Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases

A method for determining the amount of a specific binding pair (sbp) member in a sample suspected of containing said sbp member, which comprises the steps of:

• (a) combining in an assay medium:
• (1) said sample,
• (2) a conjugate of an enzyme and an analog of said sbp member, wherein said enzyme is a mutant bacterial glucose-6-phosphate dehydrogenase (G6PDH) derived from the bacterial genus Leuconostoc and having at least two amino acid mutations per subunit as compared to precursor G6PDH, wherein said at least two amino acid mutations per subunit are by deletion of, or substitution for, at least two lysine amino acids per subunit, or substitution of or insertion of at least two cysteine amino acids per subunit, and
• (3) an sbp partner of said sbp member capable of binding said conjugate, and
• (b) determining the activity of said enzyme.

A method for determining the presence or amount of an analyte in a sample suspected of containing said analyte, which method comprises the steps of:

• a) combining in an assay medium:
• 1) said sample,
• 2) a conjugate of an analyte and an enzyme, wherein said enzyme is a mutant recombinant NAD.sup.+ dependent glucose-6-phosphate dehydrogenase (G6PDH) derived from an organism selected from the group consisting of Leuconostoc mesenteroides, Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc dextranicus wherein said G6PDH has at least one amino acid mutation per subunit as compared to precursor G6PDH wherein at least one of said mutations comprises the introduction of a cysteine residue proximate to an epitope recognized by an inhibitory anti-G6PDH antibody,
• 3) an antibody capable of binding said analyte and said analyte analogs, and
• 4) substrates for said enzyme;
• and
• b) measuring the activity of said enzyme.

A method for determining the presence or amount of an analyte in a sample suspected of containing said analyte, which method comprises the steps of:

• a) combining in an assay medium:
• 1) said sample,
• 2) a mutant glucose-6-phosphate dehydrogenase (G6PDH) derived from the bacterial genus Leuconostoc and having at least one amino acid mutation per subunit as compared to precursor G6PDH wherein at least one of said mutations comprises the introduction of a cysteine proximate to an epitope recognized by an inhibitory anti-G6PDH antibody, wherein said mutant G6PDH is bound to a ligand through the sulfur atom of said cysteine,
• 3) an antibody capable of binding said ligand, and
• b) measuring the activity of said mutant enzyme.

In a method for determining the presence of a ligand in a sample suspected of containing said ligand, which comprises the steps of:

• a) bringing together in an aqueous medium:
• 1) said sample,
• 2) enzyme-bound-ligand, and
• 3) receptor capable of binding to said ligand and said enzyme-bound-ligand, wherein said receptor is at a concentration sufficient to substantially change the enzymatic activity of said enzyme-bound-ligand in the absence of said ligand;
• b) determining the enzymatic activity of said enzyme-bound-ligand in said medium,
• the improvement which comprises, employing as said enzyme a mutant glucose-6-phosphate dehydrogenase (G6PDH) derived from the bacterial genus Leuconostoc and having at least two amino acid mutations as compared to precursor G6PDH wherein said at least two amino acid mutations are by deletion of, or substitution for lysine amino acids, or substitution of or insertion of cysteine amino acids.