Girard D, Raymond Y, Labbé P, Senécal J L
Division of Rheumatology, Hôpital Notre-Dame, Montréal, Québec, Canada.
Clin Immunol Immunopathol. 1995 Nov;77(2):149-61. doi: 10.1006/clin.1995.1138.
Recent reports have used bovine fibronectin (Fn) as source of antigen to study human anti-Fn autoantibodies. We have characterized a novel human antibody (Ab) reactive with bovine and marsupial Fn, but not with human Fn. Indirect immunofluorescence, wet cleaving and protein adherence assays, immunoblotting, blot-affinity purification, a cell adhesion inhibition assay, and competitive experiments with synthetic peptides were used to characterize the anti-Fn Ab in serum from a patient with an undifferentiated connective tissue disease. A characteristic Fn-like network was observed by indirect immunofluorescence on bovine MDBK and marsupial PtK2 cells, but not on various human cell lines. Double immunofluorescence revealed colocalization of the Ab with a mouse monoclonal anti-Fn Ab. A reactive polypeptide of 240 kDa corresponding to the M(r) of Fn was identified by immunoblotting using MDBK and PtK2 total cell lysates. The Ab reacted with the 240-kDa band of purified bovine Fn with an endpoint titer of 1:64,000, while no reactivity was observed with human cellular or plasma Fn. Blot-affinity purification of the Ab from the 240-kDa PtK2 region confirmed that the Fn-like fluorescent pattern observed was due to reactivity with the 240-kDa band and not with other regions of the blot. The Ab affinity-purified from the 240-kDa region also reacted with purified bovine Fn by immunoblotting. Functional analysis disclosed specific inhibition of PtK2 and MDBK cell adhesion by the affinity-purified anti-Fn Ab. Competitive experiments with synthetic peptides demonstrated that the epitope is located in the decapeptide RGDSPASSKP containing the cell-binding domain of Fn. Longitudinal analysis of the Ab revealed its persistence over 6 years. Bovine and marsupial Fn can be the focus of a highly specific and persistent human immune response. Reactivity of a human Ab with bovine Fn does not imply cross-reactivity with human Fn. In light of recent reports using bovine Fn to characterize human anti-Fn "autoantibodies," future studies on human anti-Fn should specifically employ purified human Fn as antigen.
最近的报告使用牛纤连蛋白（Fn）作为抗原来源来研究人类抗Fn自身抗体。我们鉴定了一种新型人类抗体（Ab），它能与牛和有袋动物的Fn发生反应，但不与人Fn反应。采用间接免疫荧光、湿裂解和蛋白质黏附试验、免疫印迹、印迹亲和纯化、细胞黏附抑制试验以及与合成肽的竞争实验，对一名未分化结缔组织病患者血清中的抗Fn抗体进行了鉴定。通过间接免疫荧光在牛MDBK细胞和有袋动物PtK2细胞上观察到一种特征性的Fn样网络，但在各种人类细胞系上未观察到。双重免疫荧光显示该抗体与小鼠单克隆抗Fn抗体共定位。使用MDBK和PtK2细胞总裂解物进行免疫印迹，鉴定出一条对应于Fn相对分子质量（M(r)）的240 kDa反应性多肽。该抗体与纯化的牛Fn的240 kDa条带发生反应，终点效价为1:64,000，而与人细胞或血浆Fn未观察到反应性。从240 kDa的PtK2区域对该抗体进行印迹亲和纯化，证实观察到的Fn样荧光模式是由于与240 kDa条带发生反应，而非与印迹的其他区域反应。从240 kDa区域亲和纯化的抗体通过免疫印迹也与纯化的牛Fn发生反应。功能分析揭示亲和纯化的抗Fn抗体对PtK2和MDBK细胞黏附具有特异性抑制作用。与合成肽的竞争实验表明，表位位于包含Fn细胞结合域的十肽RGDSPASSKP中。对该抗体的纵向分析显示其持续存在超过6年。牛和有袋动物的Fn可能是高度特异性和持续性人类免疫反应的焦点。人类抗体与牛Fn的反应性并不意味着与人Fn存在交叉反应。鉴于最近使用牛Fn来鉴定人类抗Fn“自身抗体”的报告，未来关于人类抗Fn的研究应特别使用纯化的人Fn作为抗原。
