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US 2018/0003720 A1Published application
Methods and kits for measuring and quantifying dna double-stranded breaks using gamma-h2ax and h2ax
AssigneeThe United States of America as represented by the Secretary, Department of Health and Human Service
InventorsChristophe E. Redon, William M. Bonner, Yiping Zhang, Jiuping Ji
Life of this patent
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Abstract
Disclosed are methods of measuring the amount of exposure of a host to a DNA double-stranded break (DSB)-causing agent by determining the ratio of the quantity of γ-H2AX to the quantity of total H2AX in a biological sample from the host as compared to the ratio of the quantity of γ-H2AX to the quantity of total H2AX in a positive control biological sample that has been exposed to a known amount of a DSB-causing agent. Related kits and methods of quantifying DSBs in a test biological sample are also disclosed.
Claims22
A method of measuring the amount of exposure of a host to a DNA double-stranded break (DSB)-causing agent, the method comprising:
- contacting (i) a first portion of a positive control biological sample and (ii) a first portion of a biological sample from the host with an antibody or antigen binding fragment thereof that specifically binds to γ-H2AX and forms a first complex with the γ-H2AX;
- contacting (i) a second portion of the positive control biological sample and (ii) a second portion of the biological sample from the host with an antibody or antigen binding fragment thereof that specifically binds to an H2A core protein and forms a second complex with the H2A core protein,
- wherein the positive control biological sample has been exposed to a known amount of the DSB-causing agent;
- contacting the first and second complexes of the positive control biological sample and the biological sample from the host with an antibody or antigen binding fragment thereof that specifically binds to H2AX and forms a third complex with H2AX in the first complex and forms a fourth complex with H2AX in the second complex;
- detecting the third and fourth complexes in the positive control biological sample and the biological sample from the host;
- quantifying the third and fourth complexes in the positive control biological sample and the biological sample from the host;
- determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the positive control biological sample;
- determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the biological sample from the host;
- and
- comparing the ratio of the quantity of the third complex to the quantity of the fourth complex in the positive control biological sample to the ratio of the quantity of the third complex to the quantity of the fourth complex in the biological sample from the host to determine the amount of exposure of the host to the DNA DSB-causing agent.
7 more elements
A method of quantifying DNA DSBs in a test biological sample, the method comprising:
- contacting (i) a first portion of a positive control biological sample and (ii) a first portion of a test biological sample with an antibody or antigen binding fragment thereof that specifically binds to γ-H2AX and forms a first complex with the γ-H2AX;
- contacting (i) a second portion of the positive control biological sample and (ii) a second portion of the test biological sample with an antibody or antigen binding fragment thereof that specifically binds to an H2A core protein and forms a second complex with the H2A core protein,
- wherein the positive control biological sample has a known quantity of DSBs;
- contacting the first and second complexes of the positive control biological sample and the test biological sample with an antibody or antigen binding fragment thereof that specifically binds to H2AX and forms a third complex with H2AX in the first complex and forms a fourth complex with H2AX in the second complex;
- detecting the third and fourth complexes in the positive control biological sample and the test biological sample;
- quantifying the third and fourth complexes in the positive control biological sample and the test biological sample;
- determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the positive control biological sample;
- determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the test biological sample;
- and
- comparing the ratio of the quantity of the third complex to the quantity of the fourth complex in the positive control biological sample to the ratio of the quantity of the third complex to the quantity of the fourth complex in the test biological sample to determine the quantity of DSBs in the test biological sample.
7 more elements
A method of testing compounds for ability to cause DNA DSBs in a host, the method comprising:
- (a) contacting (i) a first portion of a control biological sample and (ii) a first portion of a host biological sample with an antibody or antigen binding fragment thereof that specifically binds to γ-H2AX and forms a first complex with the γ-H2AX, wherein the host biological sample is from a host which has been administered a test compound;
- (b) contacting (i) a second portion of the control biological sample and (ii) a second portion of the host biological sample with an antibody or antigen binding fragment thereof that specifically binds to an H2A core protein and forms a second complex with the H2A core protein,
- wherein the control biological sample has not been exposed to the test compound;
- (c) contacting the first and second complexes of the control biological sample and the host biological sample with an antibody or antigen binding fragment thereof that specifically binds to H2AX and forms a third complex with H2AX in the first complex and forms a fourth complex with H2AX in the second complex;
- (d) detecting the third and fourth complexes in the control biological sample and the host biological sample;
- (e) quantifying the third and fourth complexes in the control biological sample and the host biological sample;
- (f) determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the control biological sample;
- and
- (g) determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the host biological sample;
- (h) comparing the ratio of the quantity of the third complex to the quantity of the fourth complex in the control biological sample to the ratio of the quantity of the third complex to the quantity of the fourth complex in the host biological sample,
- wherein an increase in the ratio in the host biological sample as compared to the ratio in the control biological sample indicates that the test compound has the ability to cause DNA DSBs in the host.
8 more elements
A method of screening test compounds for ability to cause DNA DSBs, the method comprising:
- (a) dividing a population of test biological samples into more than one test sub-population;
- (b) treating each test sub-population with a test compound, wherein each test sub-population is treated with a different test compound;
- (c) contacting (i) a first portion of each treated test sub-population and (ii) a first portion of a control biological sample with an antibody or antigen binding fragment thereof that specifically binds to γ-H2AX and forms a first complex with the γ-H2AX;
- (d) contacting (i) a second portion of each treated test sub-population and (ii) a second portion of a control biological sample with an antibody or antigen binding fragment thereof that specifically binds to an H2A core protein and forms a second complex with the H2A core protein;
- wherein the control biological sample has not been treated with a test compound;
- (e) contacting the first and second complexes of the control biological sample and the treated test sub-populations with an antibody or antigen binding fragment thereof that specifically binds to H2AX and forms a third complex with H2AX in the first complex and forms a fourth complex with H2AX in the second complex;
- (f) detecting the third and fourth complexes in the control biological sample and the treated test sub-populations;
- (g) quantifying the third and fourth complexes in the control biological sample and the treated test sub-populations;
- (h) determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the control biological sample;
- (i) determining a ratio of the quantity of the third complex to the quantity of the fourth complex in the treated test sub-populations;
- and
- (j) comparing the ratio of the quantity of the third complex to the quantity of the fourth complex in the control biological sample to the ratio of the quantity of the third complex to the quantity of the fourth complex in the treated test sub-populations,
- wherein an increase in the ratio in a treated test sub-population as compared to the ratio in the control biological sample indicates that the test compound has the ability to cause DNA DSBs.
10 more elements
A kit comprising:
- at least one substrate;
- an antibody or antigen binding fragment thereof that specifically binds to γ-H2AX;
- an antibody or antigen binding fragment thereof that specifically binds to H2A core protein;
- an antibody or antigen binding fragment thereof that specifically binds to H2AX;
- and
- a detecting agent comprising a label.
3 more elements
Dependencies are inferred from the claim text.