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231 Quantitative Assay for O6-Carboxymethyl Guanine DNA Adducts
Number of anticipated awards: 1
(Fast-Track proposals will not be accepted.)
Budget (total costs): Phase I: $150,000;
Phase II: $750,000
The deadline for receipt of all contract proposals submitted in response to this solicitation was:
5:00 p.m. Eastern Standard Time
Monday, November 6, 2006
The goal of this project is for the small business to develop and commercialize a kit for quantitatively measuring O6-carboxymethyl guanine adducts in human DNA samples.
Nitrosamines are a class of toxic and carcinogenic agents that can form mutagenic DNA adducts. Humans are exposed to preformed nitrosamines through tobacco smoke and other routes, but a substantial source of exposure is endogenous formation in the gut and/or mouth. One import class of nitrosamines is the N-methyl-N-nitroso compounds that can be metabolized to DNA-reactive intermediates. Epidemiologic studies of the association between nitrosamine exposure and cancer have been hampered by the lack of good exposure metrics for nitrosamines. Recently, the O6-carboxymethyl guanine DNA adduct has been identified as a potentially useful marker of N-methyl-N-nitrosocompound exposure because humans seem to lack an efficient repair mechanism for this adduct. Therefore, a very sensitive, high-throughput assay for O6-carboxymethyl guanine would facilitate epidemiologic studies of nitrosamine exposure and cancer risk.
To date, a single group has reported the development of a polyclonal antibody and associated immunoassay for O6-carboxymethyl guanine. But, the assay has not been successfully applied to the type and number of samples required for epidemiologic studies. Several aspects of the assay could be optimized. First, the assay is based on a single generation of a polyclonal antibody and therefore a novel polyclonal antibody or selection of a best monoclonal antibody may improve assay sensitivity. Alternatively, a non-traditional specific labeling agent, such as an aptamer, may improve sensitivity. Second, although the immuno-slot blot technique previously used might be most sensitive under some conditions, alternative assays should be investigated, such as chemiluminescence enhanced immunoassays, with attention to optimization of plates, blocking agents, titers, and signal boosting system to improve detection and quantitation.
Phase I Activities and expected deliverables: The successful respondent shall devise, create, and test a novel O6-carboxymethyl guanine identifying system such as, but not limited to, a polyclonal antibody, monoclonal antibody, or aptamer for use on human DNA samples. Other completely novel systems not based on antibodies or aptamers may also be proposed as long as they fulfill the assay requirements. The accuracy, precision, and sensitivity of the novel method shall be tested by (1) measurement of standards with known concentrations of O6-carboxymethyl guanine such as in vitro modified DNA; (2) measurement of standards mixed with extracted human DNA collected from subjects using standard field study methods; (3) the specificity of the reagent should be demonstrated by examining the cross-reactivity with any carriers or links used in reagent production, normal DNA bases, and similar adducts. These demonstrations should show that the novel method has limits of detection and quantitation which exceed current methods. Furthermore, the respondent should demonstrate that the novel method can be assembled as a relatively low cost kit which can be used by a laboratory with the equipment and experience necessary for a typical ELISA or other routine assays (e.g. slot blots). The respondent will supply all required equipment and reagents. The National Cancer Institute will supply limited assistance to the respondent in obtaining normal human DNA for assay development if requested.
Phase II Activities and expected deliverables: The respondent will produce kits for large-scale testing for O6-carboxymethyl guanine concentrations in human DNA samples from epidemiologic studies, instructions for use of the kits, and instructions for interpretation of the data. The kits shall demonstrate reliable performance characteristics when stored under conditions and for time intervals similar to those required for comparable reagents.
