Production and immunohistochemical application of antiserum against Tyr-D-Ala-Phe, a N-terminal tripeptide common to dermorphin/deltorphin family☆ ,1

Abstract

Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 μg of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin.