Katona E E, Ajzner E, Tóth K, Kárpáti L, Muszbek L
Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Centre, University of Debrecen, P.O. Box 40, H-4012, Debrecen, Hungary.
J Immunol Methods. 2001 Dec 1;258(1-2):127-35. doi: 10.1016/s0022-1759(01)00479-3.
A new one-step ELISA was developed for the determination of the concentration of blood coagulation factor XIII subunit A (FXIII-A) in plasma and in cell lysates. Monoclonal antibodies directed against different epitopes on FXIII-A were used for the assay. The capture antibody was biotinylated on its carbohydrate moiety and the detection antibody was labelled with horseradish peroxidase. The antigen-antibody reaction was carried out in the well of a streptavidin-coated microplate. Complex formation with FXIII subunit B (FXIII-B) and association to fibrinogen did not influence the accessibility of the antibodies to FXIII-A. The method could be performed within 2 h and demonstrated good reproducibility, recovery and sensitivity. Plasma samples could be assayed after storage at -20 degrees C for at least 6 months. However, in the case of platelet lysates freezing and rethawing resulted in a significant loss of FXIII-A. FXIII-A concentrations measured in the plasma samples of healthy individuals and patients correlated well with the concentrations of complexed plasma FXIII (A2B2) and with the results of FXIII activity measurements. A reference range of 46-82 fg/platelet was established for platelet FXIII-A.
开发了一种新的一步酶联免疫吸附测定法（ELISA），用于测定血浆和细胞裂解物中凝血因子 XIII 亚基 A（FXIII-A）的浓度。针对 FXIII-A 不同表位的单克隆抗体用于该测定。捕获抗体在其碳水化合物部分进行了生物素化，检测抗体用辣根过氧化物酶标记。抗原 - 抗体反应在链霉亲和素包被的微孔板孔中进行。与 FXIII 亚基 B（FXIII-B）形成复合物以及与纤维蛋白原结合并不影响抗体与 FXIII-A 的结合。该方法可在 2 小时内完成，具有良好的重现性、回收率和灵敏度。血浆样本在 -20℃储存至少 6 个月后仍可进行测定。然而，对于血小板裂解物，冷冻和解冻会导致 FXIII-A 显著损失。在健康个体和患者的血浆样本中测得的 FXIII-A 浓度与复合血浆 FXIII（A2B2）的浓度以及 FXIII 活性测量结果相关性良好。为血小板 FXIII-A 建立了 46 - 82 fg/血小板的参考范围。
