构建兔抗人凝血因子XIIIA亚单位多克隆抗体及特异性鉴定

Construction of rabbit polyclonal antibody against human FXIII A subunit and its specificity identification

背景:关于凝血因子XIII(FXIII)抑制物作用于FXIII的具体结合表位,目前尚无报道。目的:用免疫动物方法制备针对FXIIIA亚单位多克隆抗体,并对其特异性和活性进行鉴定。方法:用人工合成的FXIIIA亚单位多肽免疫新西兰兔,制备多克隆抗体;应用Dotblot鉴定抗体特异性,并用抗体中和/尿素溶解实验检测多克隆抗体活性;Westernblot方法检测2例获得性FXIII缺乏症患者的血浆FXIII水平,再应用Dotblot分析这2例患者血浆FXIII抑制物作用的抗原表位。结果与结论:Dotblot证实兔血清中已产生抗FXIII抗体,此抗体不仅可特异性与人FXIII及转氨酶活性位点多肽结合,且在体外可抑制人FXIII的活性;用此抗体可检测出获得性FXIII缺乏症患者的血浆FXIII缺乏,分析出转氨酶活性位点是病例2血浆FXIII抑制物的作用位点。证实动物免疫法可成功制备兔抗人FXIIIA亚单位转氨酶活性位点的多克隆抗体,此抗体可用于人FXIII的检测和抗原表位分析。

BACKGROUND：There have been no reports regarding the precise FXIII binding epitopes for blood coagulation factor XIII（FXIII） acting on FXIII inhibitor.OBJECTIVE：To prepare rabbit polyclonal antibody against human FXIII A subunit by immunologic method,and to identify antibody specificity and activity.METHODS：The polyclonal antibody was prepared by immunizing rabbits using artificially synthesized FXIIIA subunit.Antibody specificity was identified by Dot blot method,and antibody activity was determined by antibody-neutralizing/urea-lysis test.Serum level of FXIII in two patients with acquired FXIII deficiency was determined by western blot assay.The FXIII binding epitopes of these two patients were analyzed by Dot blot method.RESULTS AND CONCLUSION：Dot blot results showed that the polyclonal antibody against FXIII A subunit had already been produced in rabbit serum.The antibody not only binded to purified human FXIII and transglutaminase active site,but also inhibited the activity of FXIII in vitro.The FXIII antigen level in two patients with acquired FXIII deficiency could be detected using this antibody and active site of transglutaminase on FXIII was the binding epitope of FXIII inhibitor in case 2,but not in case 1.The successful preparation of polyclonal antibody,which is applied to detect FXIII deficiency and determine FXIII binding epitopes,provides basis for studying the mechanism of acquired FXIII deficiency and clinical diagnosis.