# US 10,502,740 B2 · Enzyme detection device > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,502,740 B2 - Document ID: US10502740B2 - Status: Granted - Kind: B2 - Application number: 14/987,314 - Filed: 2016-01-04 - Published: 2016-07-07 - Granted: 2019-12-10 - Earliest claimed: 2007-08-23 - Assignees: Mologic Limited - Applicants: Mologic Limited - Inventors: Paul James Davis - Web page: https://patentgrep.com/patent/US10502740B2 ## CPC - G01N 33/573: for enzymes or isoenzymes - C12Q 1/37: involving peptidase or proteinase - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract An enzyme detection device (1) for detecting the presence, in a sample, of an enzyme capable of modifying a provided substrate (10). The device (1) comprises a substrate which has a modification region (14) that is sensitive to modification by the enzyme from an unmodified state to a modified state. The device (1) further comprises a substrate recognition molecule (16) which binds the modification region (14) in either the modified or the unmodified state. The modification region 14 of the substrate is preferentially bound by the substrate recognition molecule (16) as compared with the enzyme when mixed. The device further comprises a detectable label (18) coupled to the substrate recognition molecule (17). ## Claims ### Claim 1 A kit for detecting the presence in a sample of an enzyme capable of modifying a provided substrate comprising: (i) a chromatographic medium; (ii) a substrate comprising an attachment region and a modification region sensitive to modification by the enzyme from an unmodified state to a modified state, wherein the attachment region is a ligand that is not sensitive to modification by the enzyme; (iii) a first capture recognition molecule immobilized on the chromatographic medium capable of specifically binding the attachment region of the substrate, and capable of binding the attachment region of the substrate when the modification region of the substrate is either in the unmodified or modified state; (iv) a substrate recognition molecule capable of specifically binding the modification region in the unmodified state which does not bind the modification region in the modified state, the binding site of the substrate recognition molecule being such that the modification region of the substrate is preferentially bound by said substrate recognition molecule as compared with the enzyme when mixed, wherein the substrate recognition molecule and the enzyme competitively bind to the modification region when mixed; and (v) a detectable label coupled to the substrate recognition molecule. ### Claim 2 (depends on 1) The kit according to claim 1 wherein the substrate recognition molecule has a lower dissociation rate and a higher association rate for the substrate than the enzyme for the substrate. ### Claim 3 (depends on 1) The kit according to claim 1 wherein the first capture recognition molecule and the attachment region are each two halves of a binding pair wherein the binding pair is: an antigen and antibody or antigen-binding fragment specific therefor; biotin and avidin, streptavidin, neutravidin, or captavidin; protein A and G; a carbohydrate and a lectin; two complementary nucleotide sequences; an effector and a receptor molecule; a hormone and a hormone binding protein; an enzyme cofactor and an enzyme; an enzyme inhibitor and an enzyme; a cellulose binding domain and cellulose fibres; immobilised aminophenyl boronic acid and cis-diol bearing molecules; xyloglucan and cellulose fibres and analogues, derivatives and fragments thereof. ### Claim 4 (depends on 1) The kit according to claim 1 wherein the substrate recognition molecule is an antibody or antigen binding fragment thereof; a lectin; a nucleotide sequence; a receptor molecule; a hormone binding protein capable of binding the modification region in either the modified or the unmodified state; or is avidin, streptavidin, neutravidin, or captavidin capable of binding unmodified biotin. ### Claim 5 (depends on 1) The kit according to claim 1 wherein the enzyme is a hydrolase, a peptidase, lipase, nuclease, carbohydrase, phosphatase, sulphatase, neuraminidase, esterase, DNAase, RNAase, a kinase, a glycosyl transferase, an oxidase, a reductase or a transaminase. ### Claim 6 (depends on 1) The kit according to claim 1 wherein the detectable label is covalently bound to the substrate recognition molecule. ### Claim 7 (depends on 6) The kit according to claim 6 wherein the label is a fluorophore, a gold particle, a chromogen, a luminescent compound; a radioactive compound; a visible compound, a liposome or other vesicle containing signal producing substances, an electroactive species or a combination of an enzyme and its substrate. ### Claim 8 A kit for detecting the presence in a sample of an enzyme capable of modifying a provided substrate comprising: (i) a chromatographic medium; (ii) a substrate comprising an attachment region and a modification region sensitive to modification by the enzyme from an unmodified state to a modified state, wherein the attachment region is a ligand that is not sensitive to modification by the enzyme; (iii) a first capture recognition molecule immobilized on the chromatographic medium capable of specifically binding the attachment region of the substrate, and capable of binding the attachment region of the substrate when the modification region of the substrate is either in the unmodified or modified state; (iv) a substrate recognition molecule capable of specifically binding the modification region in the unmodified state which does not bind the modification region in the modified state, the binding site of the substrate recognition molecule being such that the modification region of the substrate is preferentially bound by said substrate recognition molecule as compared with the enzyme when mixed, wherein the substrate recognition molecule and the enzyme competitively bind to the modification region when mixed; (v) a detectable label coupled to the substrate recognition molecule; and (vi) a second capture recognition molecule, immobilised on the chromatographic medium, and capable of binding the substrate recognition molecule. ### Claim 9 (depends on 8) The kit according to claim 8 wherein the substrate recognition molecule has a lower dissociation rate and a higher association rate for the substrate than the enzyme for the substrate. ### Claim 10 (depends on 8) The kit according to claim 8 wherein the second capture recognition molecule that is immobilised on the chromatographic medium and that is capable of binding the substrate recognition molecule is present in combination with a fragment of the substrate. ### Claim 11 (depends on 10) The kit according to claim 10 wherein the first capture recognition molecule and the attachment region and/or the second capture recognition molecule and the substrate recognition molecule are each two halves of a binding pair wherein the binding pair is: an antigen and antibody or antigen-binding fragment specific therefor; biotin and avidin, streptavidin, neutravidin, or captavidin; protein A and G; a carbohydrate and a lectin; two complementary nucleotide sequences; an effector and a receptor molecule; a hormone and a hormone binding protein; an enzyme cofactor and an enzyme; an enzyme inhibitor and an enzyme; a cellulose binding domain and cellulose fibres; immobilised aminophenyl boronic acid and cis-diol bearing molecules; xyloglucan and cellulose fibres and analogues, derivatives and fragments thereof. ### Claim 12 (depends on 8) The kit according to claim 8 wherein the substrate recognition molecule is an antibody or antigen binding fragment thereof; a lectin; a nucleotide sequence; a receptor molecule; a hormone binding protein capable of binding the modification region in either the modified or the unmodified state; or is avidin, streptavidin, neutravidin, or captavidin capable of binding unmodified biotin. ### Claim 13 (depends on 8) The kit according to claim 8 wherein the enzyme is a hydrolase, a peptidase, lipase, nuclease, carbohydrase, phosphatase, sulphatase, neuraminidase, esterase, DNAase, RNAase, a kinase, a glycosyl transferase, an oxidase, a reductase or a transaminase. ### Claim 14 (depends on 8) The kit according to claim 8 wherein the detectable label is covalently bound to the substrate recognition molecule. ### Claim 15 (depends on 14) The kit according to claim 14 wherein the label is a fluorophore, a gold particle, a chromogen, a luminescent compound; a radioactive compound; a visible compound, a liposome or other vesicle containing signal producing substances, an electroactive species or a combination of an enzyme and its substrate. ### Claim 16 A kit for detecting the presence in a sample of an enzyme capable of modifying a provided substrate comprising: (i) a chromatographic medium; (ii) a substrate comprising an attachment region and a modification region sensitive to modification by the enzyme from an unmodified state to a modified state, wherein the attachment region is a ligand that is not sensitive to modification by the enzyme; (iii) a first capture recognition molecule immobilized on the chromatographic medium capable of specifically binding the attachment region of the substrate, and capable of binding the attachment region of the substrate when the modification region of the substrate is either in the unmodified or modified state; (iv) a substrate recognition molecule capable of specifically binding the modification region in the unmodified state which does not bind the modification region in the modified state, the binding site of the substrate recognition molecule being such that the modification region of the substrate is preferentially bound by said substrate recognition molecule as compared with the enzyme when mixed, wherein the substrate recognition molecule and the enzyme competitively bind to the modification region when mixed, and wherein the substrate recognition molecule has an affinity for the substrate with a dissociation rate (kd) between 10−4·s−1 and 10−7·s−1 and an association rate (ka) between 105·s−1 and 109·s−1; (v) a detectable label coupled to the substrate recognition molecule; and (vi) a second capture recognition molecule, immobilised on the chromatographic medium, and capable of binding the substrate recognition molecule.