Radioimmunoassay (RIA)

The procedure was described elsewhere (Christensson-Nylander et al., 1985; Merg et al., 2006). Briefly, 1 M hot acetic acid was added to finely powdered frozen tissues, and samples were boiled for 5 min, ultrasonicated and centrifuged. Tissue extract was run through SP-Sephadex ion exchange C-25 column, and peptides were eluted and analyzed by RIA. Cross-reactivity of Leu-enkephalin-Arg (LER) antiserum with dynorphin B and Leu- and Met-enkephalin was <0.1% molar, with α-neoendorphin 0.5% molar, with Dynorphin A (1–8) 0.7% molar, with Met-enkephalin-Arg-Phe (MEAP) 1% molar and with Met-enkephalin-Arg 10% molar. Cross-reactivity of MEAP antiserum with Met-enkephalin, Met-enkephalin-Arg, Met-enkephalin-Arg-Gly-Leu, Leu-enkephalin and LER was <0.1% molar (Nylander et al., 1997). This RIA variant readily detected LER in wild-type mice (Nguyen et al., 2005) but not in Pdyn knock-out mice; thus the assay was highly specific and not sensitive to the presence of contaminants. The peptide content is presented in fmol/mg tissue. No normalization per amount of tissue was performed for calculation of the LER/MEAP ratio that therefore was free of any bias because of differences in amount of tissue between the analyzed animal groups, and between the left and right spinal halves.