A microtiter assay for factor XIII using fibrinogen and biotinylcadaverine as substrates.
A microtiter assay for factor XIII using fibrinogen and biotinylcadaverine as substrates.
复制标题
使用纤维蛋白原和生物素尸胺作为底物对 XIII 因子进行微量滴定测定。
DOI：
10.1006/abio.1994.1551
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发表时间：
1994
影响因子：
2.9
通讯作者：
Matsueda,GR
中科院分区：
文献类型：
--
作者：
Song,YC;Sheng,D;Taubenfeld,SM;Matsueda,GR
努力开发一种改进的测定血浆和组织转氨酶导致我们一个方便，灵敏的微量滴定板测定凝血因子XIII使用人纤维蛋白原作为固定化底物。在钙、凝血酶和固定化纤维蛋白原的存在下激活因子XIII，然后通过加入生物素基尸胺进行测定。通过加入EDTA终止反应，并用链霉亲和素-β-半乳糖苷酶测量掺入的生物素水平。在本试验中，人血小板因子XIII的分析范围为0.01-100 ng，豚鼠肝转氨酶的分析范围为1-100 ng。与N，N-二甲基酪蛋白包被的微量滴定板相比，纤维蛋白原包被的板的灵敏度增加了100倍以上。批内变异系数小于5%（n = 12），批间变异系数小于6%（n = 4）。该测定法的灵敏度降低了所需的血浆样品体积，从而消除了从此类测试样品中去除纤维蛋白原的需要。正如预期，因子XIII活性可被腐胺和抗因子XIII抗体以及与人纤维蛋白γ链羧基末端结合的单克隆抗体抑制。该测定法提供了一种灵敏、简便、快速的测定因子XIII的方法。
Efforts to develop an improved assay for plasma and tissue transglutaminase have led us to a convenient, sensitive microtiter plate assay for coagulation factor XIII using human fibrinogen as an immobilized substrate. Factor XIII was activated in the presence of calcium, thrombin, and immobilized fibrinogen and then assayed by adding biotinylcadaverine. The reaction was terminated by adding EDTA and the level of incorporated biotin was measured with streptavidin-β-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0.01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinogen-coated plates gave more than 100-fold increase in sensitivity compared with N,N-dimethylcasein-coated microtiter plates. The intraassay coefficient of variation was less than 5% (n = 12) and interassay less than 6% (n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin γ-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII.