凝血因子 VIII 蛋白的固相免疫放射分析

Solid-phase immunoradiometric assay of factor-VIII protein.

作者信息

Counts R B

出版信息

Br J Haematol. 1975 Dec;31(4):429-36. doi: 10.1111/j.1365-2141.1975.tb00878.x.

DOI:10.1111/j.1365-2141.1975.tb00878.x

Abstract

A solid phase immunoradiometric assay for factor-VIII protein has been developed using 125I-labelled rabbit antibody made against highly purified factor VIII. Antibody specific for high molecular weight factor-VIII protein was isolated using an immunoadsorbent consisting of highly purified factor VIII bound to diazotized m-aminobenzyl (oxymethyl)-cellulose. The purified anti-factor VIII antibody was labelled with 125I while bound to the immunoadsorbent and then eluted at pH 2.9. Dilutions of plasma samples for assay were incubated for 48 h in anti-factor VIII antibody-coated tubes. The tubes were washed to remove unbound proteins and the 125I-labelled, purified antibody was added. After 48 h incubation the tubes were washed to remove unbound antibody and counted. The concentration of immunoreactive factor-VIII protein in pooled normal human plasma was determined to be 8 microgram/ml. The minimum amount of factor VIII measured by the assay is less than 0.16 ng. Factor-VIII protein was present in normal concentration in haemophilia A plasma, and in reduced concentration in von Willebrand's disease plasma. The method has the advantages of improved sensitivity, specificity, efficiency and economy as compared with previous factor-VIII immunoassays.

摘要

已开发出一种用于检测因子 VIII 蛋白的固相免疫放射分析方法，该方法使用针对高度纯化的因子 VIII 制备的 125I 标记兔抗体。使用由与重氮化间氨基苄基（羟甲基）纤维素结合的高度纯化因子 VIII 组成的免疫吸附剂，分离出对高分子量因子 VIII 蛋白具有特异性的抗体。纯化的抗因子 VIII 抗体在与免疫吸附剂结合时用 125I 标记，然后在 pH 2.9 条件下洗脱。用于检测的血浆样品稀释液在抗因子 VIII 抗体包被的试管中孵育 48 小时。洗涤试管以去除未结合的蛋白质，然后加入 125I 标记的纯化抗体。孵育 48 小时后，洗涤试管以去除未结合的抗体并进行计数。测定正常人混合血浆中免疫反应性因子 VIII 蛋白的浓度为 8 微克/毫升。该检测方法能检测到的因子 VIII 的最小量小于 0.16 纳克。在甲型血友病血浆中，因子 VIII 蛋白浓度正常；在血管性血友病血浆中，因子 VIII 蛋白浓度降低。与以前的因子 VIII 免疫测定方法相比，该方法具有灵敏度提高、特异性增强、效率更高和更经济等优点。