Identification of continuous epitopes of HuD antibodies related to paraneoplastic diseases/small cell lung cancer

Abstract 

HuD antibodies are associated with small cell lung cancer. To identify relevant epitopes of HuD antibodies, patient sera and a monoclonal antibody were analyzed for their reactivity to linear 20mer peptides spanning the human HuD protein. The HuD monoclonal antibody recognized a single fragment located in the first RNA recognition motif. Thorough analysis identified VRDKITQGSL as the actual epitope. Screening of anti-HuD positive patients and healthy controls identified eight peptides as potential subdominant epitopes. The majority of these peptides were located in the N-terminal end as well as in the linker region between the second and third RNA recognition motifs.

Abbreviations: AP, alkaline phosphatase, DCM, dichloromethane, DIC, N,N′-diisopropylcarbodiimide, DMAP, 4-dimethylaminopyridine, ELISA, enzyme-linked immunosorbent assay, eq, equivalent, Fmoc, 9-fluorenylmethoxycarbonyl, HMBA, 4-(hydroxymethyl)benzoic acid, HOAt, 1-hydroxy-7-aza-benzotriazole, h, hour, HPLC, high-performance liquid chromatography, mAb, monoclonal antibody, NaOH, sodium hydroxide, NMP, N-methyl-2-pyrrolidone, PEM, paraneoplastic encephalomyelitis, PIP, piperidine, pNPP, para-nitrophenylphosphate, PNS, paraneoplastic neurological syndromes, PSN, paraneoplastic sensory neuronopathy, RRM, RNA recognition motif, RT, room temperature, SCLC, small cell lung cancer, SPPS, solid-phase peptide synthesis, TFA, trifluoroacetic acid, THIO, thioanisole, TIS, triisopropylsilane, TTN, Tris–Tween–NaCl

Keywords: Epitope mapping, Monoclonal antibody, Solid-phase peptide synthesis, HuD, Small cell lung cancer