Compounds for enzyme amplification assay

• US 4,067,774 A
• Filed: 09/13/1976
• Issued: 01/10/1978
• Est. Priority Date: 05/14/1971
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syva Co.
• Inventors: Ullman, Edwin F., Rubenstein, Kenneth E.
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US05/722,964
• Time in Patent Office: 484 Days
• Field of Search: 195/63, 195/DIG. 11
• US Class Current: 435/188
• CPC Class Codes: C07J 19/00 Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
• CPC Class Codes: C07J 41/0016 Oximes
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines
• CPC Class Codes: G01N 33/948 Sedatives, e.g. cannabinoids, barbiturates opiates G01N33/9486
• CPC Class Codes: G01N 33/9486 Analgesics, e.g. opiates, aspirine
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 930/26 Containing cys-cys disulfide bridge between nonadjacent cysteine residues

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.

Claims

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

##STR61##wherein;

any one of the W groups other than W⁴⁴ and W⁴⁵ can be -X* or an H of any of the W groups other than W⁴⁴ or W⁴⁵ 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 n ligands, wherein n is in the range of 1 to the molecular weight of -A* divided by 2,000;

W⁴⁰ and W⁴¹ are hydrogen or alkyl of from 1 to 3 carbon atoms;

W⁴² is hydrogen, alkyl of from 1 to 3 carbon atoms, or may be taken together with W⁴⁰ to form a ring having 6 annular members with a nitrogen as the only heteroatom;

W⁴³ is hydrogen, hydroxyl, carbomethoxy, or may be taken together with W⁴⁰ to form a morpholine ring, with the proviso that W⁴³ is carbomethoxy, when W⁴⁰ and W⁴² are taken together; and

W⁴⁴ and W⁴⁵ are hydrogen, hydroxyl, or alkoxyl of from 1 to 3 carbon atoms.

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

##STR62##wherein;

any one of the W groups other than W^46'"'"' can be -X* or an H of any of the W groups other than W^46'"'"' 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 n ligands wherein n is in the range of 1 to the molecular weight of A* divided by 2,000;

W^40'"'"' and W^41'"'"' are hydrogen or alkyl of from 1 to 3 carbon atoms;

W^42'"'"' is hydrogen, methyl, or may be taken together with W^40'"'"' to form a piperidine ring;

W^43'"'"' is hydrogen, hydroxyl, or carbomethoxy with the proviso that W^43'"'"' is carbomethoxy when W^42'"'"' is taken together with W^40'"'"' ; and

W^46'"'"' is hydrogen.

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

##STR63##wherein;

one of W^40"'"'"', and W^42"'"'"' is -X**;

when other than -X**,

W^40"'"'"' is hydrogen;

W^42"'"'"' is methyl; and

W^44"'"'"' is hydrogen;

W^41"'"'"' is hydrogen or methyl;

X** is -Z-C═

O wherein Z is hydrocarbylene of from 1 to 7 carbon atoms, with the proviso that when W^44"'"'"' is -X**, -X** is ##STR64##and A** is an enzyme having n'"'"' ligands, wherein n'"'"' is in the range of 1 to 20, and said enzyme has a molecular weight in the range of 10,000 to 300,000.

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

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

6. An enzyme-bound-ligand according to claim 3, which is N-carboxymethylamphetamine conjugate to lysozyme, having from 2 to 4 of said amphetamine groups.

7. An enzyme-bound-ligand according to claim 3, which is N-carboxymethylamphetamine conjugate to malate dehydrogenase, having from 2 to 22 of said amphetamine groups.

8. An enzyme-bound-ligand according to claim 3, which is N-carboxymethylamphetamine conjugate to glucose 6-phosphate dehydrogenase, having from 2 to 22 of said amphetamine groups.