Methods and kits for measuring and quantifying DNA double-stranded breaks using gamma-H2AX and H2AX

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 gamma-H2AX (γ-H2AX, phosphorylated H2AX (histone family member X)) forms a first complex with the γ-H2AX that is present in each one of the positive control biological sample and the biological sample from the host;
• 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 a histone H2A core protein and forms a second complex with the H2A core protein that is present in each one of the positive control biological sample and the biological sample from the host,
• 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 the H2AX in each of the first complex of the positive control biological sample and the first complex of the biological sample from the host and forms a fourth complex with the H2AX in each of the second complex of the positive control biological sample and the second complex of the biological sample from the host;
• detecting each of the third and fourth complexes in the positive control biological sample and the biological sample from the host comprising a detectable label, comprising determining an amount of detectable label present in each of the positive control biological sample and the biological sample from the host;
• quantifying each of 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, wherein an increase in the ratio in the host biological sample as compared to the ratio in the positive control biological sample indicates that the DSB-causing agent has the ability to cause DNA DSBs in the host.