Discovery and properties of a monoclonal antibody targeting 8-oxoA, an oxidized adenine lesion in DNA and RNA.

8-oxoA, a major oxidation product of adenosine, is a mispairing, mutagenic lesion that arises in DNA and RNA when •OH radicals or one-electron oxidants attack the C8 adenine atom or polymerases misincorporate 8-oxo(d)ATP. The danger of 8-oxoA is underscored by the existence of dedicated cellular repair machinery that explicitly excise it from DNA, the attenuation of translation induced by 8-oxoA-mRNA or damaged ribosomes, and its potency as a TLR7 agonist. Here we present the discovery, purification, and biochemical characterization of a new mouse IgGk1 monoclonal antibody (6E4) that specifically targets 8-oxoA. Utilizing an AchE-based competitive ELISA assay, we demonstrate the selectivity of 6E4 for 8-oxoA over a plethora of canonical and chemically modified nucleosides including 8-oxoG, A, m6A, 2-oxoA, and 5-hoU. We further show the ability of 6E4 to exclusively recognize 8-oxoA in nucleoside triphosphates (8-oxoATP) and DNA/RNA oligonucleotides containing a single 8-oxoA. 6E4 also binds 8-oxoA in duplex DNA/RNA antigens where the lesion is either paired correctly or base mismatched. Our findings define the 8-oxoAde nucleobase as the critical epitope and indicate mAb 6E4 is ideally suited for a broad range of immunological applications in nucleic acid detection and quality control.

Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B4.

Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.