# US 7,745,153 B2 · Pyrocatechol violet-metal protein assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,153 B2 - Document ID: US7745153B2 - Status: Granted - Kind: B2 - Application number: 12/027,065 - Filed: 2008-02-06 - Published: 2009-08-06 - Granted: 2010-06-29 - Earliest claimed: 2008-02-06 - Assignees: Pierce Biotechnology, Inc. - Inventors: A. Krishna Mallia; Babu S. Antharavally - Web page: https://patentgrep.com/patent/US7745153B2 ## CPC - G01N 33/683: {involving metal ions} - 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 A method for protein determination utilizing a pyrocatechol violet-metal complex, a composition used in the protein determination assay, and a kit. ## Claims ### Claim 1 A protein assay reagent composition comprising pyrocatechol violet at a concentration from about 0.1 mM to about 0.5 mM; a metal at a concentration from about 0.05 mM to about 0.4 mM; polyvinyl alcohol at a concentration from about 0.05% w/v to about 0.4% w/v; optionally methanol at a concentration from about 0% v/v to about 10% v/v, polyvinyl pyrrolidone at a concentration from about 0.05% w/v to about 0.2% w/v, a buffer at a concentration from about 25 mM to about 200 mM, and at least one of a chelator at a concentration from about 0.05 mM to about 2 mM, or sodium benzoate at a concentration from about 1 mM to about 6 mM, wherein the pH of the protein assay reagent composition is from about pH 1 to about pH 4. ### Claim 2 (depends on 1) The composition of claim 1 wherein the metal is selected from the group consisting of molybdate, tungsten, bismuth, thorium, uranium, vanadium, copper, and combinations thereof. ### Claim 3 (depends on 1) The composition of claim 1 wherein the metal is present as a sodium, potassium, lithium, or ammonium salt. ### Claim 4 (depends on 1) The composition of claim 1 wherein the buffer is a mono- or di-aliphatic or aromatic carboxylic acid or an inorganic acid. ### Claim 5 (depends on 1) The composition of claim 1 wherein the chelator is selected from the group consisting of sodium oxalate, phytic acid, oxalic acid, ethylenediaminetetraacetic acid (EDTA), and combinations thereof. ### Claim 6 (depends on 1) The composition of claim 1 further comprising α-cyclodextrin at a concentration from about 25 mM to about 100 mM. ### Claim 7 A protein assay reagent composition comprising pyrocatechol violet at a concentration of 0.26 mM; sodium molybdate at a concentration of 0.16 mM; polyvinyl alcohol at a concentration of about 0.2% w/v; methanol at a concentration of about 4%; succinic acid at a concentration of about 50 mM; sodium oxalate at a concentration of about 1.0 mM; sodium benzoate at a concentration of 3.5 mM; and polyvinyl pyrrolidone at a concentration of about 0.1% w/v, wherein the pH of the protein assay reagent composition is about pH 2.5. ### Claim 8 (depends on 7) The composition of claim 7 further comprising α-cyclodextrin at a concentration of about 50 mM. ### Claim 9 A method for determining protein concentration in a sample, the method comprising (a) combining the sample with a protein assay reagent composition comprising pyrocatechol violet at a concentration from about 0.1 mM to about 0.5 mM; a metal at a concentration from about 0.05 mM to about 0.4 mM; polyvinyl alcohol at a concentration from about 0.05% w/v to about 0.4% w/v; optionally methanol at a concentration from about 0% v/v to about 10% v/v, polyvinyl pyrrolidone at a concentration from about 0.05% w/v to about 0.2% w/v, a buffer at a concentration from about 25 mM to about 200 mM, and at least one of a chelator at a concentration from about 0.05 mM to about 2 mM, or sodium benzoate at a concentration from about 1 mM to about 6 mM, wherein the pH of the protein assay reagent composition is from about pH 1 to about pH 4, and wherein combining the sample with the protein assay reagent composition results in a protein determination mixture; (b) incubating the protein determination mixture for a time sufficient to form a colored complex; (c) measuring the absorbance of the colored complex; and (d) determining the protein concentration in the sample by comparing the measured absorbance with the absorbance obtained from at least one sample containing a known concentration of protein. ### Claim 10 (depends on 9) The method of claim 9 wherein the metal is selected from the group consisting of molybdate, tungsten, bismuth, thorium, uranium, vanadium, copper, and combinations thereof. ### Claim 11 (depends on 9) The method of claim 9 wherein the metal is present as a sodium, potassium, lithium, or ammonium salt. ### Claim 12 (depends on 9) The method of claim 9 wherein the buffer is a mono- or di-aliphatic or aromatic carboxylic acid or an inorganic acid. ### Claim 13 (depends on 9) The method of claim 9 wherein the chelator is selected from the group consisting of sodium oxalate, phytic acid, oxalic acid, and ethylenediaminetetraacetic acid (EDTA). ### Claim 14 (depends on 9) The method of claim 9 wherein the protein assay reagent composition further comprises α-cyclodextrin at a concentration from about 25 mM to about 100 mM. ### Claim 15 (depends on 9) The method of claim 9 wherein at least one of (a), (b), or (c) occurs in a test tube or a well of a multi-well plate. ### Claim 16 A method to enhance compatibility of a protein determination assay with a sample containing at least one interfering substance, the method comprising (a) combining the sample with a protein assay reagent composition comprising pyrocatechol violet at a concentration from about 0.1 mM to about 0.5 mM; a metal at a concentration from about 0.05 mM to about 0.4 mM; polyvinyl alcohol at a concentration from about 0.05% w/v to about 0.4% w/v; optionally methanol at a concentration from about 0% v/v to about 10% v/v, polyvinyl pyrrolidone at a concentration from about 0.05% w/v to about 2% w/v, a buffer at a concentration from about 25 mM to about 200 mM, and at least one of a chelator at a concentration from about 0.05 mM to about 2 mM, or sodium benzoate at a concentration from about 1 mM to about 6 mM, wherein the pH of the protein assay reagent composition is from about pH 1 to about pH 4, and wherein combining the sample with the protein assay reagent composition results in a protein determination mixture; (b) incubating the protein determination mixture for a time sufficient to result in the formation of a colored complex; (c) measuring the absorbance of the colored complex; and (d) determining the protein concentration in the sample by comparing the measured absorbance with the absorbance obtained from at least one sample containing a known concentration of protein, wherein the interfering substance is selected from the group consisting of detergents, reducing agents, chelating agents, buffers, reagents, solvents, and combinations thereof, and wherein the method enhances the compatibility of the protein determination assay with a sample containing at least one interfering substance. ### Claim 17 (depends on 16) The method of claim 16 wherein the metal is selected from the group consisting of molybdate, tungsten, bismuth, thorium, uranium, vanadium, copper, and combinations thereof. ### Claim 18 (depends on 16) The method of claim 16 wherein the metal is present as a sodium, potassium, lithium, or ammonium salt. ### Claim 19 A kit for determining protein concentration, the kit comprising a protein assay reagent composition comprising pyrocatechol violet, a metal, polyvinyl alcohol, optionally methanol, polyvinyl pyrrolidone, a buffer, and at least one of a chelator, or sodium benzoate, and instructions for determining protein concentration using the kit components. ### Claim 20 (depends on 19) The kit of claim 19 wherein the metal is selected from the group consisting of molybdate, tungsten, bismuth, thorium, uranium, vanadium, copper, and combinations thereof. ### Claim 21 (depends on 19) The kit of claim 19 wherein the metal is present as a sodium, potassium, lithium, or ammonium salt. ### Claim 22 (depends on 19) The kit of claim 19 wherein the buffer is a mono- or di-aliphatic or aromatic carboxylic acid or an inorganic acid. ### Claim 23 (depends on 19) The kit of claim 19 wherein the chelator is selected from the group consisting of sodium oxalate, phytic acid, oxalic acid, and ethylenediaminetetraacetic acid (EDTA). ### Claim 24 (depends on 19) The kit of claim 19 further comprising α-cyclodextrin, either as a component of the protein assay reagent composition or separately. ### Claim 25 (depends on 9) The method of claim 9 wherein up to about 2000 μg/ml protein is determined. ### Claim 26 (depends on 16) The method of claim 16 wherein up to about 2000 μg/ml protein is determined. ### Claim 27 (depends on 9) The method of claim 9 wherein protein precipitation in the protein determination mixture is prevented by polyvinyl alcohol and polyvinyl pyrrolidone. ### Claim 28 (depends on 16) The method of claim 16 wherein protein precipitation in the protein determination mixture is prevented by polyvinyl alcohol and polyvinyl pyrrolidone. ### Claim 29 (depends on 9) The method of claim 9 wherein the protein determination mixture is incubated for about two minutes to about twenty minutes to form a colored complex. ### Claim 30 (depends on 16) The method of claim 16 wherein the protein determination mixture is incubated for about two minutes to about twenty minutes to form a colored complex. ### Claim 31 (depends on 9) The method of claim 9 wherein the sample is a biological fluid selected from the group consisting of urine, serum, plasma, cerebrospinal fluid, and cell lysates. ### Claim 32 (depends on 16) The method of claim 16 wherein the sample is a biological fluid selected from the group consisting of urine, serum, plasma, cerebrospinal fluid, and cell lysates.