放射免疫双抗体法检测缓激肽含量的研究

The Research of Radioimmunoassay Using Double Abs for Bradykinin

缓激肽（ＢＫ）是哺乳动物和人体内重要的舒血管物质，具有很高的生理活性并与许多疾病有关。关于ＢＫ的含量分析，虽早已建立了放射免疫测定法（ＲＩＡ），但尚存在干扰因素多、误差大和费时等缺点。本研究采用碳二亚胺连接ＢＫ与血蓝蛋白制成免疫原，免疫家兔获得了高质量的ＢＫ抗体，以氧化还原法和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５柱层析技术制成￣（１２５）Ⅰ－Ｔｙｒ￣８ＢＫ标记物。应用驴抗兔血清和ＰＥＧ_（６０００）进行分离。标准曲线检测范围为２５～１６００ｐｇ，ＮＳＢ为３．１％，亲和常数ｋ＝０．８×１０￣（１０）Ｌ／ｍｏｌ，与多种肽类激素无交叉反应，血样加ｐｏｌｙｂｒｅｎｅ（抑制剂），用ＰＥＧ沉淀血浆蛋白除去ＢＫ干扰物。实验检测人和大鼠血浆ＢＫ含量：１０名男性为１５８４±３４７ｐｇ／ｍｌ，１０名女性为１６４２±３０２ｐｇ／ｍｌ，雌性大鼠为１８０５±２２５ｐｇ／ｍｌ。本法回收率为９５％，批内误差ＣＶ＝５％，批间ＣＶ＝９．２％，研究表明该法灵敏、精确、准确、特异、快速和简便。

Bradykinin(BK)is a potent vasodilative substance,and plays great physiological andpathological roles in animals and human beings.To measure the quantity of BK,the radioimmunoassay(RIA) has been devised,but the traditional RIA method has certain defects,such as presence of numerows interfering factors and errors and time consuming.Now,we produce anti-BK serum in rabbits by using BK-ovalbumin conjugate as an immunogen,and the ￣(125)Ⅰlabeled Tyr￣8-BK by using a modified chloramine-T method. High specificactivity has been obtained after purification with DEAE-Sephadex A-25 column chromatography. We use the donkey anti-rabbit Ab and PEG 6000 to separate the bound from the free￣(125)Ⅰ-Tyr￣8-BK. The limitation range of standard curve is from 25 to 1600 pg,NSB is 3.1%,affinity constant(K) is 0.8× 10￣(10)L/mol,and there is no significant interference with otherbiological BK analogues. The blood samples are treated by adding Polybrene(inhibitor)andPEG 6000 to deposit the big serum proteins in order to reduce the disturbing substances.This method has been shownto be a sensitive,specific,reliable,simple and convenientmeasure of the serum BK level.By this method,the serum BK quantities in men,womenand rats are respectively 1584±347 pg/ml,1642±302 pg/ml and 1805±225 pg/ml,the recycling rate is 95%,the intergroup CV is 5.0%and outergroup CV=9.2%.