Immunoassays

Since the development in the 1970s of antisera against carcinogen-DNA adducts, immunological assays have been widely used for detection and quantitative assessment of DNA damage.1024-26 Immunoassays may generally be applied for measurement of any structural DNA alteration caused by carcinogens or mutagens, for which a specific antibody is available. The inception of antibodies recognizing BPDE-DNA adducts has opened new and growing avenues for monitoring human exposure to this ubiquitous carcinogen by directly assessing damage in DNA.80 Furthermore, antibodies against many different types of DNA lesions have been elicited and utilized for measurements in vivo. Usually, two types of immunogens are used for antibody development, either adducted unhydrolyzed DNA electrostatically complexed to methylated carrier protein (bovine serum albumin) or monoadducts coupled to a carrier protein (keyhole limpet hemocyanin). Polyclonal and monoclonal antibodies have been used for biomonitoring studies. The advantage of polyclonal antibodies is the rapid generation and easy handling of antisera. Monoclonal antibodies are more difficult to develop and maintain but the major and hard-to-match advantage of their use is the unlimited supply of antibody achievable by propagating the immortalized hybrid clones. The specificity of antibodies is important, since cross-reactivity with undamaged DNA or structurally related adducts may complicate the results.37 This feature may actually become advantageous if there is a need to detect a whole group of structurally similar damages, e.g., total aromatic or PAH DNA adducts. A major limitation of immunoassays is the need to develop and characterize antibodies for each DNA adduct of interest. However, the progress in the area of new anti-DNA adduct antibody development has been remarkable and more specific clones have become available. Once the antibody is fully characterized and, importantly, validated by independent techniques, it may be used in comparative experiments using human samples. Besides quantitative assays, antibodies have been employed in damage visualization techniques including immunohis-tochemistry and immuno-electron microscopy, and for immuno-affinity purification. Quantitative measurements were primarily accomplished by competitive radioimmunoasssay (RIA) and enzyme-linked immunosorbent assay (ELISA) and by noncompetitive immunoslot blot (ISB) analysis. Non-competitive immuno-slot or -dot blot methods have been developed for adduct detection in DNA immobilized directly onto nitrocellulose membrane surfaces.81 Incubation with primary antibody is followed by incubation with enzyme-conjugated secondary antibody, or an intermediate avidin-biotin binding step is performed.82 The signal is produced with either colored8183 or chemiluminescent1384 substrates. Using a polyclonal antiserum recognizing BPDE-DNA adducts, we were able to reach the sensitivity of 2 adducts/107 nucleotides with a colored endpoint.85

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