RADIOIMMUNOASSAY FOR ENKEPHALINS
摘要
Since leu-enkephalin (LEK) and met-enkephalin (MEK) were discovered in 1975, great progress has been made in the research on brain opiate pentapeptides. The preferable estimation method for these peptides is radioimmunoassay (RIA). We suggested and prepared immunogens by condensation of LEK or MEK with poly-Llysine semisuccinic acid, the rate of conjugation was 81% or 90%, one LEK or MEK molecule per 3.2 or 2.6 lysine residues. Rabbits were immunized by multiple intradermal inoculation. Specific antiserum (ALS or AMS) was obtained 6 or 3 months afterwards respectively, the working dilution of which was 1:5000, K_(eff) about 10~9 liters per mole. ~(125)I labelled LEK or MEK with specific activity of about 1,600μ Ci/μg were prepared by a modified chloramine-T method and purified by DEAE-Sephadex A-25 column chromatography, and were shown to possess a high immunoreactivity. The double-antibody technique was used to separate the bound from free ~(125)I-enkephalins. In the competitive inhibition curves, the detection limit of LEK was～ 11pg and that of MEK～130pg, whereas the precision was 1.8 or 1.4% respectively. No significant interference by β-endorphin, 8 peptide hormones, morphine and naloxone was found. The cross reaction rate was low, i. e., MEK vs. ALS was 3.3% and LEK vs. AMS was less than 1%.Brain enkephalins were extracted with 0.1N HCl. After neutralization, the supernatant portions were used directly for RIA. Several tests for validating the methodology has been processed: non-specific combination was found to bo less than 0.4～1.8%; a linear relation was found between the amounts of authentic MEK added to brain extract and the values estimated (p0.001); the recovery of authentic LEK added to brain tissue was 84.2±8.2% (C.V.=9.7%, n=6): increasing the volumes of added brain extract increases the estimated quantity of enkephalins proportionally (p0.001); the intraassay coefficient of variation of brain extracts measured was 3.4～6.2%, whereas the interassay coef