Ananieva-Jordanova R, Evans M, Nakamatsu T, Premawardhana L D K E, Sanders J, Powell M, Chen S, McGrath V, Belton C, Arnold C, Baker S, Betterle C, Zanchetta R, Smith B Rees, Furmaniak J
FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK.
J Autoimmun. 2005 Jun;24(4):337-45. doi: 10.1016/j.jaut.2005.03.003.
A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.
利用EB病毒感染1型糖尿病合并格雷夫斯病患者的外周淋巴细胞，随后与小鼠/人杂交细胞系融合，制备出一种分泌针对胰岛细胞自身抗原IA-2的人单克隆自身抗体的杂交瘤。该单克隆抗体（M13）为IgG1/κ，在免疫荧光试验中，1μg/mL的M13显示出与40个JDF单位相当的胰岛细胞抗体反应性。在免疫沉淀试验中，M13 IgG结合了（35）S标记的IA-2（100μg/mL时为26%）和（125）I标记的IA-2（100μg/mL时为34%），并且在蛋白质印迹分析中与IA-2反应良好。在使用修饰的（35）S标记的IA-2蛋白进行的分析中，发现IA-2的PTP结构域中的氨基酸777-808对M13结合很重要。M13 V区基因来自VH1-3、D3-22、JH4b、VκI DPK8/Vδ+和JK3基因，重链（11.0）和轻链（6.0）基因均显示出高置换/沉默突变率。还获得了与IA-2第604-686位氨基酸（对应近膜结构域）内至少三个不同表位反应的小鼠单克隆抗体（mMAbs）。来自mMAbs的F（ab'）2或Fab抑制了22份糖尿病血清中20份血清中IA-2自身抗体与IA-2的结合，而M13 F（ab'）2仅在22份血清中的6份中引起抑制。M13代表了一些患者血清IA-2自身抗体，因此为研究针对IA-2的自身免疫反应提供了一个有用的工具。
