Jahn S, Roggenbuck D, Niemann B, Ward E S
Institute for Medical Immunology, Medical Faculty (Charité), Humboldt University, Berlin, Germany.
Immunobiology. 1995 Aug;193(5):400-19. doi: 10.1016/S0171-2985(11)80427-4.
A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.
一种产生多特异性人单克隆IgM抗体（命名为CB03）的杂交瘤，是由小鼠骨髓瘤细胞与从一名患有慢性特发性血小板减少症的自身免疫患者获取的人脾淋巴细胞融合而成。发现该抗体由体细胞突变的VHI和VlambdaIII基因编码。为了研究突变互补决定区（CDR）对抗体特异性的影响，将纯化的完整IgM抗体的抗原结合特性与以下两者进行了比较：（i）通过热胰蛋白酶消化得到的Fab片段，以及（ii）在大肠杆菌中表达的重组单价片段。具体而言，构建了编码以下内容的载体：（i）rFab03和单链Fv03片段，其包含通过接头序列连接的VH和VL基因；（ii）scFc1.1片段，其包含VH种系等效物和CB03野生型CDR3区域；（iii）scFv片段，其包含种系构型的CDR1和CDR2以及在产生多特异性IgM抗体的CB253人胎儿B细胞杂交瘤中表达的CDR3。表达载体在3'端含有一个（His）6基序，以便在Ni(2+)-琼脂糖上进行纯化，或者含有一个c-myc标签，用于通过鼠单克隆抗体特异性检测表达产物。对表达产物的蛋白质印迹和ELISA分析表明：（i）在细菌周质中发现重组Fab片段的量极低（从1升细菌培养物中获得1 - 10微克）；（ii）从细菌周质中获得了适量的scFv片段（800 - 1000微克/升）；（iii）单价重组片段以及通过胰蛋白酶消化获得的Fab反映了完整IgM抗体的多特异性抗原结合特性，但与抗原的结合亲和力低得多；（iv）发现CDR3对于这种特定IgM的抗原结合模式至关重要。我们讨论了在大肠杆菌中表达重组scFv片段作为研究体细胞突变在自身抗体产生中的作用的一种合适方法。
