【摘要】 1975年发现亮氨酸脑啡肽（LEK）和甲硫氨酸脑啡肽（MEK）。其测定以放射免疫（RIA）最佳。作者提议并合成了多聚赖氨酸琥珀酰脑啡肽免疫原,EK结合率81～90%,每2.3或3.2个赖氨酸残基连上一个EK。免疫3或6个月时获得抗EK血清（ALS或AMS）,工作稀度可达1:5000,K_eff10⁹升/克分子。试用改进的氯胺T标记法及DEAE-Sephadex A-25柱层析制备高比放射性¹²⁵I-EK免疫活性良好。双抗体法分离抗体结合的和游离的¹²⁵I-EK。竞争抑制曲线:灵敏度LEK～11微微克,MEK～130微微克;精密度1.8%及1.4%。β-内啡肽,八种肽类激素、吗啡、纳洛酮无明显干扰;交叉反应MEK对ALS为3.3%、LEK对AMS为<1%。脑组织用0.0N盐酸抽提,中和后,直接作RIA。本方法已通过多种方法学考验。正常大鼠脑中EK浓度以下丘脑为最高,尾核次之。本法是灵敏、可靠和特异的。更多还原

【Abstract】 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 10⁹ liters per mole. ¹²⁵I 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" ¹²⁵I-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 （p<0.001）; 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 （p<0.001）; the intraassay coefficient of variation of brain extracts measured was 3.4～6.2%, whereas the interassay coefficient of variation was 6.0～19.3%. In the normal rat brain, the highest amount of enkephalins, in terms of unit wet weight, was in the hypothalamus, with the caudate the next. These two assays have been shown to be sensitive, reliable and specific in our routine assay.更多还原