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.
An enzyme-bound-ligand according to the formula: ##STR61## wherein: any one of the W groups can be --X*, or an H of any 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 n ligands, wherein n is in the range of 1 to the molecular weight of A* divided by 2,000; W 90 is hydrogen or hydroxyl; W 91 and W 92 are hydrogen or hydroxyl, at least one being hydroxyl, when both are hydroxyl, oxo is intended; W 93 is hydrogen or hydroxyl; W 94-97 are hydrogen or hydroxyl, at least one of W 94 and W 95 is hydroxyl; W 98 is methyl or formyl; and W 99 is hydrogen or two W 99 's may be taken together to form a double bond.
Claim 2
Dependent Claim
146 words
An enzyme-bound-ligand according to the formula: ##STR61## wherein: any one of the W groups can be --X*, or an H of any 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 n ligands, wherein n is in the range of 1 to the molecular weight of A* divided by 2,000; W 90 is hydrogen or hydroxyl; W 91 and W 92 are hydrogen or hydroxyl, at least one being hydroxyl, when both are hydroxyl, oxo is intended; W 93 is hydrogen or hydroxyl; W 94-97 are hydrogen or hydroxyl, at least one of W 94 and W 95 is hydroxyl; W 98 is methyl or formyl; and W 99 is hydrogen or two W 99 's may be taken together to form a double bond.
Claim 3
Dependent Claim
30 words
An enzyme-bound-ligand according to claim 1, wherein --X* is of not more than 8 carbon atoms, and not more than 4 heteroatoms, and said enzyme is an oxidoreductase or hydrolase.
Claim 4
Dependent Claim
15 words
An enzyme-bound-ligand according to any of claims 1 or 2, wherein said ligand is cortisol.
Claim 5
Dependent Claim
12 words
An enzyme-bound-ligand according to claim 3, wherein said enzyme is glucose-6-phosphate dehydrogenase.
Claim 6
Dependent Claim
31 words
An enzyme-bound-ligand according to claim 3, wherein said enzyme is bound to said ligand at the position defined by W 94 and W 95 , wherein the linking group is carboxymethyloxime.