Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X.
Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X.
复制标题
酶联凝血测定。
DOI：
10.1016/0003-2697(87)90014-5
复制
发表时间：
1987
影响因子：
2.9
通讯作者：
Doellgast,GJ
中科院分区：
文献类型：
--
作者：
Doellgast,GJ
固相凝血试验利用包被在微滴板孔上的纤维蛋白原和溶液中的过氧化物酶-纤维蛋白原作为凝血酶的底物（酶联凝血试验，ELCA），已被改进为一种免疫试验。多克隆（兔）抗体直接抑制因子II、VII和X，单克隆抗体直接抑制因子X，这些抗体在高稀释和使用ELCA检测特定因子时已被证明。利用包被第二抗体（山羊抗小鼠IgG）和纤维蛋白原的板，将活性因子结合到板上，用ELCA检测结合因子，检测因子X和因子VII的单克隆抗体。该试验非常敏感，允许检测到低至0.2 ng/ml （30 pg/assay）的单克隆抗体，或小于0.4 ng/ml （60 pg/assay）的Xa因子。当板上涂有X因子单克隆抗体和纤维蛋白原时，通过在溶液中添加的Xa因子与板结合的单克隆抗体的不同竞争，该检测允许鉴定两种X因子单克隆抗体的不同表位特异性。该方法可广泛应用于凝血因子的免疫分析，并可作为免疫分析扩增系统应用。
The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.