Malate dehydrogenase conjugates for enzyme immunoassays

Claim 2

An enzyme conjugate of malate dehydrogenase bonded to on the average with from about 1 to 18 haptens of molecular weight of from about 125 to 1,200 and having at least one heteroatom, wherein said conjugated enzyme retains at least about 2% of the original enzyme activity prior to conjugation and wherein the enzymatic activity of the conjugate is reduced by at least 30% when said haptens are bound to antibodies for said haptens.

Claim 3

An enzyme conjugate of malate dehydrogenase of the formula: MDH (X--R--Y).sub.n wherein: MDH intends malate dehydrogenase; n is on the average in the range of 1 to 18; R is a bond, a hydrocarbon linking group of from 1 to 8 carbon atoms or a substituted hydrocarbon linking group of from 1 to 8 carbon atoms and from 0 to 3 heteroatoms which are oxygen, nitrogen and sulfur; X is a bond, a non-oxocarbonyl group, including the nitrogen and sulfur analogs thereof or diazo, when bonded to an aromatic annular carbon atom; and Y is a hapten of from about 125 to about 1,000 molecular weight and having at least one heteroatom.

Claim 4

An enzyme conjugate of malate dehydrogenase of the formula: MDH (X--R--Y).sub.n wherein: MDH intends malate dehydrogenase; n is on the average in the range of 1 to 18; R is a bond, a hydrocarbon linking group of from 1 to 8 carbon atoms or a substituted hydrocarbon linking group of from 1 to 8 carbon atoms and from 0 to 3 heteroatoms which are oxygen, nitrogen and sulfur; X is a bond, a non-oxocarbonyl group, including the nitrogen and sulfur analogs thereof or diazo, when bonded to an aromatic annular carbon atom; and Y is a hapten of from about 125 to about 1,000 molecular weight and having at least one heteroatom.

Claim 5

An enzyme conjugate according to claim 2, wherein n is of from two 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.

Claim 6

An enzyme conjugate according to claim 2, wherein said malate dehydrogenase is derived from heart.

Claim 7

An enzyme conjugate of the formula ##STR10## wherein: n 2 is on the average in the range of 1 to 14; T 1 and T 2 are hydrocarbon of from 1 to 7 carbon atoms and 0 to 1 site of aliphatic unsaturation and are both phenyl when m is 0; one of W and W 1 is --R 2 --X 2 -- and the other is hydrogen; Z is oxygen or H 2 ; m is 0 or 1; R 2 is an aliphatic group of from 1 to 8 carbon atoms and 0 to 3 heteroatoms having from 0 to 1 site of aliphatic unsaturation, wherein the heteroatoms are oxygen, sulfur and nitrogen or aromatic hydrocarbon of from 6 to 9 carbon atoms; and X 2 is a bond, non-oxocarbonyl including the nitrogen and thioanalogs thereof or diazo when bounded to an aromatic annular carbon atom.

Claim 8

An enzyme conjugate of the formula ##STR10## wherein: n 2 is on the average in the range of 1 to 14; T 1 and T 2 are hydrocarbon of from 1 to 7 carbon atoms and 0 to 1 site of aliphatic unsaturation and are both phenyl when m is 0; one of W and W 1 is --R 2 --X 2 -- and the other is hydrogen; Z is oxygen or H 2 ; m is 0 or 1; R 2 is an aliphatic group of from 1 to 8 carbon atoms and 0 to 3 heteroatoms having from 0 to 1 site of aliphatic unsaturation, wherein the heteroatoms are oxygen, sulfur and nitrogen or aromatic hydrocarbon of from 6 to 9 carbon atoms; and X 2 is a bond, non-oxocarbonyl including the nitrogen and thioanalogs thereof or diazo when bounded to an aromatic annular carbon atom.

Claim 9

An enzyme conjugate according to claim 5 wherein m is 0 and T 1 and T 2 are phenyl.

Claim 10

An enzyme conjugate according to claim 5, wherein m is 1, Z is oxygen, R 2 is of from 1 to 6 carbon atoms and from 0 to 2 heteroatoms and X 2 is non-oxocarbonyl.

Claim 11

An enzyme conjugate according to claim 7, wherein Z 2 is oxygen.

Claim 12

An enzyme conjugate of the formula ##STR11## wherein: n 3 is on the average in the range of 1 to 14; one of Z 1 , Z 2 and Z 3 is --R 3 --X 3 --, wherein R 3 may be singly or doubly bonded to the annular carbon atom, and R 3 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; when other than --R 3 --X 3 --, Z 2 and Z 3 are hydrogen; X 3 is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R 3 is aromatic hydrocarbon, diazo; with the proviso that when the steroid is a gestogen, there is from 0 to 1 site of ethylenic unsaturation in the Δ 4 or Δ 5 position; and when other than --R 3 --X 3 --, Z 1 is hydroxyl or oxo; Y 1 is acetyl; and Y 2 and Y 3 are hydrogen; when the steroid is an androgen, when other than --R 3 --X 3 --, Z 1 is oxo; Y 1 is hydroxyl; and Y 2 and Y 3 are hydrogen; and when the steroid is an adrenocortical hormone, when other than --R 3 --X 3 --, Z 1 is oxo; Y 1 is hydroxyacetyl; Y 2 is hydrogen or hydroxyl; and Y 3 is hydroxy or oxo.

Claim 13

An enzyme conjugate of the formula ##STR11## wherein: n 3 is on the average in the range of 1 to 14; one of Z 1 , Z 2 and Z 3 is --R 3 --X 3 --, wherein R 3 may be singly or doubly bonded to the annular carbon atom, and R 3 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; when other than --R 3 --X 3 --, Z 2 and Z 3 are hydrogen; X 3 is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R 3 is aromatic hydrocarbon, diazo; with the proviso that when the steroid is a gestogen, there is from 0 to 1 site of ethylenic unsaturation in the Δ 4 or Δ 5 position; and when other than --R 3 --X 3 --, Z 1 is hydroxyl or oxo; Y 1 is acetyl; and Y 2 and Y 3 are hydrogen; when the steroid is an androgen, when other than --R 3 --X 3 --, Z 1 is oxo; Y 1 is hydroxyl; and Y 2 and Y 3 are hydrogen; and when the steroid is an adrenocortical hormone, when other than --R 3 --X 3 --, Z 1 is oxo; Y 1 is hydroxyacetyl; Y 2 is hydrogen or hydroxyl; and Y 3 is hydroxy or oxo.

Claim 14

An enzyme conjugate according to claim 9, wherein Z 2 and Z 3 are hydrogen and Y 1 and Y 2 are taken together to define oxo.

Claim 15

An enzyme conjugate according to claim 9, wherein Y 2 and Y 3 are hydrogen and Y 1 is hydroxyl.

Claim 16

An enzyme conjugate of the formula ##STR12## wherein: one of Z 4 , Z 5 and Z 6 is --R 4 --X 4 --, wherein when Z 4 or Z 6 is --R 4 --X 4 --, R 4 may be singly or doubly bonded to the annular carbon atom and wherein R 4 is an aliphatic radical of from 1 to 8 carbon atoms, and from 0 to 3 heteroatoms, which are oxygen, nitrogen and sulfur, or aromatic hydrocarbon of from 6 to 9 carbon atoms; X 4 is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R 4 is aromatic hydrocarbon, diazo; when other than --R 4 --X 4 --, Z 4 , Z 5 and Z 6 are hydrogen; Y 4 is hydrogen or hydroxyl; and n 4 is on the average in the range of from 1 to 14.

Claim 17

An enzyme conjugate of the formula ##STR12## wherein: one of Z 4 , Z 5 and Z 6 is --R 4 --X 4 --, wherein when Z 4 or Z 6 is --R 4 --X 4 --, R 4 may be singly or doubly bonded to the annular carbon atom and wherein R 4 is an aliphatic radical of from 1 to 8 carbon atoms, and from 0 to 3 heteroatoms, which are oxygen, nitrogen and sulfur, or aromatic hydrocarbon of from 6 to 9 carbon atoms; X 4 is a bond, non-oxocarbonyl including the nitrogen and sulfur analogs thereof or, when R 4 is aromatic hydrocarbon, diazo; when other than --R 4 --X 4 --, Z 4 , Z 5 and Z 6 are hydrogen; Y 4 is hydrogen or hydroxyl; and n 4 is on the average in the range of from 1 to 14.

Claim 18

An enzyme conjugate of the formula ##STR13## wherein: one of Z 7 , Z 8 , and Z 9 is --R 6 --X 6 --, wherein when Z 8 or Z 9 is --R 6 --X 6 --, R 6 may be singly or doubly bonded to the annular carbon atom; R 6 is an aliphatic radical of from 1 to 8 carbon atoms and 0 to 3 heteroatoms having from 0 to 1 site of aliphatic unsaturation, wherein said heteroatoms are oxygen, sulfur or nitrogen, or a mono- or disaccharide residue modified for linking to an amino group; X 6 is non-oxocarbonyl including the nitrogen and sulfur analogs thereof or a bond; when other than R 6 --X 6 --, Z 7-9 are hydrogen; Y 5 is hydrogen or hydroxyl; and n 6 is on the average in the range of from about 1 to 14.

Claim 19

An enzyme conjugate of the formula ##STR13## wherein: one of Z 7 , Z 8 , and Z 9 is --R 6 --X 6 --, wherein when Z 8 or Z 9 is --R 6 --X 6 --, R 6 may be singly or doubly bonded to the annular carbon atom; R 6 is an aliphatic radical of from 1 to 8 carbon atoms and 0 to 3 heteroatoms having from 0 to 1 site of aliphatic unsaturation, wherein said heteroatoms are oxygen, sulfur or nitrogen, or a mono- or disaccharide residue modified for linking to an amino group; X 6 is non-oxocarbonyl including the nitrogen and sulfur analogs thereof or a bond; when other than R 6 --X 6 --, Z 7-9 are hydrogen; Y 5 is hydrogen or hydroxyl; and n 6 is on the average in the range of from about 1 to 14.

Claim 20

An enzyme conjugate of a formula: ##STR14## wherein: one of W 5 and W 6 is --R 8 --X 8 --; R 8 is an aliphatic group of from about 2 to 10 carbon atoms having from 0 to 2 heteroatoms which are oxygen and nitrogen and from 0 to 1 site of aliphatic unsaturation; R 8 is a bond or nonoxocarbonyl, including the nitrogen and sulfur analogs thereof; when other than --R 8 --X 8 --, W 5 is hydrogen and W 6 is hydroxyl, alkoxy of from 1 to 3 carbon atoms or amino; and n 8 is on the average in the range of from about 1 to 14.

Claim 21

An enzyme conjugate of a formula: ##STR14## wherein: one of W 5 and W 6 is --R 8 --X 8 --; R 8 is an aliphatic group of from about 2 to 10 carbon atoms having from 0 to 2 heteroatoms which are oxygen and nitrogen and from 0 to 1 site of aliphatic unsaturation; R 8 is a bond or nonoxocarbonyl, including the nitrogen and sulfur analogs thereof; when other than --R 8 --X 8 --, W 5 is hydrogen and W 6 is hydroxyl, alkoxy of from 1 to 3 carbon atoms or amino; and n 8 is on the average in the range of from about 1 to 14.

Claim 22

A testosterone conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 testosterone groups.

Claim 23

A testosterone conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 testosterone groups.

Claim 24

An estradiol conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 estradiol groups.

Claim 25

An estradiol conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 estradiol groups.

Claim 26

A triiodothyronine conjugate to malate dehydrogenase, linked at the amino group having from about 1 to 14 triiodothyronine groups.

Claim 27

A triiodothyronine conjugate to malate dehydrogenase, linked at the amino group having from about 1 to 14 triiodothyronine groups.

Claim 28

A digoxigenin conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 digoxigenin groups.

Claim 29

A digoxigenin conjugate to malate dehydrogenase linked at the 3 position having from about 1 to 14 digoxigenin groups.