# US 10,501,774 B2 · Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,774 B2 - Document ID: US10501774B2 - Status: Granted - Kind: B2 - Application number: 15/215,307 - Filed: 2016-07-20 - Published: 2016-11-10 - Granted: 2019-12-10 - Earliest claimed: 2013-02-22 - Assignees: Promega Corporation - Applicants: Promega Corporation - Inventors: Michael P. Valley; James J. Cali; Brock Binkowski; Christopher Todd Eggers; Keith V. Wood - Web page: https://patentgrep.com/patent/US10501774B2 ## CPC - C12Q 1/66: involving luciferase - C12Q 1/26: involving oxidoreductase - 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 Methods, kits and compositions containing a mixture of D-luciferin and L-luciferin for light generation with luciferase are disclosed that have improved stability when stored over time. The mixture of D-luciferin and L-luciferin can be used to detect the presence or amount of ATP or of luciferase in a sample. ## Claims ### Claim 1 A luciferase reaction composition comprising: a luciferase; and a combination of D-luciferin and L-luciferin present in the composition in a ratio from about 0.25:1 to about 4:1 of D-luciferin to L-luciferin; wherein the composition is substantially free of adenosine triphosphate (ATP); and wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C. ### Claim 2 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C. as compared to a composition that has substantially no L-luciferin. ### Claim 3 (depends on 1) The composition of claim 1, wherein the ratio of D-luciferin to L-luciferin is from about 0.5:1 to about 2:1. ### Claim 4 (depends on 1) The composition of claim 1, wherein the ratio of D-luciferin to L-luciferin is from about 1:1 to about 4:1. ### Claim 5 (depends on 1) The composition of claim 1, wherein the ratio of D-luciferin to L-luciferin is about 1:1. ### Claim 6 (depends on 1) The composition of claim 1, wherein the composition has a pH of at least about 5 and less than about 9. ### Claim 7 (depends on 1) The composition of claim 1, wherein the luciferase is a beetle luciferase. ### Claim 8 (depends on 1) The composition of claim 1, wherein the D-Luciferin is D-5′fluoroluciferin and wherein the L-luciferin is L-5′fluoroluciferin. ### Claim 9 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 35° C. ### Claim 10 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 30° C. ### Claim 11 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 20° C. to about 25° C. ### Claim 12 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 3 days at a temperature from about 0° C. to about 35° C. ### Claim 13 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 5 days at a temperature from about 0° C. to about 35° C. ### Claim 14 (depends on 1) The composition of claim 1, wherein the composition retains at least 50% luciferase activity after being stored for at least about 10 days at a temperature from about 0° C. to about 35° C. ### Claim 15 (depends on 1) The composition of claim 1, wherein the composition further comprises at least one additional component selected from: a buffer, protein stabilizer, magnesium ions, a metal chelator, a detergent, a defoamer, inorganic phosphate, pyrophosphate, AMP, coenzyme A, a reducing agent, or a combination thereof. ### Claim 16 (depends on 1) The composition of claim 1, wherein the composition comprises magnesium sulfate and trans-1,2-Cyclohexanediaminetetraacetic Acid (CDTA). ### Claim 17 (depends on 1) The composition of claim 1, wherein the composition further comprises at least one additional component selected from: 1,4-Piperazinediethanesulfonic acid, 1,2-Diaminocyclohexanetetraacetic acid, Tergitol detergent, Mazu defoamer, pyrophosphate, DTT, sulfite, thiosulfate, sodium fluoride, dodecyltrimethylammonium, hydroxypolyethoxydodecane, potassium phosphate, ethylenediaminetetraacetic acid (EDTA), or a combination thereof. ### Claim 18 (depends on 1) A kit comprising the composition of claim 1 packaged in at least one container. ### Claim 19 (depends on 18) The kit of claim 18, wherein the composition retains at least 50% luciferase activity after being stored for at least about 1 day at a temperature from about 0° C. to about 35° C. as compared to a composition that has substantially no L-luciferin. ### Claim 20 (depends on 1) A method for determining the presence or amount of ATP in a sample comprising contacting the composition of claim 1 with a sample and detecting luminescence generated, thereby determining the presence or amount of ATP in the sample. ### Claim 21 (depends on 20) The method of claim 20, wherein the sample comprises intact cells and/or a purified enzyme which generates or uses ATP. ### Claim 22 (depends on 20) The method of claim 20, wherein the sample comprises a crude cell lysate or a clarified cell lysate.