Vittecoq O, Brard F, Jovelin F, Le Loet X, Tron F, Gilbert D
INSERM U519, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides, Hôpital Charles Nicolle et Faculté Mixte de Médecine et de Pharmacie, Rouen, France.
Clin Exp Immunol. 1999 Oct;118(1):122-30. doi: 10.1046/j.1365-2249.1999.00995.x.
Two IgM, kappa anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB x NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb.
两种IgM κ链抗髓过氧化物酶(MPO)单克隆抗体6D6和9B5,在固相酶联免疫吸附测定中与MPO结合,它们分别来源于(NZB×NZW)F1小鼠和MRL/lpr - lpr小鼠的脾细胞。6D6在乙醇固定的人中性粒细胞上呈现特征性的核周免疫荧光染色模式,通过免疫印迹法与天然形式的MPO结合,并且如实时特异性相互作用所示,对MPO具有高恒定亲和力。9B5产生胞质免疫荧光染色模式,与MPO的重链反应,对MPO具有低恒定亲和力。对单克隆抗体(mAb)6D6和9B5的重链和轻链可变区基因进行测序,发现它们与种系基因高度同源,并且在互补决定区含有带负电荷的氨基酸。在大多数BW和MRL/lpr - lpr小鼠血清中观察到IgM MPO结合活性,这可能对应于B细胞的多克隆激活,而IgG抗MPO抗体很少能被检测到。因此,本研究表明:(i)BW和MRL/lpr - lpr小鼠不会清除分泌IgM抗MPO的B细胞,不会使这些B细胞处于无反应状态,但大多数个体不能自发诱导这种自身抗体群体的类别转换;(ii)如两种狼疮小鼠来源的mAb的免疫化学特性所示,IgM抗MPO抗体可以识别MPO上的不同表位,并在乙醇固定的人中性粒细胞上产生不同的免疫荧光染色模式。