US5538857A
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Assay for enzyme activity from a red blood sample using a direct microfluorometric assay
Assignee: Isolab Inc
•
Filing: 1994-06-01
•
Granted: 1996-07-23
•
Expires: 2014-06-01
Abstract
Abstract The present invention is a method for assaying enzyme activity in a red blood sample. The method comprises these steps: (a) placing the following in a sample well: (1) a red blood sample containing an enzyme, (2) a substrate or substrates for the enzyme, (3) water, and (4) a buffer; (b) incubating the contents of the sample well for sufficient time and at sufficient temperature to allow for the formation of a fluorescent enzyme product should the enzyme be present in the red blood sample; (c) precipitating the hemoglobin; and (d) measuring the fluorescence of any fluorescent enzyme product formed in the sample well, directly from that sample well. The method of the invention may be used for assaying the activity of an enzyme, such as galactose-1-phosphate uridyl transferase (GALT) or biotinidase, in a red blood sample.
Inventors
- 👤 Murray A. Rosenthal
- 👤 Ronald A. Simkins
- 👤 Ranjan Akhaury
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 03:04
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/48; G; G01; G01N; G01N2333/00; G01N2333/795; G01N2333/805; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333; Y10T436/144444
Claims Analysis
Total Claims: 45
Claim 1
Independent Claim
122 words
A method for assaying enzyme activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a red blood sample containing an enzyme; (ii) a substrate, said substrate and enzyme being able to react to form a fluorescent enzyme product; (iii) water; and (iv) a buffer; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of said fluorescent enzyme product should said enzyme be present in said red blood sample; followed by (c) precipitating said hemoglobin; and (d) measuring the fluorescence of any said fluorescent enzyme product formed in said sample well, directly from said sample well.
Claim 2
Independent Claim
122 words
A method for assaying enzyme activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a red blood sample containing an enzyme; (ii) a substrate, said substrate and enzyme being able to react to form a fluorescent enzyme product; (iii) water; and (iv) a buffer; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of said fluorescent enzyme product should said enzyme be present in said red blood sample; followed by (c) precipitating said hemoglobin; and (d) measuring the fluorescence of any said fluorescent enzyme product formed in said sample well, directly from said sample well.
Claim 3
Dependent Claim
47 words
A method according to claim 1 wherein said red blood sample is placed in said sample well in a form selected from the group consisting of whole blood, washed red blood cells, a hemolysate made from red blood cells, and dried whole blood on a physical support.
Claim 4
Dependent Claim
29 words
A method according to claim 1 wherein said buffer is selected from the group consisting of phosphate, tris(hydroxyethyl)aminomethane, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, N-ethylmorpholine, 5,5'-diethylbarbituric acid, N-2-hydroxyethylpiperazine-propanesulfonic acid, glycine, and mixtures thereof.
Claim 5
Dependent Claim
15 words
A method according to claim 1 wherein said hemoglobin is precipitated by addition of ethanol.
Claim 6
Dependent Claim
38 words
A method according to claim 1 wherein said sample well is also provided with at least one component selected from the group consisting of lysing reagents, surfactants, ethylenediaminetetraacetic acid, dithiothreitol, magnesium enzymes, proteins, anti-microbial agents and enzyme stablizers.
Claim 7
Dependent Claim
21 words
A method according to claim 1 wherein the formation of any said fluorescent enzyme product is halted prior to step (d).
Claim 8
Dependent Claim
40 words
A method according to claim 6 wherein the formation of any said fluorescent enzyme product is halted by a method selected from the group consisting of the application of heat, dilution, change of pH and addition of an organic solvent.
Claim 9
Dependent Claim
29 words
A method according to claim 7 wherein said hemoglobin is precipitated and the formation of any said fluorescent enzyme product is halted substantially simultaneously and prior to step (d).
Claim 10
Independent Claim
143 words
A method for assaying the activity of an enzyme in the presence of hemoglobin, said method comprising the steps: (a) placing the following in a sample well: (i) a physical support bearing a sample to be analyzed, said sample containing said enzyme and hemoglobin; and (ii) at least one substrate of said enzyme, said substrate and enzyme being able to react to form a fluorescent enzyme product; and (iii) water; whereby said enzyme, said hemoglobin and said at least one substrate are placed in solution; (b) incubating said sample and at least one said substrate for sufficient time and at sufficient temperature to allow the formation of a fluorescent enzyme product should said enzyme be present in said sample; followed by (c) precipitating said hemoglobin onto said physical support; and (d) measuring the fluorescence of said fluorescent product directly from said sample well.
Claim 11
Independent Claim
143 words
A method for assaying the activity of an enzyme in the presence of hemoglobin, said method comprising the steps: (a) placing the following in a sample well: (i) a physical support bearing a sample to be analyzed, said sample containing said enzyme and hemoglobin; and (ii) at least one substrate of said enzyme, said substrate and enzyme being able to react to form a fluorescent enzyme product; and (iii) water; whereby said enzyme, said hemoglobin and said at least one substrate are placed in solution; (b) incubating said sample and at least one said substrate for sufficient time and at sufficient temperature to allow the formation of a fluorescent enzyme product should said enzyme be present in said sample; followed by (c) precipitating said hemoglobin onto said physical support; and (d) measuring the fluorescence of said fluorescent product directly from said sample well.
Claim 12
Dependent Claim
13 words
A method according to claim 9 wherein said physical support comprises filter paper.
Claim 13
Dependent Claim
26 words
A method according to claim 9 wherein said sample is placed in said sample well in a form of dried whole blood on a physical support.
Claim 14
Dependent Claim
15 words
A method according to claim 9 wherein said hemoglobin is precipitated by addition of ethanol.
Claim 15
Dependent Claim
38 words
A method according to claim 9 wherein said sample well is also provided with at least one component selected from the group consisting of lysing reagents, surfactants, ethylenediaminetetraacetic acid, dithiothreitol, magnesium, enzymes, proteins, anti-microbial agents and enzyme stabilizers.
Claim 16
Dependent Claim
20 words
A method according to claim 9 wherein the formation of said fluorescent enzyme product is halted prior to step (d).
Claim 17
Dependent Claim
39 words
A method according to claim 14 wherein the formation of said fluorescent enzyme product is halted by a method selected from the group consisting of the application of heat, dilution, change of pH and addition of an organic solvent.
Claim 18
Dependent Claim
32 words
A method according to claim 14 wherein said hemoglobin is precipitated onto said physical support and the formation of said fluorescent enzyme product is halted substantially simultaneously and prior to step (d).
Claim 19
Independent Claim
212 words
A method for assaying the activity of an enzyme in a red blood target sample and comparing same to that of a red blood control sample, said samples containing hemoglobin, said method comprising the steps: (a) assaying said activity of said enzyme both in a red blood target sample and in a red blood control sample comprising non-human mammal blood, in separate sample wells, each of said sample wells containing contents comprising respective said samples and: (i) at least one substrate of said enzyme, said substrate and enzyme being able to react to form a fluorescent enzyme product; (ii) water; and (iii) a buffer; (b) incubating said contents of each of said sample wells for sufficient time and at sufficient temperature to allow for the formation of a fluorescent enzyme product should said enzyme be present in said samples; followed by (c) precipitating said hemoglobin in each of said sample wells; (d) measuring the fluorescence of any said fluorescent enzyme product formed in each of said sample wells, respectively, directly from each of said sample wells; and (e) comparing the fluorescence of any said fluorescent enzyme product formed in said red blood target sample well to the fluorescence of any said fluorescent enzyme product formed in said red blood control sample well.
Claim 20
Independent Claim
212 words
A method for assaying the activity of an enzyme in a red blood target sample and comparing same to that of a red blood control sample, said samples containing hemoglobin, said method comprising the steps: (a) assaying said activity of said enzyme both in a red blood target sample and in a red blood control sample comprising non-human mammal blood, in separate sample wells, each of said sample wells containing contents comprising respective said samples and: (i) at least one substrate of said enzyme, said substrate and enzyme being able to react to form a fluorescent enzyme product; (ii) water; and (iii) a buffer; (b) incubating said contents of each of said sample wells for sufficient time and at sufficient temperature to allow for the formation of a fluorescent enzyme product should said enzyme be present in said samples; followed by (c) precipitating said hemoglobin in each of said sample wells; (d) measuring the fluorescence of any said fluorescent enzyme product formed in each of said sample wells, respectively, directly from each of said sample wells; and (e) comparing the fluorescence of any said fluorescent enzyme product formed in said red blood target sample well to the fluorescence of any said fluorescent enzyme product formed in said red blood control sample well.
Claim 21
Dependent Claim
42 words
A method according to claim 17 wherein said red blood target sample and said red blood control sample are placed in said respective sample wells on a physical support, and wherein said hemoglobin is precipitated onto said physical support in step (c).
Claim 22
Dependent Claim
26 words
A method according to claim 17 wherein said control sample comprises a non-human mammal blood selected from the group consisting of horse blood and sheep blood.
Claim 23
Independent Claim
162 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a red blood sample; (ii) galactose-1-phosphate; (iii) uridine diphosphoglucose; (iv) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (v) water; and (vi) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; (c) precipitating said hemoglobin; and (d) measuring the fluorescence of any said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said sample well, directly from said sample well.
Claim 24
Independent Claim
162 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a red blood sample; (ii) galactose-1-phosphate; (iii) uridine diphosphoglucose; (iv) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (v) water; and (vi) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; (c) precipitating said hemoglobin; and (d) measuring the fluorescence of any said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said sample well, directly from said sample well.
Claim 25
Dependent Claim
47 words
A method according to claim 20 wherein said red blood sample is placed in said sample well in a form selected from the group consisting of whole blood, washed red blood cells, a hemolysate made from red blood cells, and dried whole blood on a physical support.
Claim 26
Dependent Claim
29 words
A method according to claim 20 wherein said buffer is selected from the group consisting of phosphate, tris(hydroxyethyl)aminomethane, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, N-ethylmorpholine, 5,5'-diethylbarbituric acid, N-2-hydroxyethylpiperazine-propanesulphonic acid, glycine, and mixtures thereof.
Claim 27
Dependent Claim
15 words
A method according to claim 20 wherein said hemoglobin is precipitated by addition of ethanol.
Claim 28
Dependent Claim
37 words
A method according to claim 20 wherein said sample well is also provided with at least one component selected from the group consisting of: lysing reagents, surfactants, EDTA, dithiothreitol, magnesium, enzymes, proteins, anti-microbial agents and enzyme stabilizers.
Claim 29
Dependent Claim
27 words
A method according to claim 20 wherein the formation of said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted prior to step (d).
Claim 30
Dependent Claim
46 words
A method according to claim 25 wherein the formation of said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted by a method selected from the group consisting of the application of heat, dilution, change of pH and addition of an organic solvent.
Claim 31
Dependent Claim
35 words
A method according to claim 25 wherein said hemoglobin is precipitated and the formation of said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted substantially simultaneously and prior to step (d).
Claim 32
Independent Claim
174 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a dried red blood sample on a physical support; (ii) galactose-1-phosphate; (iii) uridine diphosphoglucose; (iv) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (v) water; and (vi) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; followed by (c) precipitating said hemoglobin onto said physical support; and (d) measuring the fluorescence issuing from any said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said sample well, directly from said sample well.
Claim 33
Independent Claim
174 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood sample, said sample containing hemoglobin, said method comprising the steps: (a) placing the following contents in a sample well: (i) a dried red blood sample on a physical support; (ii) galactose-1-phosphate; (iii) uridine diphosphoglucose; (iv) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (v) water; and (vi) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of said sample well for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; followed by (c) precipitating said hemoglobin onto said physical support; and (d) measuring the fluorescence issuing from any said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said sample well, directly from said sample well.
Claim 34
Dependent Claim
28 words
A method according to claim 28 wherein said red blood sample is placed in said sample well in a form of dried whole blood on a physical support.
Claim 35
Dependent Claim
13 words
A method according to claim 28 wherein said physical support comprises filter paper.
Claim 36
Dependent Claim
29 words
A method according to claim 28 wherein said buffer is selected from the group consisting of phosphate, tris(hydroxyethyl)aminomethane, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, N-ethylmorpholine, 5,5'-diethylbarbituric acid, N-2-hydroxyethylpiperazine-propanesulphonic acid, glycine, and mixtures thereof.
Claim 37
Dependent Claim
15 words
A method according to claim 28 wherein said hemoglobin is precipitated by addition of ethanol.
Claim 38
Dependent Claim
38 words
A method according to claim 28 wherein said sample well is also provided with at least one component selected from the group consisting of lysing reagents, surfactants, ethylenediaminetetraacetic acid, dithiothreitol, magnesium, enzymes, proteins, anti-microbial agents and enzyme stabilizers.
Claim 39
Dependent Claim
27 words
A method according to claim 28 wherein the formation for said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted prior to step (d).
Claim 40
Dependent Claim
46 words
A method according to claim 34 wherein the formation of said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted by a method selected from the group consisting of the application of heat, dilution, change of pH and addition of an organic solvent.
Claim 41
Dependent Claim
35 words
A method according to claim 34 wherein said hemoglobin is precipitated and the formation of said reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is halted substantially simultaneously and prior to step (d).
Claim 42
Independent Claim
258 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood target sample and comparing same to that of a red blood control sample, said samples containing hemoglobin, said method comprising the steps: (a) assaying galactose-1-phosphate uridyl transferase activity both in a red blood target sample and in a red blood control sample comprising non-human mammal blood, in separate sample wells, each of said sample wells containing contents comprising respective said samples and: (i) galactose-1-phosphate; (ii) uridine diphosphoglucose; (iii) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (iv) water; and (v) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of each of said sample wells for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; followed by (c) precipitating said hemoglobin in each of said sample wells; (d) measuring the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in each of said sample wells, respectively, directly from each of said sample wells; and (e) comparing the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said red blood sample well to the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said control sample well.
Claim 43
Independent Claim
258 words
A method for assaying galactose-1-phosphate uridyl transferase activity in a red blood target sample and comparing same to that of a red blood control sample, said samples containing hemoglobin, said method comprising the steps: (a) assaying galactose-1-phosphate uridyl transferase activity both in a red blood target sample and in a red blood control sample comprising non-human mammal blood, in separate sample wells, each of said sample wells containing contents comprising respective said samples and: (i) galactose-1-phosphate; (ii) uridine diphosphoglucose; (iii) a substance selected from the group consisting of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; (iv) water; and (v) a buffer adapted to maintain the pH within a range of from about 7.0 to about 8.5; (b) incubating said contents of each of said sample wells for sufficient time and at sufficient temperature to allow for the formation of a substance selected from the group consisting of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate should said galactose-1-phosphate uridyl transferase be present in said red blood sample; followed by (c) precipitating said hemoglobin in each of said sample wells; (d) measuring the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in each of said sample wells, respectively, directly from each of said sample wells; and (e) comparing the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said red blood sample well to the fluorescence of any reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate formed in said control sample well.
Claim 44
Dependent Claim
42 words
A method according to claim 37 wherein said red blood target sample and said red blood control sample are placed in said respective sample wells on a physical support, and wherein said hemoglobin is precipitated onto said physical support in step (c).
Claim 45
Dependent Claim
26 words
A method according to claim 37 wherein said control sample comprises a non-human mammal blood selected from the group consisting of horse blood and sheep blood.
References Cited (Backward Citations)
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