US3875011A

US3875011A - Enzyme immunoassays with glucose-6-phosphate dehydrogenase

Info

Publication number: US3875011A
Application number: US438890A
Authority: US
Inventors: Kenneth Edward Rubenstein; Edwin F Ullman
Original assignee: Syva Co
Current assignee: Syva Co
Priority date: 1972-11-06
Filing date: 1974-02-01
Publication date: 1975-04-01
Anticipated expiration: 1992-04-01

Abstract

Novel conjugated enzyme compositions are provided for use in homogeneous enzyme immunoassays. A wide variety of haptenic compounds, particularly drugs of abuse, and drugs used in repetitive therapeutic applications, and steroids are conjugated to glucose-6-phosphate dehydrogenase. The resulting product has a higher turnover rate, so as to provide a high multiplication factor when employed in a homogeneous enzyme immunoassay.

Description

United States Patent [191 Rubenstein et al.

[ Apr. 1, 1975 ENZYME IMMUNOASSAYS WITH GLUCOSE-6-PHOSPHATE DEHYDROGENASE [75] Inventors: Kenneth Edward Rubenstein, Palo Alto; Edwin F. Ullman, Atherton, both of Calif.

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

[22] Filed: Feb. 1, 1974 [21] Appl. No.: 438,890

Related US. Application Data [63] Continuation-impart of Ser. No. 304.157, Nov. 6.

1972, which is a continuation-in-part of Ser. No. 143,609, May 14, I971, abandoned.

[52] U.S. Cl. 195/99, 195/1035 R [51] Int. Cl C07g 7/02 [58] Field of Search... 195/63, 99, 103.5 R, DIG. ll

[56] References Cited UNITED STATES PATENTS 3,791,932 2/1974 Schuurs et a1. 195/1035 R Primary Examiner-Alvin E. Tanenholtz [57] ABSTRACT Novel conjugated enzyme compositions are provided for use in homogeneous enzyme immunoassays. A wide variety of haptenic compounds, particularly drugs of abuse, and drugs used in repetitive therapeutic applications, and steroids are conjugated to gluc0se-6-phosphate dehydrogenase. The resulting product has a higher turnover rate, so as to provide a high multiplication factor when employed in a homogeneous enzyme immunoassay.

21 Claims, No Drawings ENZYME IMMUNOASSAYS WITH GLUCOSE-6-PHOSPHATE DEHYDROGENASE CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application Ser. No. 304,157, filed Nov. 6, 1972, which is in turn a continuation-in-part of application Ser. No. 143,609, filed May 14, 1971, now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention lmmunoassays have shown themselves to be extremely versatile in allowing for methods to determine the presence of a particular substance, even when a wide variety of other materials of similar or different structure are present in the unknown sample. The immunoassays rely on the ability of an antibody to specifically detect or bind to an haptenic organic compound, while not interacting with other compounds. The divalent nature of the antibody and its high molecular weight, 150,000 or greater, allow for a sufficient change in the compound or environment of the compound to permit a discrimination between a compound which is bound and a compound which is not bound to antibody. Among various immunoassays involving antibodies are radioimmunoassay, spin immunoassays, available under the trademark FRAT, supplied by Syva Company. homogeneous enzyme immunoassay, available under the trademark EMIT, supplied by Syva Company, and hemeagglutination.

The enzyme immunoassay is extremely versatile in permitting spectrophotometric determinations. The immunoassay employs an enzyme to which the organic compound to be determined is conjugated. The organic compound is conjugated at a position where when bound to antibody, the activity of the enzyme is substantially reduced. To the extent that the unknown sample contains the same organic compound, the amount of antibody available for binding to the organic compound conjugated to the enzyme is reduced. Therefore, by analyzing for enzymatic activity, a significant increase in enzymatic activity over the enzymatic activity in the absence of the unknown indicates the presence of the organic compound in the unknown.

The sensitivity of the homogeneous enzyme immunoassay is based to a substantial degree on the activity of the enzyme when conjugated and the degree of inhibitability when antibody is bound to the organic compound conjugated to the enzyme. It is, therefore, desirable to have an enzyme which not only has a high turnover rate initially, but retains a substantial proportion of this turnover rate after conjugation, and is strongly inhibited when antibody is bound to the organic compound which is conjugated to the enzyme. Also, the enzyme should allow for strong specific binding of antibody to the conjugated organic compound.

2. Description of the Prior Art An homogeneous enzyme immunoassay system has been sold under the trademark EMIT employing haptens conjugated to lysozyme, where the enzymatic activity is determined by the reduction in turbidity as a result of lysis of bacterial walls. Numerous publications concerning the system have issued since June of 1971, see for example, Rubenstein, et al., Biochem & Biophysical Res. Comm. 47 846 (1972). US. Pat. No. 3,654,090 teaches a heterogeneous immunoassay em- SUMMARY OF THE INVENTION Haptenic conjugates to glucose-6-phosphate dehydrogenase are provided for employment in homogeneous enzyme immunoassays to provide high sensitivity in detecting extremely small amounts of organic materials. One or more of the haptens (hereinafter referred to as ligands) are conjugated by relatively short chains or linking groups to the glucose-6-phosphate dehydrogenase to provide a product still retaining a substantial proportion of the original enzyme activity and having a high degree of inhibitability, usually in excess of 50% of the original activity of the conjugated glucose-6- phosphate dehydrogenase. The linking chains normally employ a non-oxocarbonyl group or a covalent bond to saturated carbon as the linking group to the enzyme.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Haptenic conjugated glucose-6-phosphate dehydrogenase is provided having from about 1 to 18, usually from about 2 to 14, and more usually from about 2 to 12 ligands, normally the majority or all of the ligands being bonded to amino groups, particularly of lysine. The haptens or ligands will normally have molecular weights of at least and generally not exceeding 1,000, more usually not exceeding 800, and frequently not exceeding 600 molecular weight. The ligands will have at least one heteroatom and may have two or more heteroatoms, which will normally be oxygen, nitrogen and sulfur, although halogen, particularly chlorine and iodine may also be present. The ligands for the most part will be naturally occurring. physiologically active compounds and synthetic drugs, which will be modified to the extent necessary for conjugation to the glucose-6-phosphate dehydrogenase.

The enzyme conjugates of this invention will for the most part have the following formula:

wherein:

G6PDH intends glucose-6-phosphate dehydrogenase;

11 indicates the average number of groups bonded to the G6PDH and will generally be in the range of l to 18, more usually in the range of 2 to 14, and particularly in the range of 2 to 12;

R is a bond or a hydrocarbon (aliphatic, alicyclic or aromatic), particularly aliphatic, linking group, either branched or straight chain, of from 0 to 1 rings and of from 1 to 8 carbon atoms, more usually of from 1 to 6 carbon atoms, and preferably of from 1 to 4 carbon atoms, usually having from 0 to 1 site of aliphatic unsaturation, and more usually aliphatically saturated, or substituted hydrocarbon group having from 0 to 3 heteroatoms, more usually 0 to 2 heteroatoms, which are oxygen, sulfur and nitrogen, more usually oxygen and nitrogen (atomic number 7-8); and

Y is a ligand of at least 125 molecular weight, usually not greater than 1,000 molecular weight, more usually not greater than 800 molecular weight, and generally not exceeding 600 molecular weight, and has at least one common epitope to a naturally occurring physiologically active compound or synthetic drug, usually differing from the naturally occurring physiologically active compound or synthetic drug by replacement of a hydrogen or modification of a functionality such as an olefin, oxo or the like, to provide a site for bonding of R to the ligand; and

X is a bond, a non-oxocarbonyl group, including the nitrogen and sulfur analogs thereof, i.e. imino and thiocarbonyl, or diazo when R is arylene, aralkylene or a bond and the nitrogen of the diazo group is bonded to an aromatic annular carbon atom.

X may be bonded to R through carbon or a heteroatom, particularly nitrogen. Since sulfur bonds and certain oxygen bonds, e.g. esters will tend to be reactive, these will usually be avoided. Oxygen will normally be present in the linking group as a carbonyl (x0 or nonoxo) or oxyether. Sulfur will normally be present in the linking group as thiocarbonyl or thioether. Nitrogen will normally be present in the linking group as tertiary or quaternary amino, diazo or bonded to a nonoxocarbonyl, including the amino and thioanalogs thereof.

Usually, when R is aromatic (aromatic includes arylene, aralkylene or alkarylene), R will be bonded to Y through a heteroatom, particularly ethereal oxygen, i.e. oxy. R groups of particular interest are methylene or polymethylene, i.e. (CH- where p is an integer in the range of l to 6, alkyleneoxyalkylene, i.e. (CH ),,O(CH where q and r are the same or different and are integers in the range of l to 3, there being at least two methylene groups between heteroatoms, or (CH ,NH, where s is an integer in the range of l to 6, usually 1 to 4, there being at least two methylene groups between heteroatoms.

When X is other than a bond, X will normally have one of the following formulae:

and will preferably be either the oxygen or the imino non-oxocarbonyl.

The groups for R-X will include ethylene,

propylene, butylene, hexylene, phenylene, pbenzylylene, a-carboxymethine, carbamoylmethylene (-NHCOCH iminoxyacetyl (=NOCH CO-), thrioacetyl, p-oxybenzyl, maleidioyl, succindioyl, oxoethylene (-OCCH loxobutylene(-OCCH CH CH ethyleneoxyacetyl, propyleneoxyacetyl, N-methyl 3-aza-l-imino-pentyethylenecarbamoyl (-(O=C)NHCH CH propylenethiocarbamoyl ((S-C)NHCH CH C- H ethyleneoxyacetimidate, ethyleneoxyethylenethiocarbamoyl, propyleneoxypropylenecarbamoyl and diethyleneoxyacetimidoyl,

Turning now to consideration of individual compounds, the first group of compounds are the alkaloids. Of particular interest among the alkaloids are the opiate alkaloids which will have for the most part the following formula:

G- 6 -PDH -X R O wherein:

T is hydrogen or acetyl, usually hydrogen,

n is on the average 1 to 14, usually 1 to 12, more usually 2 to 12;

R may be the same as R, but will usually be either (1) an aliphatic group, either branched or straight chain, having from O to 1 site of aliphatic unsaturation, e.g. ethylenic and of from 1 to 8 carbon atoms, more usually of from 1 to 6 carbon atoms, and preferably of from 1 to 4 carbon atoms and has from O to 3, usually 0 to 2 heteroatoms, which are oxygen, sulfur or nitrogen, usually oxygen and nitrogen, and bonded to X with other than sulfur and oxygen, and bonded to oxygen through carbon, wherein the oxygen is present as oxocarbonyl or oxy, particularly ether, and the nitrogen is present as tertiary amino; or (2) aromatic hydrocarbon, e.g. arylene, alkarylene or aralkylene of from 6 to 9 carbon atoms; and

X is a bond, non-oxocarbonyl (including thio and imino analogs thereof), or diazo, when bonded to an aromatic annular carbon atoms, i.e. when R is aromatic hydrocarbon.

Illustrative groups for -R-X include carboxymethyl, imidoylmethyl, thiocarbamoylethyl, diazophenyl, ethylene, ethyleneoxyethylene, carboxymethyleneoxyethyl, 2-(l-carboxypropylene) and N-methyl imidoylmethylaminoethyl.

The next group of compounds are cyclic lactams or urea compounds of the following formula: I

wherein:

n is on the average from 1 to 14, usually 1 to 12, more usually of from 2 to 12; T and T are hydrocarbon of from 1 to 7 carbon atoms, more usually of from 1 to 6 carbon atoms,

and from 0 to 1 site of aliphatic unsaturation, e.g.

ethylenic, including ethyl, n-butyl, a-methylbutyh;

5 isoamyl, allyl, hexyl, A-cyclohexenyl and phenyl, and when m is phenyl;

one of W and W is R X and the other is hydrogen.

Z is oxygen, with the proviso that Z may be H; when one of T and T is phenyl, e.g. primidone.

m is 0 when the compound is diphenylhydantoin, and 1 when the compound is a barbiturate or Z is H R may be the same as R but is usually an aliphatic group of from 1 to 8 carbon atoms, usually of from 1 to 6 carbon atoms, and preferably of from 1 to 4 carbon atoms, and from 0 to 3 heteroatoms, usually of from O to 2 heteroatoms, and from 0 to 1 site of aliphatic unsaturation, where the heteroatoms are oxygen, sulfur and nitrogen, usually oxygen and nitrogen, R being bonded to nitrogen through an aliphatically saturated carbon atom and to X with other than oxygen and sulfur; or aromatic hydrocarbon of from 6 to 9 carbon atoms; and

X is a bond, non-oxocarbonyl including the nitrogen and thioanalogs thereof, or diazo when bonded to an aromatic annular carbon atom.

lllustrative groups for R X include diazo, methylene, ethylene, butylene, ethyleneoxyethyl, acetyl. imidoylmethyl, propyleneoxyacetimidoyl, carboxyvinylene, carboxypropylene, imidoylbutylene, N- methyl ethyleneaminoethyl, N-methyl ethyleneaminoacetyl, and l-( l-carboxyethylene).

The next group of compounds are the steroids, which include the estrogens, gestogens, androgens, adrenocortical hormones (glucocorticoids and mineral corticoids and bile acids). Of particular interest are the sex hormones and the adrenocortical hormones. The steroids will be divided into two groups depending on whether the A ring is aromatic or cycloaliphatic.

For the most part, those compounds which are gestogens, androgens, or adrenocortical hormones will come within the following formula:

wherein:

one of Z, Z, and Z is RX", wherein the R may be singly or doubly bonded to the annular carbon atom. R" may be the same as R, but is usually an aliphatic group having from O to 1 site of aliphatic unsaturation and of from 1 to 8 carbon atoms, usually of from 1 to 6 carbon atoms, and more usually of from 1 to 4 carbon atoms, having from 0 to 3 heteroatoms which are oxygen, nitrogen and sulfur, usually oxygen and nitrogen, R being bonded to X at other than oxygen and sulfur; or atomatic hydrocarbon of from 6 to 8 carbon atoms;

X is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof, or diazo, when bonded to an aromatic annular carbon atom;

when other than RX, Z and Z are hydrogen;

when the compound is' a gestogen, there is from 0 to 1 site of ethylenic unsaturation in the A or A posi- 5 tion, and when other than R X Z is hydroxyl or oxo;

Y is acetyl; and

Y and Y are hydrogen;

when the compound is an androgen, when other than -R X Z is 0x0;

Y is hydroxyl; and Y and Y are hydrogen; when the compound is an adrenocortical hormone: when other than -R -X Z is oxo; Y is hydroxyacetyl;

Y is hydrogen or hydroxyl; and Y is hydroxy or 0x0; 11 on the average will be in the range of 1 to 14, usually l to 12, more usually in the range of 2 to 12.

When the compound is an estrogen and the A ring is aromatic, the compounds will for the most part have the following formula:

G-6-PDH l l 5 z o 6 4 Z I I I/ wherein:

one of Z, Z and Z is RX", wherein when Z or Z is RX, R may be singly or doubly bonded to the annular carbon atom, wherein R may be the same as R, but is usually an aliphatic radical having from O to 1 site of aliphatic unsaturation and of from 1 to 8 carbon atoms, usually of from 1 to 6 carbon atoms, and more usually of from 1 to 4 carbon atoms and from O to 3 heteroatoms which are oxygen, nitrogen and sulfur, more usually oxygen and nitrogen, or aromatic hydrocarbon of from 6 to 8 carbon atoms, and X is non- .oxocarbonyl including the nitrogen and sulfur analogs thereof, or diazo when bonded to an aromatic annular carbon atom;

when other than RX, Z, Z and Z will be hydrogen;

Y is hydrogen or hydroxyl; and

n on the average is in the range of from 1 to 14, usually 1 to 12, more usually in the range of from 2 to 12.

lllustrative groups for R X and R X- include ethylene, ethyleneoxyacetyl, iminoxyacetyl, pphenylenediazo, ethylenethiocarbamoyl, carboxybutylene, imidoylpropylene, p-diazobenzyl and thioetheracetyl.

The next compounds are methadone derivatives which will, for the most part, have the following formula:

(CH NCH CH c() dwn RX5- G-6-PDH wherein:

R may be the same as R, but is usually an aliphatic radical of from 1 to 8 carbon atoms, usually of from 1 to 6 carbon atoms, and more usually of from 1 to 4 carbon atoms; and from 0 to 3 heteroatoms which are oxygen, sulfur and nitrogen, particularly oxygen and nitrogen;

X is non-oxocarbonyl including the nitrogen and sulfur analogs thereof; and

If is on the average in the range of from 1 to 14, usually l to 12, more usually in the range of from 2 to 12.

The next group of compounds are associated with steroids and are cardiac glycosides of which digoxigenin and digoxin are well known members. For the most part, the compounds will have the following formula:

wherein:

one of Z Z", and Z is R"-X"', wherein when Z and Z are R, R may be singly or doubly bonded to the annular carbon atoms, wherein R may be the same as R, but is usually an aliphatic radical having from 0 to 1 site of aliphatic unsaturation and of from 1 to 8 carbon atoms, usually of from I to 6 carbon atoms, and more usually of from 1 to 4 carbon atoms and from O to 3 heteroatoms, usually of from 0 to 2 heteroatoms, which are oxygen, sulfur or nitrogen, preferably oxygen and nitrogen, and X is non-oxocarbonyl including the nitrogen and sulfur analogs thereof, or atomatic hydrocarbon of from 6 to 9 carbon atoms.

when other than R"'-X, Z, Z and Z are hydrogen;

Y is hydrogen or hydroxyl; and

n is on the average in the range of from 1 to 14, usually l to 12, more usually in the range of from 2 to 12.

The same groups illustrative of -R X" are also illustrative of R"--X"'.

While various sources of glucose-6-phosphate dehydrogenase may be employed, a particularly desirable source for the primary use for the subject compounds is the bacterium L.mesenteroides. The particular value of the G6PDH from this bacterium is that it is able to utilize both NADP and NAD. Since G6PDH from animal sources normally is able to utilize only NADP, one can limit interference from endogenous G-6PDH by employing NAD as the co-factor, when the subject compounds are used in immunoassays.

In preparing the conjugates, it is desirable that at least 20, preferably at least 40 and particularly preferred at least 50% of the original enzyme activity is retained. Furthermore, the enzyme is substituted in such a manner so that when one or more groups are bonded to the enzyme, and are bound by antibody, the activity of the enzyme is reduced by at least 30% of its original activity after conjugation, usually at least 40%, and preferably by at least 50%.

Various ways can be employed for conjugating the various compounds or ligands to the glucose-6- phosphate dehydrogenase. The conditions employed will normally reflect the particular functionality which is employed in forming a bond to the glucose-6- phosphate dehydrogenase. The functionalities which find primary use are the mixed anhydride employing an alkyl chloroformate, acyl azide, the imidate ester, thioimidate, isothiocyanate, reductive alkylation with an aldehyde, or an isocyanate. Normally, the groups will be bonded to available amino groups of lysine as the major mode of conjugation, and therefore amides, amidines, ureas, thioureas, and alkylamines will be formed.

The reaction mixture will normally be buffered to a pH in the range of 5 to 10, more usually in the range of 6 to 9. Various buffers may be used, such as phosphate, carbonate, Tris, and the like. An aqueous solvent will normally be used, and a preferred solvent includes from about 10 to 40 weight percent of an oxyethylene alcohol or ether having from 1 to 3 oxyethylene units. Particularly useful is carbitol. The temperatures will normally be at or above 5C and generally less than about 40C, usually from about 0 to 25C.

The concentration of the enzyme will vary widely, generally ranging from about 0.05 to 5, more usually from about 0.1 to lOmg/ml. The amount ofligand to be conjugated will vary, depending on the ligand enzyme ratio which is desired.

EXPERIMENTAL The following examples are offered by way of illustration and not by way of limitation.

(All temperatures not otherwise indicated are in centigrade).

EXAMPLE I A survey was carried out employing the isobutyl I chloroformate mixed anhydride of O -carboxymethylagainst 0.05M sodium phosphate, pH 7.5 and diluted with that buffer to a concentration of 2 mg/ml. The pH was adjusted to 7.0 with 1M HCl. To one ml of this cooled (4) stirred enzyme solution was added in five portions during 5 minutes, 37.5111 ofa 0.2M solution of the mixed anhydride (N-methyl- C) in dimethylformamide. After each addition, the pH rose slightly and was readjusted to 7 with 1M HCl. The solution was then maintained for 5 hours at 4 and dialyzed exhaustively against 0.55M Tris-HCl, pH 7.9. The resulting solution was diluted to 2ml with dialysis buffer. Scintillation counting was then employed to determine the number of ligands conjugated to the enzyme on the average.

To determine the activity of the enzyme and its inhibition, immunoassays were carried out. The assay mixture had a total volume of lml and was prepared from 20;).1 of 0. l M NAD in water (pH 5-6), 50,1.1 of 0066M glucose-6-phosphate in assay buffer, and enzyme solution. The remaining volume was made up by the assay buffer which was 0.055M Tris-HCl, pH 7.9. The mixture was incubated for 60 seconds in a spectrophotometer flow cell at 30, and the increase in absorbance at 350nm was then read over a 1 minute interval. .tl of the above enzyme solution diluted 1:100 in assay buffer containing 0.1% RSA (rabbit serum albumin) gave a rate of 0.160 optical density units per min. (OD/min) This corresponded to 52% of the activity of the native enzyme. Addition of a large excess of an antiopiate gamma-globulin preparation (5p.l of a solution that was 8 X 10 "M in binding sites) prior to addition of the enzyme solution to the assay mixture reduced the activity by 78% (0.0350D/min.) When 5011.1 of 10"M morphine and water was added to the substrates prior to EXAMPLE II A general conjugation procedure was employed as follows: Commercial G-6-PDH 4.9mg/ml (Beckman Microbics) was dialyzed against 0055M Tris-HCl buffer, pH 7.9. The concentration subsequent to dialysis was adjusted to 2mg/ml or 1 X 10 moles of enzyme per milliter. A 0.5ml aliquotof the enzyme solution was placed in a glass vial equipped with a micromagnetic stirring bar and a pH electrode and cooled in an ice bath. To the stirring solution was added 20mg NADH (0.026mm0le) and 11mg of glucose-o-phosphate (0.034mmole) as crystalline solids. To the cold stirring solution was added slowly by means of a syringe needle below the liquid surface, sufficient carbitol to provide 25% by volume (approximately 125,ul). To this solution was then added by means of a syringe, the ligand at approximately 0.1M in carbitol. The reaction mixture was then incubated and aliquots withdrawn, diluted and the rates determined as to deactivation and inhibition.

The assay procedure was as follows. Zparts of a solution 0.1M NAD in water at pH 5-6 was combined with 3 parts by volume of 0.11M glucose-o-phosphate in 0055M Tris-HCl buffer, pH 7.9. An aliquot from the conjugation reaction mixture was diluted 111,000 with the above indicated buffer. An assay solution was formed from 50p.l of the G6P/NAD solution, 750ml of buffer, 50; .1 of buffer or buffer containing addition of the antibody and enzyme, the total enzyme antibody, depending on whether the deactivation or activity was recovered. inhibitability was being determined, and 50,u.l of the en- A number of preparations were carried out using difzyme conjugate or enzyme control. Portions of buffer fering buffers, pHs, and mole ratios. Also, in some inwere employed to insure quantitative transfers. The sostances, the enzyme substrates were included to deterlution was aspirated into a spectrometer and the rate of mine their effect on deactivation. It was found that NADH production was followed at 340nm at 37. The above 14 ligands per enzyme molecule on the average, change in OD per min. was determined between the substantial deactivation of the enzyme had occurred. second and third minutes.

The following table indicates the results: The following table indicates the results obtained.

TABLE I Reagent Buffer" pH G6PDH Reagent Deactivation Inhibition Ligand" moles X l0 moles 10 7t 71 G6-PDH a l P-C 9 3.85 1.25 56 52 7.5

d 2* T-M 8.5 3.85 15 75 9.5

e 2+ T-M 8.5 3.85 15 48 82 11.5 f 2 T-M 8.5 3.85 15 63 87 12.0

Prior to conjugation added G-6-P to mM and NAD to 40mM. The pH required adjustment to 8.5

G-fi-PDH had activit of 561 lLlnig. while other G-o-PDH had activity M460 lU/mg.

+ Prior to conjugation added G-o-P to 5(1mM and NAD to 40mN.

l- O-carhoxymethylmorphine; 2- methyl O"-morphinoxyacetimidate P-C l).5M sodium phosphate-0.5M sodium carbonate; P -0.5M sodium phosphate; T M -0.055mTris-HCl-0.003M

magnesium chloride of original enzyme activity after conjugation and dialysis Maximum inhibition by excess antimorphine Both ligands contain "C. Determined by liquid scintillation counting of aliquots of product.

* No NAD or G6P were included "Dialyzed against four changes of 0.05514 Tris-HCI, pH 7. 9

EXAMPLE III A number of experiments were carried out following the following procedure. A 500p.l solution at a concenover a 5 minute period. A syringe was used for the addition of the ligand, with the tip of the syringe kept underneath the surface of the reaction mixture near the stirring bar.

As soon as the addition of the ligand was complete,

an aliquot was taken out and diluted 1:1,000 with the above buffer containing 0.1 weight RSA. Subsequently, additional aliquots were removed and assayed.

TABLE II i Ligand Carbitol ii Reagent Solution Time Temp. Dialysis Deactivation Inhibition M ul hrs. C

24 4 l2 15 96 4 27 36 a I 0.218 69 192 4 30 48 24 4 57 16 b* I 0.218 69 9G 4 92 29 24 20 36 39 48 20 48 49 c I 0.218 69 72 20 58 24 4 24 56 :1 II 0.133 37.5 48 4 Yes 34 73 24 4 44 66 e III 0.25 60 48 4 Yes 54 83 5* IV 0.128 25 24 4 Yes 76 64 The assay procedure was as follows. Approximately, a l0,u.l aliquot of the reaction mixture was added to a 0.5m] portion of the buffer containing 0.1 weight 7a- RSA and then further diluted, by taking 2011.] of this solution and adding it to 0.4ml of the same buffer. This provides a 1:1,000 dilution.

Sufficient buffer was initially added to the cup to provide a final volume of lml. To the buffer was then added p.l of 2:3 parts by volume of 0066M G6P in 0.055M Tris, pH 7.9, 50;1 of stock antimorphine solution and 50a] of the above diluted enzyme reaction mixture. The solution was aspirated into a spectrometer and followed at 340nm for the first 40 secs.

The following table indicates the results obtained with a variety of haptens.

TABLE III i Moles Reagent Carbitol Time iii Deactivaticn Inhibition Reagent Mo e Lysine vol. hr. pH Cofactor a V 6.6 i 17 3 8.0 40 86 3 8.0 23 69 b VI 6.6 17 4.5 8.0 59 3 8.0 25 67 0 VII 6.4 17 4.5 8.0 41 79 24 8.0 61 19 6 VIII 6.6 17 24 8.0 Yes l0 16 e VIII 6.6 25 72 8.0 Yes 24 31 f VIII 13.2 25 72 8.0 Yes 33 52 i 11 IX 6.7 8.5 72 7.0 Yes 42 41 Table Ill-Continued RO a v -ca i:oMe

EH v1 -cn SMe Jble H v11 -CH CO5'. Bu no VIII -CI-I2CH2NCS 1x -ca 0110 EXAMPLE IV 5-(N-Phenobarbityl)pentanoic acid conjugation to glueose-6-phosphate dehydrogenase A. Into a reaction vessel was introduced 0.5m] of dimethylformamide (DMF), 16.6mg of 5-(N- phenobarbityl)pentanoic acid and 6.95u1 of triethylamine and cooled to -l0. To the solution was then added 8.5ul of carbityl chloroformate, the mixture warmed to 0 and allowed to stand for 45 minutes, at which time it was ready for use.

Based on 57 lysine residues per molecule of G-6-PDH Reagent added in a. 1:1 by volume water carbitol solution I TABLE IV Phenobarbityl 0.1M aq. Enzyme mixed Anhydride Carbitol carbonate Time P/E 7( Buffer pl #1 p.1 p hrs. mole-ratio D I 1. T 10 65 50 9.0 0.5 200 63.6 54.1 2. T 2.5 72.5 50 7.9 50 16.6 10 3. T 5 70 9.0 100 50.6 34.4 4. T 2.5 72.5 7.9 50 16.6 10 5. T 10 50 9.0 2 200 55 6. T 2.5 72.5 50 7.9 2 50 10 7. T 5 -35 9.0 2 37 8. T 2.5 72.5 50 7.9 2 50 7 9. P 3.75 71.3 50 10 50.5 75' 22 10. P 7.5 67.5 50 9 7O 11. P 3.75 71.3 50 10 2 75 26 12. P 7.5 67.5 50 9 2 150 66 1. T tris-HC10.055M.pH 8.4

P phosphate 0.01M. pH PIE phenoharhiwl/enzyme deactivation based on activity of enzyme prior to conjugation.

aura

. inhibition based on activity of enzyme in presence of excess of antiphenobarhital as compared to activity of conjugated enzyme EXAMPLE V 4-(5'-Phenylbarbituryl-5')crotonic acid conjugate to glucose-6-phosphate dehydrogenase A. Into a reaction flask fitted with a drying tube was introduced 25011.] of DMF, 15.8mg of 4-(5-pheny1barbituryl-S') crotonic acid, and 6.8p.l of triethylamine and cooled to 8. To the mixture was slowly added 9.3p.l of carbityl chloroformate while maintained at a' temperature of -4.

B. Two reactions were carried out, each reaction mixture employing one ml of 0.055M tris buffer, pH 7.9, containing 1.55mg of G6PDH, 353,ul of carbitol, and 25ul of 1N sodium hydroxide following the previously described procedure in Example IV. In the first reaction mixture, 2511.1 of the above mixed anhydride was employed, while in the second reaction mixture 29;.tl of the above mixed anhydride was employed. Upon assaying for the enzyme as described previously, the percent deactivation was found to be 69.4 and 69.9 respectively, while the percent inhibition was found to be 74.4 and 75%, respectively.

EXAMPLE VI 5-(N-Diphenylhydantoinyl)pentanoic acid conjugate to glucose-6-phosphate dehydrogenase A. Into a reaction vessel was introduced 0.125ml DMF 17.8mg of 5-(N-diphenylhydantoinyl)pentanoic acid and 6.8,u.l of triethylamine, the mixture cooled to -1 0, and 9.3,u.l of carbityl chloroformate added slowly while maintaining the temperature below 0.

B. Following the previously described procedure in Example IV, into a reaction flask was introduced 0.595ml of tris buffer, pH 7.9 containing G6PDH at a concentration of 0.955 mg/ml, 5.25mg of glucose-6- phosphate, 9.9mg of NADH and 150a] of carbitol, and the pH adjusted to 8.5 with IN sodium hydroxide. To the mixture was then added 2a] of the above mixed anhydride with the pH being brought to 9.5, the total amount of IN sodium hydroxide added being ll6ul. The following table indicates the percent deactivation and inhibitability of the resulting product.

TABLE V 71 Deactivation Inhibition EXAMPLE vn' 2-(N-Diphenylhydantoinyl)ethoxyacetic acid conjugate to glucose-6-phosphate dehydrogenase was added slowly by syringe below the surface of the solution. The pH was monitored and adjusted by the addition of 0.1N sodium carbonate to 9.14. To the solution was then added 15p] of the above mixed anhydride solution in the same manner as the carbitol, with the pH dropping to 8.98. After 10 minutes from the addition, the solution was diluted with lml Tris-HCI, pH 7.9 buffer and assayed. Following the procedure described previously in Example IV, the percent deactivation was found to be 29, while the percent inhibitability was found to be 47.

EXAMPLE VIII 2-(N-Diphenylhydantoinyl)propionic acid conjugate to glucose-6-phosphate dehydrogenase A. To a flask containing 162mg of 2-(N- diphenylhydantoinyl)propionic acid was added under nitrogen 250g] of DMF and 7.05p.l of triethylamine. After cooling the mixture to -4, 9.25p.l of carbityl chloroformate was added, and the mixture allowed to react at -3 for 1 hour.

B. To 0.5m] of Tris-HCl buffer, pH 7.9, containing 0.732mg of glucose-6-phosphate dehydrogenase, 19.8mg of NADH and 10.5mg of G6P was added slowly by syringe below the surface of the reaction mixture, 9.5,ul of the above prepared mixed anhydride. During the addition, the pH was maintained at 9.0 by the addition of 0.lN NaOH. Employing the procedure described previously in Example IV, the enzyme was then assayed and was shown to be 59% deactivated and 53% inhibitable.

Reagents were prepared as follows. Where the preparation is not available in the literature, an exemplary preparation is provided.

EXAMPLE A Carbityl N-phenobarbitylacetimidate l. To a solution of 2g sodium phenobarbital in 10ml of DMSO, 500p] of chloroacetonitrile in 10ml of DMSO was added over a l-hour period under nitrogen.

After stirring for an additional 30 minutes, the mixture was poured into ml 5 weight sodium carbonateand washed with chloroform. The chloroform was washed once with 5 weight sodium carbonate and the combined carbonate solutions acidified with 6N HCl, followed by extracting three times with chloroform. After drying the combined chloroform extracts,

dride under nitrogen and the mixture was allowed to stand until the sodium hydride had reacted. To the alkoxide was added 118mg of the above product and the mixture stirred at room temperature under nitrogen for 24 hours.

EXAMPLE B Carbityl 2-(N-phenobarbityl)ethoxyacetimidate 1. To a solution of 635mg of sodium phenobarbital in 7ml of dry DMSO heated to 60 under nitrogen was added 345ul 2-chloroethoxyacetonitrile and mg potassium iodide and the mixture stirred overnight under nitrogen at 60-70. The reaction mixture was then stripped of volatiles at 60 at 0.2mm Hg, the residue dissolved in ethyl ether and extracted 5 times with 5 weight sodium carbonate. The alkaline extracts were acidified, extracted three times with ethyl ether and the combined ether extracts washed with brine, dried, and stripped to yield an oil. The oil was purified on preparative TLC with 4:1 ethyl ether: petroleum ether. An oil weighing 237mg was isolated.

2. Into lml of carbitol was dissolved 8.5mg of sodium hydride under nitrogen. To the mixture was 42mg of the above product and the mixture allowed to stand for 2 days at room temperature under nitrogen.

EXAMPLE C Carbityl N-diphenylhydantoinylacetimidate l. A solution of 500mg of sodium diphenylhydantoin and 415mg of chloroacetonitrile in ml of DMF under nitrogen was heated at 4050 for 66 hours. At the end of this time, the mixture was evaporated to near dryness, water added and the aqueous solution extracted with chloroform. The chloroform was first washed with water followed by lN sodium hydroxide and then dried and evaporated. The resulting oil was crystallized from absolute ethanol to yield 255mg. m.p. l7.

2. Approximately 1mg of sodium hydride was dissolved in 0.5m] carbitol to which was added 25mg of the above product, which was stirred overnight under nitrogen.

EXA M PLE D Carbityl O-estradioloxyacetimidate 1. To a solution of lg of estradiol in ml of dry acetone was added 0.7ml of chloroacetonitrile and 5g of freshly powdered anhydrous potassium carbonate. The solution was refluxed under nitrogen overnight, filtered, the solids washed with acetone, and the combined acetone fractions evaporated. The residue was dissolved in chloroform, filtered through silica, discarding early fractions which were product free. The remaining fractions were evaporated, the residue taken up in ethyl ether and the resulting crystals isolated. 760mg. m.p. l10l3. Recrystallization from ethyl ether yielded a product having a m.p. l l2l 14.

2. Into lml of carbitol was dissolved 1mg sodium hydride, followed by the addition of 31mg of the above product under nitrogen. The mixture was stirred overnight at room temperature.

EXAMPLE E Methyl O-morphinoxyacetthioimidate l. o -Cyanomethylmorphine (200mg) was suspended in 2ml of methyl mercaptan. To the mixture was added 3mg sodium hydride, the flask sealed and stirred at room temperature for 24 hours. The mixture was neutralized with 3.5 .1.1 acetic acid and concentrated in vacuo. The residue was taken up in methylene chloride, filtered through celite and concentrated in vacuo to yield 220mg of a light brown foam.

EXAMPLE F O -Isothiocyanatoethy[morphine 1. In 10ml of THF freshly distilled from lithium alu- 5 minum hydride (LAH) was suspended 400mg of LAH under nitrogen. A solution of 400mg of of chlo roacetonitrile in4ml freshly distitlled THF was added over 5 minutes, followed by refluxing for 1 hour. The mixture was allowed to cool and 0.6m] water added followed by 0.6m] 10 weight sodium hydroxide and 2ml of water. After filtering the mixture, the salts were washed with THF, the THF fractions combined, dried with magnesium sulphate under nitrogen, filtered and the filtrate evaporated yielding 380mg of O -aminoethylmorphine.

2. O -Aminoethylmorphine (200 mg) in 10ml chloroform was added to a solution of thiophosgene (56;.tl) and potassium bicarbonate (482mg) in 55ml water and the mixture stirred for two hours. The phases were separated and the aqueous phase washed twice wich chloroform (20ml). The combined chloroform phases were dried by gravity filtration through three thicknesses of chloroform moistened filter paper. The choloroform solution was evaporated yielding 200mg.

EXAMPLE G O -Morphinoxyacetaldehyde 1. To 6.06g dry morphine in 40 ml degassed DMSO was added 850mg of 56 weight sodium hydride in mineral oil and the mixture stirred under nitrogen until hydrogen evolution ceased. Bromoacetaldehyde diethyl acetal (3ml) was then added and the mixture stirred at 60 under nitrogen overnight. The solvent was then removed at 40, 0.05mm Hg and the residue purified on a 350g silica gel HF-254 column, using 10% methanolchloroform as a solvent. Fractions containing the desired product were collected and the solvent removed yielding 5.35g (67%).

2. Into 80ml of degassed 1N hydrochloric acid was added 5.9g of the above product and the mixture allowed to stand overnight. The solution was then brought up to pH 4.7 by addition of 5N sodium hydroxide and the water removed at 30 at 0.05mm Hg. The residue was dissolved in hot isopropanol and cooled to precipitate NaCl and impurities. The solution was filtered, the filtrate stripped in vacuo and the residue isolated as a glass. The product could not be crystallized and was isolated as a glass.

EXAMPLE H 5-(N-Phenylbarbital)pentanoic acid Into a reaction flask was introduced 48ml of dimethylformamide, 4g of sodio phenobarbital, 368g of ethyl 5-bromopentanoate and 0.8g of potassium iodide, the mixture heated to 40C and then stirred at room temperature overnight. After evaporating to dryness under a high vacuum, the residue was washed with water, followed by dissolving the residue in methylene chloride. The organic phase was then extracted with dilute sodium hydroxide, pH 12, and the alkaline layer acidified with 6N HCl to pH 2. A mixture of starting material and product precipitated out which was chromatographed with 1% methanol in chloroform on g silica gel.

Of the 1.44g of the product which is obtained from the column, 1.24g was dissolved in a mixture of 25ml tetrahydrofuran, 25ml ofconcentrated HC1 and 16ml of water, the mixture stirred overnight, followed by evaporation of the THF. After diluting with saturated sodium chloride, the mixture was extracted with methylene chloride, the organic phase isolated and extracted with saturated bicarbonate solution. After acidifying the aqueous phase to pH 3 with concentrated HCl, the aqueous phase was extracted with methylene chloride, washed with water and then evaporated to yield 0.95g. The 0.95g was recrystallized from a mixture of diethyl ether-heptane, to yield 700mg, m.p. l22-l23.

EXAMPLE 1 4-(5-Phenylbarbituryl-5)crotonic acid Into a reaction flask was introduced 100mg (0.49mmole) of S-phenylbarbituric acid, 24mg (0.5mmole) of sodium hydride and 5ml of DMF and the mixture stirred vigorously until a clear solution was obtained. To the mixture was then added 145mg (l03p.l, 0.75mmole) of ethyl 4-bromocrotonate. After stirring the mixture overnight at room temperature, the reaction mixture was evaporated to dryness under high vacuum and the residue partitioned between ethyl acetate and dilute hydrochloric acid. After extracting the aqueous layer with ethyl acetate, the organic layers were combined, and washed twicewith water and once with brine, followed by drying over sodium sulphate. The dried solution was then evaporated to dryness, and the residue chromatographed employing a 1:1 benzene-ethyl acetate mixture.

The ester (50mg, 0.1:6mmole) prepared above was dissolved in lml of 1N aqueous sodium hydroxide at room temperature. After about minutes, the mixture was acidified with 6N HCl and the precipitate filtered and dried to give 35mg of a white powder. m.p. 226-8.

EXAMPLE .1

5-(N-Diphenylhydantoinyl)penta'noic acid To a stirring suspension of 3.4g of sodio diphenylhydantoin in ml of dry DMF was added 2.63g of ethyl 5-bromopentanoate and a trace of potassium iodide. After stirring the mixture overnight at 60, the solution was diluted with water and extracted with diethylether. After washing the ethereal solution with water, the ethereal solution was dried, filtered and the filtrate evaporated to an oil, which solidified and was recrystallized from aqueous ethanol. Total yield 3.2g.

The ester prepared above (2.0g) was dissolved in 80ml dioxane and 40ml 10 weight 70 aqueous potassium hydroxide added. The phases separated but stirring was vigorously continued for 10 minutes, at which time additional water was added and a single phase formed. The aqueous solution was extracted with ether three times, acidified and filtered to give 1.5g of acid, which was recrystallized from benzene.

EXAMPLE K 2-(N-Diphenylhydantoinyl)ethoxyacetic acid Into a reaction flask was introduced 4g of sodio diphenylhydantoin, 3ml of 2-chloroethoxyacetonitrile and 120ml of DMF and the mixture was stirred under nitrogen at 35 for 15 minutes, followed by raising the temperature to 60 and continuing the stirring for an additional 24 hours. After evaporating to dryness, the

residue was taken up in diethylether, and the ethereal solution washed twice with water, once with 0.05N aqueous sodium hydroxide and once with aqueous sodium chloride, followed by drying with magnesium sulphate. The solution was then evaporated, the residue taken up in 60ml ethanol and the solution heated to boiling to yield 3.49g of white crystals. After recrystallizing from ethanol, the product had a m.p. l25l28.

Into 15ml of ethanolic 1N sodium hydroxide was introduced 200mg of the above nitrile and the mixture stirred overnight. Water was added, and the ethanol evaporated to yield a clear solution which was acidified to pH 1 with 6N HCl. The precipitate was isolated by filtration to yield 187mg of a white powder. m.p. 160l68.

EXAMPLE L 2-(N-Diphenylhydantoinyl)propionic acid Into a reaction flask was introduced 825mg sodio diphenylhydantoin, 543mg of ethyl 2-bromopropionate, and 10ml of DMF, the mixture heated to 60 under nitrogen and 40mg potassium iodide added to the stirred solution. After heating overnight, the reaction mixture was poured into 25ml of water, the flask washed with water and ether, the liquids combined and extracted twice with a total volume of 100ml of ether, and the ethereal extracts washed three times with a total volume of ml of water and once with brine. After evaporating the ether, the solution was flashed with a mixture of benzene, chloroform and ethanol, followed by flashing with carbon tetrachloride to yield an oil. The oil was dissolved in diethyl ether, the ethereal solution washed twice with water (total volume 25ml), dried over sodium sulphate and evaporated to an oil which was flashed with carbon tetrachloride, followed by chloroform, followed by a mixture of benzene and ethanol. The residue was dissolved in hot ethanol, a portion of the ethanol evaporated, and water added to I cloud point. Upon standing, an oil formed, which was repeatedly redissolved by repeated reheating and cooling, followed by addition of ethanol, at which time crystals formed which quickly became an oil. Benzeneethanol was added and the solution evaporated to leave an oil. 1

Into approximately 12ml of THF and 4ml 1N aqueous sodium hydroxide was dissolved the oil prepared above. The two phase solution was stirred at room temperature for about 60 hours. The THF was thenstripped in vacuo, the solution acidified to pH 1 with 6N HCl, at which time a solid formed which was filtered and dried.

The solid was further purified by dissolving in 50ml diethyl ether, the ethereal solution washed with 50ml of saturated bicarbonate, the bicarbonate layer acidified to produce a precipitate which was isolated, extracted three times with diethyl ether for a total volume of ml and the diethyl ether evaporated to dryness to yield 673mg. The product could be recrystallized by dissolving in methanol and adding chloroform to the cloud point.

Because of the high turnover rate for the glucose-6- phosphate dehydrogenase, as well as the substantial degree of activity which is retained after conjugation with haptens, highly sensitive assays can be developed for a wide variety of haptens. Of course, the sensitivity of any particular enzyme conjugate in an assay is based on keeping all of the variables constant. Two significant considerations in'an immunoassay are: (l) the lowest concentration level at which the material can be detected with some degree of reliability; and (2) the spread between various concentration levels. In order to evaluate the G-6PDl-l assay, standards were prepared having varying concentrations of the drug of interest. The assay was carried out as follows. Serum (50p.l) containing the drug at the particular concentration was diluted with 250M of buffer, Tris-l-lCl, pH 8.2 in order to insure quantitative transfer to the cuvette. To the dilute serum was then added 50;.Ll of the appropriate antibody at a concentration of about 4-5 X l M in binding sites (as determined by FRAT immunoassay, supplied by Syva Company), 0.1M NAD and 0.066M G6-P. Quantitative transfer was achieved with 250g] of buffer. Finally, the appropriate enzyme conjugate was added which had sufficient enzyme to provide an activity in the absence of antibody of 200 AOD/min at 340nm in the assay mixture, and the transfer made quantitative with ZSO tl of buffer, to provide a final volume of 0.9ml. The entire mixture was allowed to stand and equilibrate for one minute and the change in optical density read for the second minute at 340nm. The following table indicates the readings for the vari- It is evident from the above results, that concentrations ofa few ng with morphine or a few ,ug with phenobarbital and diphenylhydantoin are readily determinable rapidly from serum. It is believed, that further improvement in the assays of the latter two drugs can be achieved with further refinement as to the antibodies Therefore, the use of glucose-6-phosphate dehydrogenase as the enzyme provides an opportunity for extremely selective and sensitive tests for a wide variety of different drugs having widely varying structures.

The use of glucose-6-phosphate dehydrogenase has many desirable characteristics when employed in an homogeneous enzyme immunoassay. The high turnover rate of the enzyme provides theopportunity for an extremely sensitive test for determining drugs at very low concentration. Furthermore, the enzyme can be conjugated so as to be readily inhibitable, without undesirably high deactivation of the enzyme. Furthermore, the enzyme is relatively stable so it can be prepared as a reagent and stored and shipped. In addition, the enzyme employs a substrate which is colored, having strong absorption in the far ultraviolet region, and therefore can be easily detected in a conventional spectrophotometer.

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 within the scope of the appended claims.

What is claimed is:

1. An enzyme conjugate of glucose-6-phosphate dehydrogenase of the formula:

22 G6-PDI-l} (XRY),| wherein:

'G-6PDH intends glucose-6-phosphate dehydrogenase;

n is the average number of groups bonded to the G- 6PDH and is in the range of l to 18;

X is a bond or non-oxocarbonyl, including the nitrogen and sulfur analogs thereof;

R is an aliphatic linking group of from 1 to 8 carbon atoms and O to 3 heteroatoms which are oxygen, sulfur, or nitrogen;

Y is a hapten of at least molecular weight and not greater than about 1,000 molecular weight.

2. An enzyme conjugate according to claim 1, wherein n is of from 2 to 12, X is a non-oxocarbonyl group, R is of from 1 to 4 carbon atoms and O to 2 heteroatoms, and Y is a hapten of from about 125 to about 650 molecular weight.

3. An enzyme conjugate according to claim 1 wherein said glucose-6-phosphate dehydrogenase is derived from the bacterium L.mesenteroides.

4. An enzyme conjugate of the formula:

wherein:

T is hydrogen or acetyl;

n is on the average 1 to 14;

X is a bond, non-oxocarbonyl, including the thio and nitrogen analogs thereof or, when R is aromatic hydrocarbon, diazo; and

R is an aliphatic group of from 1 to 8 carbon atoms and 0 to 3 heteroatoms which are oxygen, sulfur or nitrogen or aromatic hydrocarbon of from 6 to 9 carbon atoms.

5. An enzyme conjugate according to claim 4, wherein X is non-oxocarbonyl, R is a saturated aliphatic group of from 1 to 4 carbon atoms and T is hydrogen. I

6. An enzyme conjugate of the formula:

wherein:

n is on the average from 1 to 14;

T and T are hydrocarbon of from 1 to 7 carbon atoms having from to 1 sites of aliphatic unsaturation;

one of W and W is -R"-X and the other is hydrogen;

m is 0 or I, with the proviso that when m. is O, T and T are phenyl;

Z is oxygen with the proviso that Z may be H when one of T and T is phenyl and m is l;

X is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R is aromatic hydrocarbon, diazo; and

R is an aliphatic group of from 1 to 8 carbon atoms and from O to 3 heteroatoms which are oxygen, sulfur or nitrogen, or aromatic hydrocarbon of from 6 to 9 carbon atoms.

7. An enzyme conjugate according to claim 6, wherein m is 0, Z is oxygen, and R is of from 1 to 4 carbon atoms and from 0 to 2 heteroatoms.

8. An enzyme conjugate according to claim 6, wherein m is 1, Z is oxygen, R is of from 1 to 4 carbon atoms and from 0 to 2 heteroatoms and X is nonoxocarbonyl.

9. An enzyme conjugate according to claim 8, wherein X is the oxygen moiety.

10. An enzyme conjugate according to claim 8, wherein X is the nitrogen moiety.

11. An enzyme conjugate of the formula:

3 G-6-PDH wherein:

'n is on the average in the range of l to 14;

one of Z, Z and Z is -R=X--, wherein R may be singly or doubly bonded to the annular carbon atom, and R is an aliphatic group of from 1 to 8 carbon atoms and O to 3 heteroatoms, which are oxygen, sulfur or nitrogen, or aromatic hydrocarbon of from 6 to 9 carbon atoms;

when other than -R -X, Z and Z are hydrogen;

X is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R is aromatic hydrocarbon, diazo;

with the proviso that when the steroid is a gestogen,

there is from O to 1 site of ethylenic unsaturation in the A or A position; and

when other than -R X Z is hydroxyl or 0x0;

Y is acetyl; and

Y and Y are hydrogen;

when the steroid is an androgen, when other tha- Y is hydroxyl; and

Y and Y are hydrogen; and

when the steroid is an adrenocortical hormone, when other than R -X Z is oxo;

Y is hydroxyacetyl;

Y is hydrogen or hydroxyl; and

Y is hydroxy or 0x0.

12. An enzyme conjugate of the formula:

wherein:

one of Z, Z and Z is -RX, wherein when Z or Z is R X-, R may be singly or doubly bonded to the annular carbon atom and wherein R is an aliphatic radical of from 1 to 8 carbon atoms, and from O to 3 heteroatoms which are oxygen, nitrogen and sulfur or aromatic hydrocarbon of from 6 to 9 carbon atoms;

when other than R -X, Z, Z and Z are hydrogen;

Y is hydrogen or hydroxyl; and

n" is on the average in the range of from 1 to l4.

13. An enzyme conjugate of the formula:

wherein:

R is an aliphatic radical of from 1 to 8 carbon atoms and from O to 3 heteroatoms which are oxygen, sulfur or nitrogen or aromatic hydrocarbon of from 6 to 9 carbon atoms;

n is on the average in the range of from 1 to 14.

14. An enzyme conjugate of the formula:

G-6-PDH 26 hydrocarbon, diazo;

when other than R- -X, Z, Z and Z are hydrogen;

Y is hydrogen or hydroxyl; and

n is on the average in the range of from 1 to l4.

15. A morphine conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 morphine groups, wherein the morphine is joined at the 0 position to an acetimidate linking group.

16. A morphine conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 morphine groups, wherein the morphine is joined at the 0 site to an acetyl linking group.

17. A phenobarbital conjugate to glucose-6- phosphate dehydrogenase having from 1 to 14 phenobarbital groups, wherein the phenobarbital is joined at the l nitrogen position through an acetimidate linking group.

18. A phenobarbital conjugate to glucose-6- phosphate dehydrogenase having from 1 to 14 phenobarbital groups wherein the phenobarbital is joined at the l nitrogen position to an ethyleneoxyacetimidate linking group.

19. A phenobarbital conjugate to glucose-6- phosphate dehydrogenase having from 1 to 14 phenobarbital groups wherein the phenobarbital is joined at the 5 position to a a-crotonyl linking group.

20. A diphenylhydantoin conjugate to glucose-6- phosphate dehydrogenase having from 1 to 14 diphenylhydantoin groups wherein the diphenylhydantoin is joined at the 3 nitrogen to an acetimidate linking group.

21. An estradiol conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 estradiol groups, wherein the estradiol is joined at the 0 position to an acetimidate linking group.

fiisemim l' 3,87 5,011.Ke1meth Edwcwd Rubenstein, Palo Alto, and Edwin F. U ZZman,

Atherton, Calif. ENZYME IMMUNOASSAYS WITH GLUCOSE- 6-PHOSPHATE DEHYDROGENASE. Patent dated Apr. 1, 197 5. Disclaimer filed Nov. 13, 197 5, by the assignee, Sg w Oompany.

The term of this patent subsequent to June 18, 1991 has been disclaimed.

[Ofiicial Gazette Mawch 30, 1.976.]

Claims

1. AN ENZYME CONJUGATE OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE OF THE FORMULA: G-6-PDH (X-R-Y)N WHEREIN: G-6-PDH INTENDS GLUCOSE-6-PHOSPHATE DEHYDROGENASE; N IS THE AVERAGE NUMBER OF GROUPS BONDED TO THE G-6PDH AND IS IN THE RANGE OF 1 TO 18; X IS A BOND OR NON-OXOCARBONYL, INCLUDING THE NITROGEN AND SULFUR ANALOGS THEREOF; R IS AN ALIPHATIC LINKING GROUP OF FROM 1 TO 8 CARBON ATOMS AND 0 TO 3 HETEROATOMS WHICH ARE OXYGEN, SULFUR, OR NITROGEN; Y IS A HAPTEN OF AT LEAST 125 MOLECULAR WEIGHT AND NOT GREATER THAN ABOUT 1,000 MOLECULAR WEIGHT.

2. An enzyme conjugate according to claim 1, wherein n is of from 2 to 12, X is a non-oxocarbonyl group, R is of from 1 to 4 carbon atoms and 0 to 2 heteroatoms, and Y is a hapten of from about 125 to about 650 molecular weight.

4. An enzyme conjugate of the formula:

5. An enzyme conjugate according to claim 4, wherein X1 is non-oxocarbonyl, R1 is a saturated aliphatic group of from 1 to 4 carbon atoms and T is hydrogen.

6. An enzyme conjugate of the formula:

7. An enzyme conjugate according to claim 6, wherein m is 0, Z is oxygen, and R2 is of from 1 to 4 carbon atoms and from 0 to 2 heteroatoms.

8. An enzyme conjugate according to claim 6, wherein m is 1, Z is oxygen, R2 is of from 1 to 4 carbon atoms and from 0 to 2 heteroatoms and X2 is non-oxocarbonyl.

9. An enzyme conjugate according to claim 8, wherein X2 is the oxygen moiety.

10. An enzyme conjugate according to claim 8, wherein X2 is the nitrogen moiety.

11. An enzyme conjugate of the formula:

12. An enzyme conjugate of the formula:

13. An enzyme conjugate of the formula:

14. An enzyme conjugate of the formula:

15. A morphine conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 morphine groups, wherein the morphine is joined at the O3 position to an acetimidate linking group.

16. A morphine conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 morphine groups, wherein the morphine is joined at the O3 site to an acetyl linking group.

17. A phenobarbital conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 phenobarbital groups, wherein the phenobarbital is joined at the 1 nitrogen position through an acetimidate linking group.

18. A phenobarbital conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 phenobarbital groups wherein the phenobarbital is joined at the 1 nitrogen position to an ethyleneoxyacetimidate linking group.

19. A phenobarbital conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 phenobarbital groups wherein the phenobarbital is joined at the 5 position to a Alpha -crotonyl linking group.

20. A diphenylhydantoin conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 diphenylhydantoin groups wherein the diphenylhydantoin is joined at the 3 nitrogen to an acetimidate linking group.

21. An estradiol conjugate to glucose-6-phosphate dehydrogenase having from 1 to 14 estradiol groups, wherein the estradiol is joined at the O3 position to an acetimidate linking group.