Monoclonal antibodies for the specific detection of 3-alkyladenines in nucleic acids and body fluids

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Abstract
We describe an immunoanalytical procedure for the detection and quantitation of 3-alkyladenines in biological samples with the use of anti-(3-alkyladenine) monoclonal antibodies (Mab). A new hapten-protein conjugate, 3-ethyl-8-(3-carboxy-propyl)-adenine, was used for immunization of BALB/c mice after conjugation to carrier proteins via the carboxyl group. Eighty-nine hybridomas were established which secrete anti-(3-alkyladenine) Mab with antibody affinity constants ranging from 1 × 107 to 5 × 109 l/mol for 3-ethyladenine (3-EtAde). One of these Mab (EM-6-47) had detection limits of 30 fmol for 3-EtAde, 17 fmol for 3-n-butyladenine (3-BuAde) and 475 fmol for 3-methyladenine (3-MeAde) respectively, at 25% inhibition of tracer-antibody binding. The binding pattern of Mab EM-6-47 revealed high specificity for adenine substituted at N-3 with different alkyl residues and no, or very low, cross-reactivity with other alkylated or unmodified nucleic acid components or structurally related compounds. 3-MeAde and 3-EtAde can be well separated from nucleic acids, and from rat and human urine samples, using HPLC with two successive stationary phases. Using Mab EM-6-47 in conjunction with a competitive RIA, both 3-MeAde and 3-EtAde were detected in the range of 100–300 ng (3-MeAde) and 2–10 ng (3-EtAde) in urine samples (10 ± 2 ml) of untreated rats collected over a 24 h period. Only 3-MeAde (range 1.3–24.20 μg) was found in human urine samples. The concentration of 3-EtAde in rat urine increased significantly during the 24 h following a single i.v. application of N-ethyl-N-nitrosourea. After i.p. application of known amounts of 3-MeAde and 3-EtAde, >90% of 3-MeAde and >70% of 3-EtAde were excreted in rat urine within the subsequent 24 h. The concentration of 3-alkyladenines in body fluids (urine) may thus provide a useful indicator of environmental exposure to nucleic acid-reactive agents, and the immunoanalytical procedure described here permits the sensitive determination of adenines carrying different substituents at N-3.

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