一种用于检测血浆中可溶性纤维蛋白的新型抗纤维蛋白单体和可溶性纤维蛋白单克隆抗体。

A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma.

作者信息

Hamano Akiei, Tanaka Seiji, Takeda Yukiko, Umeda Mamoru, Sakata Yoichi

机构信息

Central Research Laboratory of Nissui Pharmaceutical Co, Ltd., 1075-2 Hokunanmoro, Yuuki, Ibaraki 307-0036, Japan.

出版信息

Clin Chim Acta. 2002 Apr;318(1-2):25-32. doi: 10.1016/s0009-8981(01)00779-3.

DOI:10.1016/s0009-8981(01)00779-3

PMID:11880109

Abstract

BACKGROUND

Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF.

METHODS

We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody.

RESULTS

The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the alpha-chain N-terminal region exposed upon removal of the A peptide from the Aalpha-chain because F405 was found to bind to the alpha-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1-300 microg/ml. The assay detection limit was < 0.5 microg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (< 9.4% and < 10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean +/- S.D. of the SF concentration in the DIC and control samples were 63.4 +/- 65.3 microg/ml and 1.9 +/- 1.0 microg/ml, respectively.

CONCLUSIONS

The ELISA using F405 is useful for the diagnosis of DIC.

摘要

背景

可溶性纤维蛋白（SF）由纤维蛋白单体（FM）和纤维蛋白原组成，在血栓性疾病患者的循环血液中存在，对其进行定量有助于了解血管内凝血的状态和程度。然而，此前尚无便捷的SF检测方法。

方法

我们以去A肽纤维蛋白为免疫原，在抗聚合肽（甘氨酸 - 脯氨酸 - 精氨酸 - 脯氨酸，GPRP）存在的情况下制备了一种新型抗FM和SF的单克隆抗体（MoAb）（F405），并通过蛋白质印迹分析和酶联免疫吸附测定（ELISA）对F405进行表征。我们还尝试使用一种涉及固定化F405和辣根过氧化物酶（POD）标记的抗纤维蛋白原多克隆抗体的ELISA法检测人血浆中的SF。

结果

该抗体与纤维蛋白降解产物片段X、Y和E反应，但不与纤维蛋白原或其片段X、Y、D和E或纤维蛋白D - 二聚体反应。F405识别的表位似乎是从Aα链去除A肽后暴露的α链N端区域，因为发现F405与纤维蛋白的α链N端寡肽（GPRVVERHQ）结合。由于F405不仅在GPRP肽存在下与FM反应，还与通过将凝血酶处理的FM添加到人纤维蛋白原中制备的SF复合物反应，我们尝试使用ELISA法检测人血浆中的SF。该方法的分析范围为1 - 300μg/ml。检测限<0.5μg/ml，批内和批间精密度研究结果表明该方法准确且结果可重复（分别<9.4%和<10%）。当检测56份弥散性血管内凝血（DIC）患者的血浆样本和117份健康个体的对照样本时，在DIC样本中检测到SF复合物水平升高：DIC样本和对照样本中SF浓度的平均值±标准差分别为63.4±65.3μg/ml和1.9±1.0μg/ml。