# US 8,771,975 B2 · Quantitation of GCP activity in biological samples > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,975 B2 - Document ID: US8771975B2 - Status: Granted - Kind: B2 - Application number: 13/977,966 - Filed: 2012-01-05 - Published: 2013-12-26 - Granted: 2014-07-08 - Earliest claimed: 2011-01-05 - Assignees: The Johns Hopkins University - Applicants: Barbara S. Slusher; Michael Polydefkis; Camilo Rojas; Marigo Stathis - Inventors: Barbara S. Slusher; Michael Polydefkis; Camilo Rojas; Marigo Stathis - Web page: https://patentgrep.com/patent/US8771975B2 ## CPC - C12Q 1/37: involving peptidase or proteinase - G01N 2500/00: Screening for compounds of potential therapeutic value - 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 - 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) ## Abstract Provided herein are methods for quantitating glutamate carboxypeptidase II activity in a biological sample, such as a skin biopsy sample, and for determining whether an agent is inhibiting GCP in a subject. ## Claims ### Claim 1 A method of quantitating glutamate carboxypeptidase II (GCPII) activity in a biological skin sample that contains endogenous N-acetyl-aspartate glutamate (NAAG), the method comprising the steps of: (a) reducing the amount of the endogenous NAAG in a biological skin sample; (b) contacting the biological skin sample with exogenous NAAG; and (c) detecting an exogenous NAAG cleavage product in the biological skin sample. ### Claim 2 (depends on 1) The method of claim 1, wherein the biological skin sample is a skin biopsy collected from a subject. ### Claim 3 (depends on 2) The method of claim 2, wherein the subject had been administered a GCPII inhibitor before the skin biopsy was collected. ### Claim 4 (depends on 1) The method of claim 1, wherein the step of reducing the amount of endogenous NAAG in the biological skin sample comprises the steps of: (i) homogenizing the biological sample in a buffer to create a homogenate; (ii) centrifuging the homogenate to create a sample pellet that contains the GCPII and a supernatant that contains the endogenous NAAG; and (iii) removing the supernatant from the sample. ### Claim 5 (depends on 4) The method of claim 4, wherein the sonication is used to homogenize the biological skin sample. ### Claim 6 (depends on 1) The method of claim 1, wherein the exogenous NAAG comprises a detectable label that is detected in step (c) as an indication of the presence of the exogenous NAAG cleavage product. ### Claim 7 (depends on 6) The method of claim 6, wherein the detectable label is a radioactive, fluorescent, enzymatic, colorimetric or chemiluminescent label. ### Claim 8 (depends on 1) The method of claim 1, wherein the exogenous NAAG cleavage product is glutamate or N-acetyl-aspartate (NAA). ### Claim 9 (depends on 1) The method of claim 1, wherein the exogenous NAAG cleavage product is separated from remaining exogenous NAAG before step (c). ### Claim 10 (depends on 9) The method of claim 9, wherein the exogenous NAAG cleavage product is separated from the remaining exogenous NAAG using strong cation exchange. ### Claim 11 A method of determining whether an agent is inhibiting GCPII in a subject, the method comprising the steps of: (a) administering the agent to the subject; (b) obtaining a biological skin sample from the subject; (c) reducing the amount of endogenous NAAG in the biological skin sample; (d) contacting the biological skin sample with exogenous NAAG; and (e) detecting the amount of an exogenous NAAG cleavage product in the biological skin sample; wherein a lower amount of the exogenous NAAG cleavage product in the biological skin sample compared to the amount of exogenous NAAG cleavage product in a control skin sample indicates that the agent is inhibiting GCPII in the subject. ### Claim 12 (depends on 11) The method of claim 11, wherein the control skin sample is a sample that was obtained from the subject before the agent was administered to the subject. ### Claim 13 (depends on 11) The method of claim 11, wherein the biological skin sample and the control sample are each skin biopsies obtained from the subject. ### Claim 14 (depends on 11) The method of claim 11, wherein the step of reducing the amount of endogenous NAAG in the biological skin sample comprises the steps of: (i) homogenizing the biological skin sample in a buffer to create a homogenate; (ii) centrifuging the homogenate to create a sample pellet that contains GCPII and a supernatant that contains endogenous NAAG; and (iii) removing the supernatant from the sample. ### Claim 15 (depends on 14) The method of claim 14, wherein the sonication is used to homogenize the biological skin sample. ### Claim 16 (depends on 11) The method of claim 11, wherein the exogenous NAAG comprises a detectable label that is detected in step (e) as an indication of the presence of the exogenous NAAG cleavage product. ### Claim 17 (depends on 16) The method of claim 16, wherein the detectable label is a radioactive, fluorescent, enzymatic, colorimetric or chemiluminescent label. ### Claim 18 (depends on 11) The method of claim 11, wherein the exogenous NAAG cleavage product is glutamate or N-acetyl-aspartate (NAA). ### Claim 19 (depends on 11) The method of claim 11, wherein the exogenous NAAG cleavage product is separated from remaining exogenous NAAG before step (c). ### Claim 20 (depends on 19) The method of claim 19, wherein the exogenous NAAG cleavage product is separated from the remaining exogenous NAAG using strong cation exchange.