Cloning and expression of a novel human antibody-antigen pair associated with Felty's syndrome
Cloning and expression of a novel human antibody-antigen pair associated with Felty's syndrome
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DOI：
10.1073/pnas.97.16.9234
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发表时间：
2000-08-01
影响因子：
11.1
通讯作者：
Burton, DR
中科院分区：
文献类型：
--
作者：
Ditzel, HJ;Masaki, Y;Burton, DR
越来越多的研究表明抗体在大多数系统性和器官特异性自身免疫性疾病的发病机制中的重要性，尽管对所涉及的自身抗体的确切作用存在相当大的争议。在人类中，进展的主要障碍是相关自身抗体和自身抗原的鉴定和克隆。在这里，开发了一种基于抗体噬菌体展示和抗原表达文库的顺序使用的方法，并将其应用于患有类风湿性关节炎（RA）、脾肿大和导致中性粒细胞减少症的中性粒细胞的外周破坏（Festival综合征）的供体。抗体噬菌体展示库构建从骨髓从供体和高亲和力的人单克隆抗体，ANA 15，选择通过淘选对新鲜的中性粒细胞和独立地通过淘选对固定的细胞系。该抗体显示中性粒细胞和许多细胞系的强染色。用单克隆抗体ANA 15从诱导的骨髓单核细胞系中探测λ gt 11表达文库，鉴定真核延伸因子1A-1（eEF 1A-1）为新的自身抗原。ANA 15的特异性通过与纯化的和重组的eEF 1A-1的反应性来证实，对大量血清的筛查显示，66%的Festival综合征患者的抗eEF 1A-1抗体水平升高。该抗体-抗原对的克隆应允许合理评价由相互作用产生的任何致病性及其在中性粒细胞减少症中的意义。
An increasing number of studies suggest the importance of antibodies in the pathogenesis of most systemic and organ-specific autoimmune diseases, although there is considerable controversy over the precise role of the autoantibodies involved. In humans, a major obstacle to progress is the identification and cloning of the relevant autoantibodies and autoantigens. Here, an approach based on the sequential use of antibody phage display and antigen expression libraries is developed and applied to a donor suffering from rheumatoid arthritis (RA), splenomegaly, and peripheral destruction of neutrophils leading to neutropenia (Felty's syndrome). An antibody phage display library was constructed from bone marrow from the donor and a high-affinity human mAb, ANA15, selected by panning against fresh neutrophils and independently by panning against a fixed cell line. The antibody showed strong staining of neutrophils and a number of cell lines. Probing of a lambda gt11 expression library from an induced myelomonocytic cell line with the mAb ANA15 identified the eukaryotic elongation factor 1A-1 (eEF1A-1) as a novel autoantigen, The specificity of ANA15 was confirmed by reactivity with both purified and recombinant eEF1A-1, Screening of a large panel of sera revealed that 66% of patients with Felty's syndrome had elevated levels of anti-eEF1A-1 antibodies. The cloning of this antibody-antigen pair should permit rational evaluation of any pathogenicity resulting from the interaction and its significance in neutropenia.