London Biotechnology Limited

Brian R Rabin

Christopher J Taylorson

Michael R Hollaway

Stuart Harbron

Hendrikus J Eggelte

Michael R Hollaway, Deceased

Ann Holloway, Legal Representative

Hydrolase enzymes are sensitively determined using novel substituted FAD substrates. The substituted FAD substrates are hydrolysed to FAD by the enzyme to be detected. The FAD is then combined with an apoenzyme to form a holoenzyme which is used to initiate a reaction that leads to a detectable product. When the hydrolase to be detected is phosphatase, the novel substrate is a phosphorylated derivative of FAD. A suitable apoenzyme is apo-glucose oxidase which provides exceptional sensitivity. Apo-D-amino acid oxidase, which is suitable for use in a 'single pot' assay system, can also be used.

Amplification assay for hydrolase enzymes

A small enzymically inactive peptide fragment of an enzyme (e.g. ribonuclease S-peptide) is used as the label and conjugated with the complementary fragment (S-protein) to form an enzyme which catalyses a primary reaction whose product is, or leads to, an essential coenzyme or prosthetic group for a second enzyme which catalyses a secondary reaction leading to a detectable result indicating the presence of analyte. Also disclosed are novel synthetic substrates for the primary reaction. Substrates for ribonuclease S conjugate enzyme are of the formula R-X where R is pyrimidine 3'-phosphate moiety and X is a leaving group linked to R through the 3'-phosphate group and leads to said coenzyme or prosthetic group, e.g. via riboflavin, thiamine, pyridoxal, pyridoxine or pyridoxine phosphate.

Enzymic method of detecting analytes and novel substrates therefor

A small enzymically inactive peptide fragment of an enzyme (e.g. ribonuclease S-peptide) is used as the label and conjugated with the complementary fragment (S-protein) to form an enzyme which catalyzes a primary reaction whose product is, or leads to, an essential coenzyme or prosthetic group for a second enzyme which catalyzes a secondary reaction leading to a detectable result indicating the presence of analyte. Also disclosed are novel synthetic substrates for the primary reaction. Substrates for ribonuclease S conjugate enzyme are of the formula R-X where R is a pyrimidine 3'-phosphate moiety and X is a leaving group linked to R through the 3'-phosphate group and leads to said coenzyme or prosthetic group, e.g. via riboflavin, thiamine, pyridoxal, pyridoxine or pyridoxine phosphate.

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Brian R Rabin