recognize molecular shape and distribution of polar groups in a ligand, although a portion of the ligand may be significantly modified without preventing recognition. For example, both morphine and its glucuronide can be bound to certain morphine antibodies.

An enzyme is first modified by bonding one or more morphine molecules to the enzyme; a sufficient number of morphine groups are employed so that greater than about percent inhibition, usually 50 percent inhibition, and preferably, at least 70 percent inhibition is obtained when the maximum number of ligands are conjugated to receptor. Complete inhibition is usually neither necessary or desirable. In many instances, all that is required is that there be a measurable difference between completely uninhibited and maximally inhibited enzyme-bound-ligand which would allow for a semiquantitative or quantitative determination of a ligand through a desired range of concentrations. Any convenient enzyme can be used that will catalyze the reaction of a substrate that can be easily detected and for which a substrate is available which allows for inhibition of the enzyme when bound to receptor.

A solution is prepared of the antibody at the requisite concentration. Only a few microliters of solution are required. The antibody, maintained at a pH at which it is active in binding morphine, is introduced into a solution of the enzyme-bound-morphine at the desired concentration. The reactivity of the combined antibody and enzyme-bound-morphine solution can be determined by taking an aliquot, adding it to its substrate under conditions where the enzyme is active, and determining the spectroscopic change as a function of time at a constant temperature. The rate of this change will be the result that should be obtained when there is no morphine present in the unknown solution.

Normally, the ligand and enzyme-bound-ligand reversably bind to receptor, so that the order of addition of reagents is not crucial.

A second aliquot is taken and added to the unknown solution. The unknown solution may contain the substrate and any other additives which are required for enzymatic activity. Alternatively, the unknown solution may first be combined with the antibody-(enzymebound-morphine) complex, allowed to come to equilibrium and then mixed with the substrate' In either case the rate of change in the spectrum is determined. A variant of the above method is to add combined enzyme-bound-morphine and unknown solution to the antibody and then add this solution to the substrate. Other obvious variations come readily to mind.

If all concentrations of reagents except morphine are kept constant and several standard solutions of morphine are employed, then one can relate the change in the spectrum over specified periods of time to the morphine concentrations. Obviously, the standardized sys tem can then be used to determine rpaidly, accurately, and efficiently the amount of morphine, or any other ligand in the unknown.

The manner of assaying for the enzyme can be widely varied depending on the enzyme, and to some degree the ligand and the medium in which the ligand is obtained. Conveniently, spectrophotometric measurements can be employed, where absorption of a cofactor, a substrate or the product of the substrate absorbs light in the ultraviolet or visible region. However, in many instances other methods of determination may be preferred. Such methods include fluorimetry, measuring luminescence, ion specific electrodes, viscometry, electron spin resonance spectrometry, and metering pH, to name a few of the more popular methods.

The assays will normally be carried out at moderate temperatures, usually in the range of from 10 to 50 C, and more usually. in the range of about 15 to 40 C. The pH of the assay solutions will be in the range of about 5 to 10 usually about 6 to 9. Illustrative buffers include (trishydroxymethyl)-methylamine salt, carbonate, borate and phosphate.

Whether oxygen is present or the assay is carried out in an inert atmosphere, will depend on the particular assay. Where oxygen may be an interferant, an inert atmosphere will normally be employedpNormally, hydroxylic media will be employed, particularly aqueous media, since these are the media in which the enzyme is active. However 0 to 40 vol. percent of other liquids may also be present as co-solvents, such as alcohols, esters, ketones, amides, etc. The particular choice of the cosolvent will depend on the other reagents present in the medium, the effect on enzyme activity, and any desirable or undesirable interactions with the substrate or products.

As already indicated, antibodies will frequently recognize a family of compounds, where the geometry and spatial distribution of polargroups are similar. Frequently, by devising the haptenic structure and the method of binding to the antigen when producing the antibodies, the specificity of the antibody can be varied. In some instances, it may be desirable to use two or more antibodies, usually not more than six anitbodies, so that the antibody reagent solution will be able to detect an entire group of compounds, e.g., morphine and barbiturates. This can be particularly valuable for screening a sample to determine the presence of any member of a group of compounds or determining whether a particular class of compounds is present, e.g., drugs of abuse or sex hormones. When combinations of antibodies are used, it will usually be necessary to employ corresponding combinations of enzymepounds for which receptors can be provided range from simple phenylalkylamines, e.g., amphetamine, to very high molecular weight polymers, e.g., proteins.

Among ligands which are drugs, will be compounds which act as narcotics, hypnotics, sedatives, analgesics, antipyretics, anaesthetics, psychotogenic drugs, muscle relaxant's, nervous system stimulants, anthcholinesterase agents, parasympathomimetic agents, sympathomimetic agents, a-adrenergic blocking agents, antiadrenergic agents, ganglionic stimulating and blocking agents, neuromuscular agents, histamines, antihista mines, S-hydroxy-tryptamine and antagonists, cardiovascular drugs, anitarrhythmic drugs, antihypertensive agents, vasodilator drugs, diuretics, pesticides (fungicides, antihelminthics, insecticides, ectoparasiticides, etc.), antimalarial drugs, antibiotics antimetabolites, hormones, vitamins, sugars thyroid and antithyroid drugs, corticosteroids, insulin, oral hypoglemic drugs, tumor cells, bacterial and viral proteins, toxins, blood proteins, and their metabolites.

(A drug is any chemical agent that affects living protoplasm. (Goodman and Gilman, The Pharmacological Basis of Therapeutics, 3rd Ed., Macmillan, New York (1965).) A narcotic is any agent that produces sleep as well as analgesia.)

included among such drugs and agents are alkaloids, steroids, polypeptides and proteins, prostaglandins, catecholamines, xanthines, arylalkylamines, heterocyclics, e.g., thiazines, piperazines, indoles, and thiazoles, amino acids, etc.

Other ligands of interest besides drugs are industrial pollutants, flavoring agents, food additives, e.g., preservatives, and food contaminants. v

Broadly, the ligands will be organic compounds of from 100 to 100,000 molecular weight, usually of from about 125 to 40,000 molecular weight, more usually 125 to 20,000 molecular weight. The ligand will usually have from about 8 to 5,000 carbon atoms and from about 1 to 3,500 heteroatoms.

A substantial portion of the ligands will be monomers or low order polymers, which will have molecular weights in the range of about to 2,000 more usually to 1,000. Another significant portion of the ligands will be polymers (compounds having a recurring group) which will have molecular weightsin the range of from about 750 to 100,000, usually from about 2,000 to 60,000, more usually 2,000 to 50,000. For polymers of varying molecular weight, weight average molecular weight is intended.

In some instances, high molecular weight materials will be of interest. For example, blood proteins will generally be in excess of 100,000 molecular weight. In the case of lipoproteins, the molecular weight will be in the range of 3 million to 20 million. The globulins, albumins and fibrinogens will be in the range of 100,000 to 1,000,000.

The ligands will normally be composed of carbon, hydrogen, nitrogen, oxygen, sulfur. phosphorous, halogen, and metals, primarily as their cations, such as the alkali and alkaline earth metals and the metals of Groups 18, H8, V118, and VlllB, particularly the third row of the periodic chart. Most usually, the ligands will be composed primarily of carbon, hydrogen, nitrogen, oxygen and sulfur.

Structurally, the ligands may be monomers or polymers, acyclic, mono or polycyclic, having carbocyclic or heterocyclic rings. The ligands will have a wide variety of functionalities, such as halo, oxocarbonyl, nonoxocarbonyl, amino, oxy (hydroxy, aryloxy, alyloxy and cycloalyloxy [alyl intends a monovalent aliphatic radicall), thiooxy, dithio, hydrazo, and combinations thereof.

The ligands may be divided into three different categories, based on their biological relationship to the receptor. The first category is antigens, which when introduced into the bloodstream of a vertebrate, result in the formation of antibodies. The second category is haptens, which when bound to an antigenic carrier, and the hapten bound antigenic carrier is introduced into the bloodstream of a vertebrate, elicit formation of antibodies specific for the hapten. The third categoryof ligands includes those which have naturally occuring receptors in a living organism and the receptors can be isolated in a form specific for the ligand.

Of course, biological substances which are native to one species and have naturally occurring receptors in that species, may also be haptens when bonded to a protein and a introduced into an animal of the same or a different species. Therefore, the classification is somewhat arbitrary in that the ligand may be an antigen as to one species, a hapten as to another species, and may have naturally occurring receptors in a third species.

Anitgens are for the most part protein or polysaccharide in nature and foreign to the animal into which they are injected.

The most important body of ligands for the purposes of the invention are the haptens. Substances which on injection do not give rise to antibodies, but which are able to react with antibodies specifically to produce either precipitation or to inhibit precipitation have been termed haptens. This definition has been used to include not only the simple chemical substances which are determinants of specificity when conjugated to protein, and which inhibit precipitation, but also substances obtained from natural sources such as the pneumococcal type specific polysaccharides and dextran which are not antigenic in the rabbit on primary injection." Kabat, et al., Experimental Immunochemistry, Charles C. Thomas, Springfield, Illinois (1967). In the following discussion the term hapten will be confined to groups artificially introduced into antigenic carriers which promote the formation of antibodies to those groups.

The third group of ligands are those which have naturally occurring receptors. The receptors may be proteins, nucleic acids, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or membranes associated with cells. Illustrative ligands which have naturally occurring receptors are thyroxine, many steroids, such as the estrogens, cortisone, corticosterone, and estradiol; polypeptides such as insulin and angiotensin, as well as other naturally occurring biologically active compounds. See Murphy, et al., J. Clin. Endocr., 24, 187 (1964); Murphy, ibid, 27, 973, (I967); ibid, 28, 343 (I968); BBA, 176, 626, (1969); McEwen, et al., Nature, 226, 263 (1970); Morgan, et al., Diabetes, (1966); Page, et al., J. Clin. Endocr., 28, 200, (1969).

The ligands may also be categorized by the chemical families which have become accepted in the literature. In some cases, included in the family for the purpose of this invention, will be those physiominmetic substances physiomimetic are similar in structure to a part of the naturally occurring structure and either mimic or in- .hibit the physiological properties of the natural substances. Also, groups of synthetic substances will be included, such as the barbiturates and amphetamines. In addition, any of these compounds may be modified for linking to the enzyme at a site that may cause all biological activity to be destroyed. Other structural modifications may be made for the ease of synthesis or control of the characteristics of the antibody. These modified compounds are referred to as ligand counterfeits.

A general category of ligands of particular interest are drugs and chemically altered compounds, as well as the metabolites of such compounds. The interest in assaying for drugs varies widely, from determining whether individuals have been taking a specific illicit drug, or have such drug in their possession, to determining what drug has been administered or the concentration of the drug in a specific biological fluid.

The drugs are normally of from eight carbon atoms to 40 carbon atoms, usually of from nine to 26 carbon atoms, and from 1 to 25, usually from one to 10 heteroatoms, usually oxygen, nitrogen or sulfur. A large category of drugs have from one to two nitrogen atoms.

One class of drugs has the following basic functionalwhere the lines intend a bond to a carbon atom, and wherein any of the carbon atoms and the nitrogen atom may be bonded to hydrogen, carbon or a heterofunctionality. Drugs which have this basic structure include the opiates such as morphine and heroin, meperidine, and methadone.

Another class of drugs are the epinephrine like drugs which have the following basic functionality;

where the lineintend a bond to a carbon atom and wherein any of the carbon atoms and the nitrogen atom may be bonded to hydrogen, carbon or a heterofunctionality. Drugs which have this basic structure include amphetamine, narceine, epinephrine, ephedrine and L-dopa.

The ligand analogs of drugs will usually have molecular weight in the range of to 1,200 more usually in the range of to 700.

Alkaloids The first category is the alkaloids. Included in the category of alkaloids, for the purpose of this invention, are those compounds which are synthetically prepared to physiologically simulate thenaturally occurring alkaloids. All of the naturally occurring alkaloids have an amine nitrogen as a heteroannular member. The synthetic alkaloids will normally have a tertiary amine, which may or may not be a heteroannular member. The alkaloids have a variety of functionalities present on the molecule, such as ethers, hydroxyls, esters, acetals, amines, isoxazole, olefins, all of which, depending on their particular position in the molecule, can be used as sites for bonding to the enzyme.

Opiates The opiates are morphine alkaloids. All of these molecules have the following functionality and minimum structures:

wherein the free valences are satisfied by a wide variety of groups, primarily carbon and hydrogen.

The enxyme-bound-ligand analog of these compounds will for the most part have the following mini-.

mum skeletal Stl'UClIUI'BI wherein X is a bond or a functionality such as imino, azo, oxy, thio, sulfonyl, oxocarbonyl, nonoxocarbonyl, or combinations thereof. Oxygen will be in the ortho, meta or B position. A is an enzyme which is bonded to X at other than its reactive site and retains a substantial portion of its natural enzymatic activity. There will be m ligands bonded through X to the enzyme A.

The enzyme-bound-morphine and its closely related analogs will have the following formula:

wherein: i

any one of the W groups can be X* or an H of any i of the W groups may be replaced by X*, wherein X* is a bond or a linking group;

A* is an enzyme bonded at other than its reactive site, having a number (n) of ligands in the range of 1 to the molecular weight of A* divided by 2,000, usually in the range of 2 to 40;

W is hydrogen or hydrocarbon of from one to eight carbon atoms, particularly alkyl or alkenyl of from one to four carbon atoms, cycloalkylalkyl of from four to six carbon atoms, or aralkyl, e.g., methyl, allyl, 3-methylbut-2-enyl-l, cyclopropylmethyl and ,8-phenethyl;

W is hydrogen; W is hydrogen;

W is hydrogen or taken together with W a divalent radical of from three to six carbon atoms and to 2 oxygen atoms, forming a six membered carbocyclic ring with the carbon chain to which they are attached, e.g., propylene-l ,3, l -hydroxyprop-2-enylenel ,3, l hydroxypropylenel ,3,l -acetoxypropylenel ,3, l acetoxyprop-2-enylene-l ,3 l -oxopropylene-l ,3 l oxoprop-2-enylene-l ,3;

W is hydrogen or hydroxyl;

W is hydrogen, hydroxyl or taken together with W y W is hydrogen or methyl;

W is hydrogen, methyl or hydroxyl;

W is hydrogen, hydroxy, acyloxy of from one to three carbonatorns, e.g., acetoxy, (unless otherwise indicated, acyl intends only nonoxocarbonyl), hydrocarbyloxy of from I to 3 carbon atoms, e.g., methoxy, ethoxy, 2-(N-morpholino)ethoxy and glucuronyl; and

W is hydrogen. (It is understood that in all the formulas, except when a minimum or skeletal structure is indicated, unsatisfied valences are satisfied by hydrogen).

(Hydrocarbyl is an organic radical composed solely of hydrogen and carbon and may be saturated or unsat- W oxy (-0-); and

W is hydroxy, acetoxy, or alkoxy of from one to three carbon atoms;

Those preferred compounds having the basic morphine sturcture will have the following formula:

O J i N-W\ l\/ i 1 i l 7 wherein: one of W" and W is X**; when other than X**; W" is methyl; and W is hydrogen, methyl, acetyl or glucuronyl; W is hydrogen or acetyl, usually hydrogen;

-X** is wherein Z is hydrocarbylene of from one to seven carbon atoms, preferably aliphatic, having from 0 to I site of ethylenic unsaturation; and

-Z** is an enzyme, either specifically labelled with n equal to l to 2 ligands or randomly (random as to one or more particular available reactive functionalities) labelled with n equal to 2 to 30, more usually 2 to 20, the enzyme retaining a substantial proportion of its activity. The enzyme will be of from about 10,000 to 300,000, frequently about 10,000 to l50,000 molecular weight and is preferably an oxidoreductase, e.g., malate dehydrogenase, lactate dehydrogenase, glyoxylate reductase, or glucose 6-phosphate dehydrogenase, or a glycosidase, e.g., lysozyme or amylase.

Illustrative opiates which can be bound to an enzyme include morphine, heroin, hydromorphone, oxymorphone, metopon, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodeinone, pholcodine, dextromethorphan, phenazocine, and dionin and their metabolites.

Preferred compounds have W, or W as X*-A* or have W and W taken together to provide A*X- *CHCH CH or A*X*CH-CH=CH. Methadone Another group of compounds having narcotic activity is methadone and its analogs, which for the most part have the following formula:

wherein:

any one of the W groups can be X*; X*, A*, and'n have been defined previously; p is or 1, usually being the same in both instances;

q is 2 or 3;

W is hydrogen;

W and W are hydrogen, alkyl of from one to three carbon atoms, e.g., methyl, or may be taken together to form a six-membered ring with the nitrogen atom to which they are attached, e.g., pentylene-l,5 and 3-oxa or 3-azapentylene-l,5;

W is hydrogen or methyl, only one W being methyl;

W is hydrogen; W is hydrogen or hydroxyl; W is hydrogen, acyloxy of from one to three carbon atoms, e.g., propionoxy, or hydroxy (when W and W are both hydroxy, the oxo group is intended); and

W" is hydrogen or alkyl of from one to three carbon atoms, e.g., ethyl.

Illustrative compounds which can be linked to an enzyme are methadone, dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon) and acetylmethadol.

Metabolites of methadone and methadone analogs are also included. Among the metabolites for methadone is N-methyl 2-ethyl 3,3-diphenyl-5- methylpyrroline.

Preferred compounds are when W or W is -X*.

A narrower class of methadone and its analogs are of 55 the formula:

wherein: 7 any one of the W groups can be X*;

. gen atom and the carbon atom to which W"" X*, A* and n have been defined previously;

W and W' are hydrogen;

W' and W are methyl or are taken together with the nitrogen atom to which they are attached to form a morpholino or piperidine ring;

W and W*' are hydrogen, hydroxy, acetoxy, at least one being hydroxy or acetoxy; and

W is alkyl of from one to three carbon atoms.

, The methadone derivatives will for the most part have the following formula:

w 'o-owncn,onmormw H 1 I A wherein:

one of W" or W" is X**; X**, A**, and n have been defined previously; (1) is phenyl; I

when other than X** W" is methyl; and

W"" is propyl.

The metabolites of methadone and close analogs will for the most part have the following formula:

wherein:

any one of the W groups can be X*, X*, A* and n have been defined previously;

is hydrogen,. hydroxyl, methoxyl or acetoxyl, that is of from one to two carbon atoms, and except when hydrogen of from one to two oxygen atoms;

W is hydrogen, methyl, or a free valence joined with W W is an unshared pair of electrons;

W is hydrogen or methyl; I

W is hydrogen, hydroxy, or taken together with W""' forms a double bond between the nitroand W are respectively attached; and

W""' is alkyl of from one to three carbon atoms, usually two carbon atoms, or maybe taken together with W to form alkylidenyl of from one to three carbon atoms, usually two carbon atoms.

Preferred compounds are those where W or W""' are X*, particularly W with W"' as methyl. I

phenylbenzyl( l- The third group of compounds which have narcotic activity and are meperidine or meperidine analogs, have for the most part the following formula:

wherein:

any one of the W groups can be X*;

X*, A*, and n have been defined previously;

W is hydrogen;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl, aminophenylalkyl, e.g., B-(p aminophenyl)ethyl, or phenylaminoalkyl, e.g., phenylaminopropyl, (alkyl of from two to three carbon atoms);

W is alkoxy of from one to three carbon atoms, e. g., ethoxy; and

W is hydrogen or methyl.

Illustrative compounds are meperidine, alphaprodine, alvodine and anileridine.

Preferred compounds are those where W or W is X* or a hydrogen of W is replaced with X*. indole Alkaloids A second group of ligands of interest are based on tryptamine and come within the class of indole alkaloids, more specifically ergot alkaloids. These compounds will have the following minimal structure:

wherein the free valences are satisfied by a variety of groups, primarily carbon and hydrogen, although other substituents may be present such as carboxyl groups, hydroxyl groups, keto groups, etc. The most common member of this class which finds use is lysergic acid, primarily as its diethylamide. Other members of the indole alkaloid family which can also be assayed for are the strychnine group and the indolopyridocoline group, which finds yohimbine and reserpine as members.

The enzyme substituted indole alkaloids will have the following formula:

wherein m, X and A have been defined previously.

Other groups of alkaloids include the steroid alkaloids, the iminazolyl alkaloids, the quinazoline alkaloids, the isoquinoline alkaloids. the quinoline alkaloids, quinine being the most common, and the diterpene alkaloids.

For the most part, the alkaloids bonded to an enzyme will be of from about 300 to 1,500 molecular weight, more usually of from about 400 to 1,000 molecular weight. They are normally solely composed of carbon, hydrogen, oxygen, and nitrogen; the oxygen is present as oxy and 0x0 and the nitrogen present as amino or amido. Catecholamines The first group in this category are Catecholamines of the formula:

wzm xv i w w W was I o-w '37 wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or alkyl of from one to three carbon atoms, e.g., methyl;

W is hydrogen, or alkyl of from one to three carbon 5 atoms, e.g., methyl;

Illustrative compounds include cotainine, narceine,

. noscapine and papaverine.

Preferred compounds are where W W or W are X* or have a hydrogen replaced with X*.

A group of compounds related to the Catecholamines are epinephrine, amphetamines and related compounds. These compounds have the formula:

wherein: any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen or alkyl of from one to three carbon atoms, e.g., methyl and isopropyl, preferably one is hydrogen;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl and ethyl, or may be taken together with W to form a ring having six annular members with the nitrogen as the only heteroatom;

W is hydrogen, hydroxyl, carbomethoxy, or may be taken together with W" to form a morpholine ring;

W is carbomethoxy, when W and W are taken together to form a pip'eridine ring; and

W and W are hydrogen, hydroxyl or alkoxyl of from one to three carbon atoms.

Illustrative compounds which can be bonded to an enzyme are ephedrine, epinephrine, L-dopa, benzidrine (amphetamine), paredrine, methamphetamine, methyl phenidate and norephedrine.

Illustrative compounds which can be linked to an enzyme include 3-(3,4'-dihydroxyphenyl)-3-hydroxypropionic acid, N-(B-(B,3,4-trihydroxyphen)ethyl) N- methyl glycine, N-(l-phenyl-2-propyl)oxalamic acid, l-phenyl-2-methylamino-l -propyl)glycolic acid, p-

(2-methylaminopropyl-l )phenoxyacetic acid, N-( l '-phenyl-2-propyl) glycine, 4-methylamino-4-phenylvaleric acid, para-( 2- aminopropyl-l )phenoxyacetic acid,

4-methylamino-5-phenylvaleric acid, and 3-amino-4- phenylbutyric acid.

Where W and W are hydrogen, preferred compounds will have the following formula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W' and W are hydrogen or alkyl of from one to three carbon atoms, preferably one is hydrogen;

W is hydrogen, methyl or may be taken together with W' to form a piperidine ring;

W is hydrogen, hydroxyl or carbomethoxy; and

W is hydrogen.

Where W and W" are oxy, the preferred compounds have the following formula:

W43" is hydrogen or hydroxyl; and ndcfliila e rhydwayl rr rncthq y W Closely related compounds to the amphetamines are those where a saturated five or six membered ring is substituted for the phenyl ring. These compounds will have the following formula:

wherein:

any oneof the W groups is X*; X*, A* and n have been defined previously;

W' have been defined above; W' is hydrogen or methyl; W' is hydrogen or hydroxyl;

W is hydrogen; and

thyl)barbituric b is an integer of from four to five. Of particular interest are those amphetamines bonded to enzymes of the following formula:

W is hydrogen;

W is methyl; and

W"' is hydrogen;

W is hydrogen or methyl;

X** is ZCO, wherein Z is hydrocarbylene of from one to seven carbon atoms, usually aliphatic, having from 0 to I site of ethylenic unsaturation, with the proviso that when W"' is -X**, X** is O-ZCO-;

A** and n have been defined previously. Barbiturates A wide class of synthetic drugs which finds extensive and frequent abuse are the barbiturates. These compounds are synthetically readily accessible and their use only difficultly policed. The compounds which find use will come within the following formula:

wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl or alkali metal, e.g., sodium;

W and W are hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, or aryl hydrocarbon of from one to eight, more usually one to six carbon atoms, e.g., ethyl, nbutyl, a-methylbutyl, isoamyl, allyl, A -cyclohexenyl, and phenyl;

W is hydrogen, or alkali metal, e.g., sodium;

W is oxygen or sulfur.

Illustrative compounds are veronal, medinal, luminal, prominal, soneryl, nembutal, amytal, dial, phenadorn. seconal, evipan, phenobarbital and pentothal.

Preferred compounds would have W or W or a hydrogen of W or W as --X*. Also preferred is when one of W and W is hydrocarbyl of from two to eight carbon atoms.

Illustrative compounds which may be linked to an enzyme include 5,5-diethyl-l-carboxymethylbarbituric acid, S-ethyl-S-n-butyl-l-succinoylbarbituric acid, 5- ethyl-S-phenyll N 2 '-chloroethyl )-2 '-aminoeacid, 5-(2'-carboxy-A" cyclohexenyl )-l ,S-dimethylbarbituric acid, carboxymethyl phenobarbital, S-(y-crotonic acid)-5- (2'-pentyl)-barbituric acid, 5-(p-aminophenyl)-5- ethylbarbituric acid, 5-(5'-pentanoic acid) -5-(2'-pen- 19 tyl)barbituric acid, and l-methyl--ethyl-5-(p-carboxyphenyl)barbituric acid.

Of particular interest are those barbiturates bonded to an enzyme of the formula:

wherein one of W and W is X**; when other than X**:

W is hydrogen, methyl or alkali metal, e.g., sodie; and

W is hydrocarbon of from one to eight carbon atoms, having from 0 to 1 site of ethylenic unsaturation;

W is hydrocarbon of from two to eight carbon atoms, having from O to 1 site of ethylenic unsaturation;

X** is ZCO, wherein Z is hydrocarbylene of from one to seven carbon atoms, usually aliphatic, having from O to 1 site of ethylenic unsaturation;

A** and n have been defined previously.

Glutethimide Another compound of interest is glutethimide, wherein the enzyme bound analog will have the following formula: 3 0

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen; and

W is lower alkyl of from one to three carbon atoms, e.g., ethyl. Cocaine A drug of significant importance in its amount of use is cocaine. The enzyme bound cocaine or cocaine metabolites or analogs, such as ecgonine, will for the most part have the followingformula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydroxy, methoxy, amino or methylamino;

W is hydrogen or benzoyl; and 60 W is hydrogen or alkyl of from one to three carbon atoms, e.g., methyl.

Of particular interest are those ecgonine derivatives (including cocaine derivatives) of the formula:

wherein one of W and W when other than X**:

W is hydrogen or benzoyl; and W is methyl;

W is hydroxy or methoxy; X** is wherein Z" is methylene or carbonyl; or

Z-CO wherein Z is hydrocarbylene of from one to seven carbon atoms, usually aliphatic, having from 0 to I site of ethylenic unsaturation;

A** and n have been defined previously. Diphenyl Hydantoin Another compound of interest is the antiepileptic drug diphenyl hydantoin. This compound and its analogs will have the following formula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

()5 is phenyl;

W W" and W are hydrogen. Marijuana Because of its ready availability and widespread use, tetrahydrocannabinol (the active ingredient of mari juana) and its congeners, cannabidiol and cannabinol and their metabolites are compounds of great interest, where a simple assay method would be of importance. The compounds which find use as analogs have the following formula:

Wino

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W( is hydrogen or carboxyl;

W" is hydroxyl or methoxyl;

W" is hydrogen;

W is pentyl or hydroxypentyl;

W" is hydrogen, methyl, or the two W s may be taken together to form a carbocyclic ring of from 5 to 6 annular members; and

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W" and W are amino.

The next group of tranquilizers are benzdiazocycloheptanes and are known as Librium, Valium, Diazepam, or Oxazepam. These compounds and their related analogs will have the following formula:

wherein:

any one of the W groups can be -X*; X*, A*, and n have been defined previously; W' and W are hydrogen;

W is hydrogen, lower alkyl of from one to three carbon atoms, e.g., methyl, or may be taken together with W to form a double bond between the carbon andthe nitrogen;

W is amino or lower alkylamino of from one to three carbon atoms, e.g., methylamino, or may be taken together with W" to form a carbonyl;

W" is hydrogen or hydroxyl; and

W" is oxy or an unshared pair of electrons.

The next group of compounds are the phenothiazines of which chlorpromazine is a member. These compounds will for the most part have the following forwherein:

any one of the W groups can be X*;

X*, A*, and n have been defined previously;

W" is hydrogen, alkyl of from one to six carbon atoms, dialkylaminoalkyl of from four to eight carbon atoms, e.g., 3-(dimethylamino)propyl; N-hydroxyalkyl (alkyl of from two to three carbon atoms), N'- piperazinoalkyl (alkyl of from two to three carbon atoms), e.g., N-hydroxyethyl N'-piperazinopropyl; N-

'alkyl (alkyl of from one to three carbon atoms) N'- piperazinoalkyl (alkyl of from two to three carbon atoms), e.g., N-methyl N-piperazinopropyl; and Z-(N- alkyl)-piperidinoalkyl, wherein the N-alkyl is of from one to three carbon atoms and the other alkyl is of from two to three carbon atoms, e.g., 2-(N-methyl)- piperidinoethyl, there being at least two carbon atoms between the heteroatoms;

W is hydrogen, chloro, trifluoromethyl, alkylmercapto of from one to three carbon atoms, e.g., methylmercapto and acyl of from one to three carbon atoms, e.g., acetyl; and

W" and W are hydrogen.

Amino Acids, Polypeptides and Proteins The next group of compounds are the amino acids, polypeptides and proteins. For the most part, the amino acids range in carbon content from two to 15 carbon atoms, and include a variety of functional groups such as mercapto, dithio, hydroxyl, amino, guanidyl, pyrrolidinyl, indolyl, imidazolyl, methylthio, iodo, diphenylether, hydroxyphenyl, etc. These, of course, primarily primaril the amino acids related to humans, there being other amino acids found in plants'and animals.

Polypeptides usually encompass from about 2 to amino acid units (usually less than about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associated by covalent or non-covalent bonds. Subunits are normally of from about I00 to 400 amino acid groups (-l0,000 to 50,000 molecular weight).

Individual polypeptides and protein subunits will normally have from about-2 to 400, more usually from about 2 to 300 recurring amino acid groups. Usually, the polypeptides and protein subunits of interest will be not more than about 50,000 molecular weight and greater than about 750 molecular weight. Any of the amino acids may be used in preparing the polypeptide.

Because of the wide variety of functional groups which are present in the amino acids and frequently present in the various naturally occurring polypeptides, the enzyme bonded compound can be bonded to any convenient functionality. Usually, the enzyme bonded compound can be bonded to a cysteine, lysine or arginine, tyrosine or histidine group, although serine, threonine, or any other amino acid with a convenient functionality, e.g., carboxy and hydroxy, may be used.

For the most part, the enzyme-labeled polypeptides will have the following formula:

ine, histidine, methionine, hydroxyproline, tryptophan,

tyrosine, thyroxine, omithine, phenylalanine, arginine, and lysine. Polypeptides of interest are ACTH, oxytocin, lutenizing hromone, insulin. Hence-Jones protein, chorionic gonadotropin, pituitary gonadotropin, growth honnone, rennin, thyroxine bonding globulin,

bradykinin, angiotensin, follicle stimulating hormone, etc.

' In certain instances, it will be desirable to digest a protein and assay for the small polypeptide fragments. The concentration of the fragment may then be related to the amount of the original protein.

Steroids Another important group of compounds which find use in this invention are the steroids, which have a wide range of functionalities depending on their function in the body. In addition to the steroids, are the steroidmimetic substances, which while not having the basic polycyclic structure of the steroid, do provide some of the same physiological effects.

The steroids have been extensively studied and derivatives prepared which have been bonded to antigenic proteins for the preparation of antibodies to the steroids. Illustrative compounds include: l7B-estradiol-6- (O-carboxymethyl-oxime)-BSA (bovine serum albumin) (Exley, et al., Steroids 18 593, (1971 testosterone-3-oxime derivative of BSA (Midgley, et al., Acta Endocr. 64 supplement 147, 320 (1970)); and progesterone-3-oxime derivative of BSA (Midgley, et al., ibid.)

For the most part, the steroids used have the following formula:

wherein m, X and A have been defined previously. Usually, the enzyme will be bonded to the A, B, or C rings, at the 2, 3, 4, 6 or 11 positions, or at the 16 or 17 positions of the D ring or on the side chains at the 17 position. Of particular interest is where X is bonded to the 6 position. The rings may have various substituents, particularly methyl groups, hydroxyl groups, oxocarbonyl groups, ether groups, and amino groups. Any of these groups may be used to bond the enzyme to the basic ring structure. For the most part, the steroids of interest will have at least one, usually l to 6, more usually I to 4 oxygen functionalities, e.g., alcohol, ether, esters, or keto. In addition, halo substituents may be present. The steroids will usually have from 18 to 27 carbon atoms, or as a glycoside up to 50 carbon atoms.

The rings may have one or more sites of unsaturation, either ethylenic or aromatic and may be substituted at positions such as the 6, 7 and l 1 positions with oxygen substituents. In addition, there may be methyl groups at the and 13 positions. The position marked with a Z, 17, may be and will be varied widely depending on the particular steroid. Z represents two monovalent groups or one divalent group and may be a carbonyl oxygen,an hydroxy group, an aliphatic group of from one to eight carbon atoms, including an acetyl group, an hydroxyacetyl group, carboxy or carboxyalkyl of from two to six carbon atoms, an acetylenic group of from two to six carbon atoms or halo substituted alkyl or oxygenated alkyl group or a group having more than one functionality, usually from I to 3 functionalities.

For the second valence of Z, there may be a H or a second group, particularly hydroxyl, alkyl, e.g., methyl, hydroxyalkyl, e.g., hydroxymethyl; halo, e.g., fluoro or chloro, oxyether; and the like.

These steroids find use as hormones, male and female (sex) hormones, which may be divided into oestrogens, gestogens, antrogens, adrenocortical hormones (gluco corticoids), bile acids, cardiotonic glycosides and aglycones, as well as saponins sapogenins.

Steroid mimetic sutstances, particularly sex hormones are illustrated by diethyl stilbestrol.

The sex hormones of interest may be divided into two groups; the male hormones (androgens) and the female hormones (oestrogens).

The androgens which find use will have the following formula:

\I AW...

W m w, y

we: m

0-1 site of cthylenic uusnturution wherein: I

any one of the W groups can be -X*; X*, A* and n have been defined previously; W is hydrogen, or hydroxyl; W is hydrogen, methyl or hydroxy] (when two groups bonded to the same carbon atom are hydroxyl,

oxo is intended);

W and W are hydrogen or hydroxyl, at least one of W is hydroxy (either as hydroxy or oxo);

W is hy drogen or two W 5 may be taken together to form a double bond;

W is methyl; and

W is hydrogen.

Illustrative compounds which may be bonded to an enzyme include testosterone, androsterone, isoandrosterone, etiocholanolone, methyltestosterone and de hydroisoandrosterone.

Illustrative compounds which may be linked to an enzyme include N-carboxymethoxy testosteroneimine, l7-monotestosteronyl carbonate, androsteronyl succinate, testosteronyl maleate, O -carboxymethyl 0'- methyl androst-S-ene-BB, l7B-diol, testosterone O-carboxypropyl oxime and androsteronyl carbonate.

The oestrogens have an aromatic A ring and for the most part have the following formula:

wr2 W WG-1 site of etliylcnic unsaturation W and W" are hydrogen, ethinyl, or hydroxyl (when two hydroxyls are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W is hydroxyl or alkoyl of from one to three carbon atoms;

W is hydrogen or two W s may be taken together to form a double bond; and

W is hydrogen.

Illustrative compounds which may be bonded to an enzyme are equilenin, B-estradiol, estrone, estriol, and

l7-a-ethinyl-estradiol.

Illustrative compounds which may be linked to an enzyme include 3-carboxymethyl estradiol, 2- chloromethylestrone, estrone glutarate, Ocarboxymethyloxime of 6-ketoestradiol, equilenyl N- carboxymethyl thiocarbamate.

Another class of hormones are the gestogens which have the following formula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen or hydroxyl, at least one being hydroxyl (where two hydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W and W are hydrogen or being hydroxyl; and

W is hydrogen, or two W s may be taken together to form a double bond.

Illustrative compounds which may be bonded to an hydroxyl, at least one enzyme include progesterone, pregnenolone, allopregwas Q-l site of thylonic unsatumtion wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or hydroxyl;

W and W are hydrogen or hydroxyl, at least one of which is hydroxyl (when two hydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W W W and W are hydrogen or hydroxyl, at

least one of W and W is hydroxyl;

W is methyl or formyl; and

W is hydrogen or two W s may be taken together to form a double bond.

Illustrative compounds which may be bonded to an enzyme are 17-hydroxydioxycorticosterone (Compound S), deoxycorticosterone, cortisone, corticosterone, l l-dihydrocortisone (Compound F), cortisol, prednisolone and aldosterone.

Illustrative compounds which may be linked to an enzyme include O -carbox ymethyl corticosterone, N- carboxymethyl 2l-carbamate cortisol, 2 l-cortisone succinate, 2l-deoxocorticosterone succinate, and 0"- methyl, O -carboxymethyl cortisone-i An additional steroid family is the cardiotonic glycosides and aglycones of which digitalis is an important member. The basic compound is digitoxigenin, which is also found as the glycoside. The compounds of interest have the following formula:

0-1 site of cthylenic unsaturation CO 5 l l wit-z wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W", W, W" and W are hydrogen, hydroxyl, or a glycoside, at least one being hydroxyl or a sugar, mostly as a glycoside. The sugars include xylose, glucose, cymarose, rhamnose, and galactose.

Also of interest are the saponins and sapogenins derived from plants. These compounds have a spiro ring structure at C Vitamins Sugars The next group of compounds are the sugars and saccharides. The saccharides are combinations of various -l site of ethylenic unsaturatian wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously; W is hydrogen or hydroxyl;

W and. W are hydrogen or hydroxyl (where two hydroxyl groups are bonded to the same carbon atom,

oxo is intended);

W is hydrogen or hydroxyl; and

W is hydroxyl, amino or an oxy group of from one to six carbon atoms, e.g., alkoxy.

Miscellaneous Included in this group are the antibiotics such as penicillin, chloromycetin, antinomycetin, tetracycline, terramycin, and nucleic acids or derivatives, such as nucleosides and nucleotides.

Also of interest is serotonin which is 3-(2- aminoethyl)5hydroxyindole. X* may be bonded at either of the amino nitrogen atoms or the hydroxyl group. 7

Of course, many of the compounds which are of interest undergo metabolic changes, when introduced into a vertebrate. The particular physiological fluid which is tested may have little, if any of the original compound. Therefore, the original presence of the compound might only be detectable as a metabolite. In many instances, the metabolite may be the glucuronide, either oxy or oxo derivative of the original compound. In other instances, theoriginal compound may have undergone oxidation, e.g., hydroxylation, reduction, acetylation, deamination, amination, methylation or extensive degradation. Where the metabolite still retains a substantial portion of the spatial and polar geometry of the original compound, it will be frequently possible to make the ligand analog based on either the original compound or metabolite. Where the metabolite is distinctively different than the original compound, the ligand analog will be based on the metabolite.

Of particular interest as metabolites, particularly of the steroids, are the sulfates and glucuronides.

Besides metabolites of the various drugs, hormones and other compounds previously described, of significant interest are metabolites which relate to diseased states. illustrative of such compounds are spermine, galactose, phenylpyruvic acid and porphyrin Type 1, which are believed to be diagnostic of certain tumors, galactosemia, phenylketonuria and congential porphyra, respectively.

Two compounds of interest which are metabolites of epinephrine are vanillylmandelic acid and homovanillic acid. With these compounds, either the hydroxyl and carboxyl groups can be used as the site for X*.

Another general category of interest is the pesticides, e.g., insecticides, fungicides, bacteriocides and nematocides. Illustrative compounds include phosphates such as malathion, DDVP, dibrom; carbamates, such as Sevin, etc.

Since many of the biologically active materials are active in only one stereoisomeric form, it is understood that the active form is intended or the racemate, where the racemate is satisfactory and readily available. The antibodies will be specific for whatever form is used as the hapten.

Enzymes (A) Enzymes vary widely in their substrates, cofactors, specificity, ubiquitousness, stability to temperature, pH optimum, turnover rate, and the like. Other than inherent factors, there are also the practical considerations, that some enzymes have been characterized extensively, have accurate reproducible assays developed, and are commercially available. In addition, for the purposes of this invention, the enzymes should either be capable of specific labelling or allow for efficient substitution, so as to be useful in the subject assays. By specific labelling is intended selective labelling at a site in relationship to the active site of the enzyme, so that upon binding of the receptor to the ligand, the enzyme is satisfactorily inhibited. By allowing for efficient substitution to be useful in the subject assay, it is intended that the enzyme be inhibited sufficiently when the ligand is bound to the receptor, and that the degree of substitution required to achieve this result does not unreasonably diminish the turnover rate for the enzyme nor substantially change the enzymes solubility characteristics.

From the standpoint of operability, a very wide variety of enzymes can be used. But, as a practical matter, there will be a number of groups of enzymes which are preferred. Employing the International Union of Biochemists (I.U.B.) classification, the oxidoreductases (1.) and the hydrolases (3.) will be of greatest interest, while the lyases (4.) will be of lesser interest. Of the we idoreductases, the ones acting on the CHOH group, the aldehyde or keto group, or the C1-lNl-l group as donors (1.1, 1.2, and 1.4 respectively) and those acting on hydrogen peroxide as acceptor (1.1 1) will be preferred. Also, among the oxidoreductases as preferable will be those which employ nicotinamide adenine dinucleotide, or its phosphate or cytochrome as an acceptor, namely 1.X.l and 1. .2, respectively under the 1.U.B. classification. Of the hydrolases, of particular interest are those acting on glycosyl compounds, particularly glycoside hydrolases, and those acting on ester bonds, both organic and inorganic esters, namely the 3.1 and 3.2 groups respectively, under the 1.U.B. classification. Other groups of enzymes which might find use .are the transferases, the lyases, the isomerases, and the ligases.

In choosing an enzyme for commercialization, as compared to a single or limited use for scientific investigation, there will be a number of desireable criteria. These criteria will be considered below.

The enzyme should be stable when stored for a period of at least 3 months, and preferably at least 6 months at temperatures which are convenient to store in the laboratory, normally 20 C or above.

The enzyme should have a satisfactory turnover rate at or near the pH optimum for binding to the antibody, this is normally at about pH 6 l0, usually 6.0 to 8.0. Preferably, the enzyme will have the pH optimum for the turnover rate at or near the pH optimum for binding of the antibody to the ligand.

A product should be either formed or destroyed as a result of the enzyme reaction which absorbs light in the ultra-violet region or the visible region, that is in the range of about 250750 nm, preferably 300-600 nm.

Preferably, the enzyme should have a substrate (including cofactors) which has a molecular weight in excess of 300, preferably in excess of 500, there being no upper limit. The substrate may either be the natural substrate, or a synthetically available substrate.

Preferably, the enzyme which is employed or other enzymes, with like activity, will not be present in the fluid to be measured, or can be easily removed or deactivated prior to the addition of the assay reagents. Also, one would want that there not be naturally occurring inhibitors for the enzyme present in fluids to be assayed.

Also, although enzymes of up to 600,000 molecular weight can be employed, usually relatively low molecular weight enzymes will be employed of from 10,000 to 300,000 molecular weight, more usually from about 10,000 to 150,000 molecular weight, and frequently from 10,000 to 100,000 molecular weight. Where an enzyme has a plurality of subunits the molecular weight limitations refer to the enzyme and not to the subunits.

For synthetic convenience, it is preferable that there be a reasonable number of groups to which the ligand may be bonded, particularly amino groups. However, other groups to which the ligand may be bonded include hydroxyl groups, thiols, and activated aromatic rings, e.g., phenolic.

Therefore, enzymes will preferably be chosen which are sufficiently characterized so as to assure the availability of sites for linking, either in positions which allow for inhibition of the enzyme when the ligand is bound to antibody, or there exist a sufficient number of positions as to make this occurrence likely.

A list of common enzymes may be found in Hawk, et al., Practical Physiological Chemistry, McGraw-Hill Book Company, New York (1954), pages 306 to 307. That list is produced in total as follows, including the source of the enzyme, the substrate and the end products.

Name & Class Distribution Substrate End-products Hydrolases Carbohydrases Carbohydrates l. Amylase Pancreas, sal- Starch, dex- Maltose and iva, malt, etc. trin, etc. dextrins 2. Lactase Intestinal juice Lactose Glucose and and mucosa galactose 3. Maltase Intestinal juice, Maltose Glucose yeast, etc. 4. Sucrase Intestinal juice Glucose and yeast, etc. Sucrose fructose 5. Emulsin Plants fi-Cvluco- Glucose, etc.

sides Nucleases Nucleic acid and derivatives l. Polynucleo- Pancreatic juice Nucleic Nucleotides 'tidase Intestinal juice acid etc. 2. Nucleoti- Intestinal juice Nucleotides Nucleotides and dase and other tissues phosphoric acid 3. Nucleotidase Animal tissues Nucleotides Carbohydrate and bases Amidases Amino compounds and amides l. Arginase Liver Arginine Ornithine and urea 2. Urease Bacteria, soy- Urea Carbon dioxide bean, jack bean and ammonia etc. 3. Glutami- Liver. etc. Glutamine Glutamic acid nase and ammonia 4. Transaminase Animal tissues Glutamic acid a-Ketogluturic I and oxalacetic acid, aspnrtic acid, etc. acid, etc. Purine Deaminases Purine basesa and derivatives l. Adenase Animal tissues Adenine Hypoxanthine and ammonia 2. Guanase Animal tissues Guanine Xanthine and ammonia Peptidases Peptides l. Aminopolypep- Yeast, intestines Polypeptides Simpler peptidase etc. tides and amino acids 2. Carboxypcp- Pancreas Polypeptides Simpler peptidase tides and amino acids oxidase 3. Peroxidase Enzymes Containing Coenzymes l and/or ll 1. Alcohol dehydrogenase 2. Malic dehydrogenase 3. lsocritric dehydrogenase 4. Lactic dehydrogenase 5. fl-Hydroxybutyric dehydrogenase 6. Glucose dehydrogenase 7. Robison ester dehydrogenase Glycerophosphate dehydrogenase 9. Aldehyde dehydrogenase ganisms except a few species of microorganisms Nearly all plant cells Plant and animal tissues Plant tissues Animal and plant 7 tissues Animal and plant tissues Animal and plant tissues gmimal tissues nd yeast Liver, kidneys, and heart Animal tissues Erythrocytes and yeast Animal tissues Liver tochromc C in the presence of oxygen A large number of phenols aromatic amines etc.

in the presence of H 0 Various phenolic compounds Ascorbic acid in the presence of oxygen Ethyl alcohol and other alcohols L( Malic acid L-lsocitric acid Lactic acid acid D-Glucose Robison ester (hexose--phosphate Glycerophosphate Aldehydes Name 8!. Class Distribution 'Substrate End-products 3. Dipeptidasc Plant and animal Dipeptides Amino acids tissues and bacteria 4. Prolinase Animal tissues Proline Proline and a and yeast peptides simpler pep- V v Mm, E@S

Proteinases Proteins l. Pepsin Gastric juice Proteins Proteoses,

peptones, etc. 2. Trypsin Pancreaticjuice Proteins, Polypeptides proteoses, and amino acid and peptones '3. Cathepsin Animal tissues Proteins Proteoses, V and peptones 4. Rennin Calf stomach Casein Paracasein 5. Chymotrypsin Pancreatic juice Proteins, Polypeptides proteoses and amino acid and peptones 6. Papain Papaya, other Proteins.

plants proteoses,

and peptones 7. Ficin Fig sap Proteins Proteoses,

"Ji .W Esterases Esters Alcohols and acids l. Lipase Pancreas, castor Fats Glycerol and bean, etc. fatty acids 2. Esterases Liver. etc. Ethyl buty- Alcohols and rate, etc. acids 3. Phosphatases Plant and animal Esters of Phosphate and tissues phosphoric alcohol acid 4. Sulfatases Animal and plant Esters of Sulfuric acid tissues sulfuric and alcohol acid 5. ChOlitleS Blood. tissues Acetylcho- Choline and te rase line A acetic a cid iron Enzymes l. Catalase All living or- Hydrogen Water and ganisms except a peroxide oxygen few species of microorganisms 2. Cytochrome All living or- Reduced cy- Oxidiied cytochrome C and water Oxidation product of substrate and water Oxidation product of substrate Dehydroascorbic acid Acetaldehyde and other aldehydes Oxalacetic acid Oxalosuccinic acid Pyruvic acid Acetoacetic acid D-Giuconic acid Phosphohexonic acid Phosphogylceric acid W Acids Name & Class Distribution Substrate End-products Enzymes which Reduce Cytochrome l. Succinic de- Plants, animals Succinic Fumaric acid hydrogenase and microoracid (as ordinarily) ganisms prepared) l. Warburgs old Yeast Reduced co- Oxidized co yellow enzyme enzyme ll enzyme I! and reduced yellow enzyme 2. Diaphorase Bacteria, Reduced co- Oxidized coyeasts, higher enzyme 1 enzyme 1 and plants, and anireduced yelmals low diaphorase 3. Haas enzyme Yeast Reduced co- Oxidized coenzyme ll enzyme H and reduced yel- 4. Xanthine oxidase 5. D-amino acid oxidase 6. L-Amino acid oxidases 7. TPN-Cytochrome C reductase 8. DPN Cytochrome C reductase Hydrases l. Fumarase 2. Aconitase 3. Enolase Mutases i. Oiyoxalase Desmolases 1. Zymohexase (aldolase l 2. Carboxylase 3. B-Keto carboxylases 4. Amino acid decarboxylases 5. Carbonic anhydrase Other Enzymes l. Phosphorylase 2. Phosphohexoisomerase 3. Hexokinase 4. Phosphoglucomutase Animal tissues Animal tissues Animals, snake venoms Yeast, liver Liver, yeast Living oranisms in general Animals and plants Animal tissues and yeast Living organisms in general All cells Plant tissues Animals, bacteria, plants Plants, animals, bacteria Erythrocytes Animal and plant tissues Animal and plant tissues Yeast, animal tissues Plant and animals hypoxanthine xanthine, aldehydes, reduced coenzyme l, etc.

D-Amino Acids 0 L-amino acids Reduced co-;, enzyme ll and cytochrome C Reduced c0- enzyme l and cytochrome C Fumaric acid H O Citric acid 2-Phosphoglyceric acid Methyl glyoxal and other substituted glyoxals Fructosel .6-diphosphate Pyruvic acid B-Keto acids L-Amino acids Carbonic acid Starch or glycogen and phosphate Glu c0se-6- phosphate Adenosinetriphosphate Glucose-lphosphate L-Malic acid cis-Aconitic acid and L- isocitric acid Phospyruvic acid H O D Lactic acid Dihydroxyacetone phosphoric acid and phosphoglyceric acid Acetaldehyde and C0 a-Keto acids Amines and C0, C0; H O

Glucosel phosphate Fructose-6 phosphate Adenosinediphosphate glucose- 6-phosphate Glucose-6- phosphate l. Oxidoreductases 1.1 Acting on the CH-OH group of donors 1.1.1 With NAD or NADP as acceptor l.'alcohol dehydrogenase 6. glycerol dehydrogenase 26. glyoxylate reductase 27. L-lactate dehydrogenase 37. malate dehydrogenase 49. glucose 6-phosphate dehydrogenase 17. mannitol 1-phosphate dehydrogenase 1.1.2 With cytochrome as an acceptor 3. L-lactate dehydrogenase 1.1.3 With as acceptor 4. glucose oxidase 9. galactose oxidase 1.2 Acting on the CH-Nl-l group of donors 1.4.3 with 0 as acceptor 2. L-amino acid oxidase 3. D-amino acid oxidase 1.6 Acting on reduced NAD or NADP as donor 1.6.99 With other acceptors diaphorase 1.10 Acting on diphenols and related substances as donors 1.10.3 With-O as acceptor l. polyphenol oxidase 3. ascorbate oxidase 1.1 1 Acting on H O as acceptor 1.1 1.1

6. catalase 7. peroxidase 3. Hydrolases 3.1 Acting on ester'bonds 3. l .1 Carboxylic ester hydrolases 7. cholinesterase 3.1.3 Phosphoric monoester hydrolases l. alkaline phosphatase 3.1.4 Phosphoric diester hydrolases 3. phospholipase C 3.2 Acting on glycosyl compounds 3.2.1 Glycoside hydrolases l. a-amylase 4. cellulase 17. lysozyme 23. B galactosiclase 27. amyloglucosidase 31. B-glucuronidase 3.4 Acting on peptide bonds 3.4.2 Peptidyl-amino acid hydrolase 1. carboxypeptidase A 3.4.4 Peptidyl-peptide hydrolase 5. a-chymotrypsin 10. papain 3.5 Acting on C-N bonds other than peptide bonds 3.5.1 in linear amides 5. urease 3.6 Acting on acid anhydride bonds 3.6.1 In phosphoryl-containing anhydrides l. inorganic pyrophosphatase 4. Lyases 4.1 Carbon-carbon lyase 4.1.2 Aldehyde lyases 7. aldolase 4.2 Carbon-oxygen lyases 4.2.1 Hydrolases l. carbonic anhydrase 4.3 Carbon-nitrogen lyases 4.3.1 Ammonia lyases 3. histidase ies will recognize that portion of the ligand molecule which extends from the protein, ordinarily the same linking group will be attached on the same site on the ligand, as was used in bonding the ligand to the protein to provide the antigenic substance.

The functional groups which will be present in the enzyme for linking are amino (including guanidino), hydroxy, carboxy, and mercapto. In addition, activated aromatic groups or imidazole may also serve as a site for linking.

Amino acids having amino groups available for linking include lysine, arginine, and histidine. Amino acids with free hydroxy] groups include serine, hydroxyproline, tyrosine and threonine. Amino acids which have free carboxyl groups include aspartic acid and glutamic acid. An amino acid which has an available mercapto group is cysteine. Finally, the amino acids which have activated aromatic rings are tyrosine and tryptophan.

In most instances, the preferred linking group will be the amino group. However, there will be situations with certain enzymes. where one of the other linking groups will be preferred.

The ligand, of course, will have a great diversity of functionalities which may be present. In addition, as already indicated, the functionalities which are present may be modified so as to form adifferent functionality, e.g., keto to hydroxy or an olefin to aldehyde or carboxylic acid. To that extent, the choice of groups for linking to the ligand may be varied much more widely than the choice of groups for linking to the enzyme. In both cases, however, a wide variety of different types of functionalities have been developed, specifically for linking various compounds to proteins and particularly enzymes.

Where a linking group is employed for bonding the ligand to the enzyme. it will be the more frequent procedure to bond the linking group to the ligand to provide an active site for bonding to the enzyme. This may be achieved in a single step or may require a plurality of synthetic steps, including blocking and unblocking the active groups on the ligand, other than the one involved in providing the linking group. The linking groups which are reported hereafter are solely concerned with the bridge bonding the enzyme and the ligand.

Where a linking group is used, there will normally be from one atom to 14 atoms in the chain, more usually from two atoms to eight atoms in the chain bonding the ligand to the enzyme. Where cyclic structures are involved, the cyclic structure will be equated to the number of atoms providing a similar length to the chain.

The linking group (excluding the atoms derived from the ligand and enzyme), when other than a direct bond is involved, will be of from about one to 30 atoms carbon, hydrogen, nitrogen, oxygen, phosphorous, and sulfur more usually four to atoms.

Preferably, the linking group will normally be of from zero to 14 carbon atoms, usually from one to eight carbon atoms and from one to eight heteroatoms, and frequently of from one to eight carbon atoms and from one to four heteroatoms, which are oxygen, sulfur and nitrogen, more usually oxygen and nitrogen. The most frequent heterofunctionalities present in the linking group are nonoxocarbonyl or thiocarbonyl, amino, l5

imino (oxime or imidate) diazo, or oxy.

A group of linking groups are derived from a group having a nonoxocarbonyl functionality and when a second functionality is present, the second functionality may be based on a second nonoxocarbonyl functionality, a haloalkyl, O-substituted hydroxylamine, imino, amino or diazo. The linking group will normally have from two to eight carbon atoms and from one to four heteroatoms which are usually oxygen and nitrogen (the heteroatoms of the ligandand enzyme are not included in the above range of heteroatoms). Such determination is somewhat arbitrary, so that between a carbon atom of the ligand and a carbon atom of the enzyme, there may be as many as six heteroatoms. The heteroatoms may be part of the linking group chain or branched from the chain, e.g., nonoxocarbonyl oxygen.

One group of linking groups will have from two to six carbon atoms, more usually two to four carbon atoms and be an aliphatic non-oxo carbonyl functionality. Another group of linking groups will have from two to eight carbonatoms and have from one to two heteroatoms, e.g., oxygen and nitrogen, in the chain, such as amino, oximino, diazo, oxy, and the like.

The following tabulation indicates various linking groups, varying with the functionalities present on the Ligand Enzyme Ligand Enzyme (only primary amino) ll L S ll .07,

ligand and the enzyme. Except as indicated, the linking group satisfies one to two valences on the ligand and enzyme functional groups to which it is bound.

2- bond, hydrocarbylene of from one to IO carbon atoms, more specifically alkylene of from one to six carbon atoms, alkenylene of from two to six carbon atoms, alkynylene of from two to six carbon atoms, cycloalkylene of from four to 10 carbon atoms and arylene of from six to l0 carbon atoms; oxaalkylene of from four to eight carbon atoms; and azaalkylene of from four to eight carbon atoms;

R alkyl of from one to six carbon atoms;

R hydrogen or alkyl of from one to three carbon atoms;

Z or non-0x0 carbonyl are preferred for bonding to hydroxyl, while non-0x0 carbonyl, non-0X0 thiocarbonyl and Z are preferred with amino.

Ligand Enzyme Oxocarbonyl C:O) Amino (-Nll2), hydroxy] will) or innrt'nplo (S ll).

Z" arylene of from six to 10 carbon atoms. Where the enzyme is to be linked through a carboxyl group of the ligand or a linking group bonded to the ligand, either esters or amides will be prepared. The ligand may be bonded to any of the linking groups which are appropriate to provide a link between the ligand and the alcohol or amine group of the enzyme to form the ester or amide group respectively. When the enzyme has an activated aromatic ring, the ligand may be bonded to an aromatic diazonium salt to provide the desired bridge.

Claims (11)

1. AN ENZYME-BOUND-LIGAND OF THE FORMULA:

2. An enzyme-bound-ligand of the formula:

3. An enzyme-bound-ligand according to claim 2, wherein A** is an enzyme of from 10,000 to 300,000 molecular weight and n'' is in the range of 2 to 20.

4. An enzyme-bound-ligand according to claim 3, which is an oxidoreductase.

5. An enzyme-bound-ligand according to claim 3, which is a hydrolase.

6. An enzyme-bound-ligand according to claim 3, which is O3-carboxymethylmorphine conjugate to lysozyme having from 2 to 4 O3-carboxymethylmorphine groups.

7. An enzyme-bound-ligand according to claim 3, which is O3-carboxymethylmorphine conjugate to malate dehydrogenase having from 2 to 22 O3-carboxymethylmorphine groups.

8. An enzyme-bound-ligand according to claim 3 which is O3-carboxymethylmorphine conjugate to glucose 6-phosphate dehydrogenase having from 2 to 22 O3-carboxymethylmorphine groups.

9. An enzyme-bound-ligand according to claim 3 which is O3-( Alpha -isopropyl)carboxymethylmorphine conjugate to malate dehydrogenase having from 2 to 22 O3-( Alpha -isopropyl)carboxymethylmorphine groups.

10. An enzyme-bound-ligand according to claim 3 which is O3-( Alpha -isopropyl)carboxymethylmorphine conjugate to glucose 6-phosphate dehydrogenase having from 2 to 22 O3-( Alpha -isopropyl)carboxymethyl morphine groups.

11. An enzyme-bound-ligand according to claim 3, which is O3-imidoylmethylmorphine conjugate to lysozyme, glucose 6-phosphate dehydrogenase or malate dehydrogenase.