Development of rapid screening and production technique of antibody Fv for protein chip

我们构建了HE和LE嵌合受体，分别由抗鸡蛋溶菌酶（HEL）抗体的V_H和V_L与细胞外D2和红细胞生成素受体的跨膜/胞内结构域融合。用gp130取代胞内结构域构建H_g和L_g嵌合受体。构建了一系列以抗荧光素抗体单链F_v为配体识别域的嵌合受体。基因转导后，在培养基中加入同源抗原，成功扩增出基因修饰细胞。接下来，我们随机选取V_H中hell识别位点的4个氨基酸，与gp130连接，并将合成载体转导到表达le的细胞中。经过HEL选择，突变体V_H表现出与野生型V_H相当的结合亲和力。用PCR方法从超氧化物歧化酶（SOD）免疫小鼠脾细胞中扩增出V_H文库，并与gp130融合。当细胞用该V_H文库转导时，获得了一些生长的克隆，表明使用嵌合受体进行文库选择的可能性。此外，我们构建了一个载体，其中嵌合受体基因两侧有两个loxP序列。利用该载体，我们成功构建了一个嵌合受体表达细胞依次转化为抗体产生细胞的系统，该系统通过在细胞中表达抗原选择后的Cre重组酶获得抗体产生细胞。此外，我们开发了一种技术，通过电喷雾沉积法在玻璃板（9mm x 9mm）上以150 μm的直径制作324个均匀的抗体斑点。夹心ELISA采用该抗体芯片可重复性地检测抗原浓度在0.1 ~ 1 ng/ml的检出限。

We constructed HE and LE chimeric receptors comprising V_H and V_L of anti-hen egg lysozyme (HEL) antibody fused with extracellular D2 and transmembrane/intracellular domains of erythropoietin receptor, respectively. H_g and L_g chimeric receptors were also constructed by replacing intracellular domain with that of gp130. Furthermore, a series of chimeric receptors whose ligand recognition domain is anti-fluorescein antibody single chain F_v were constructed. After the gene transduction, genetically modified cells were successfully amplified by addition of cognate antigen in the culture medium.Next, we randomized 4 amino acids of HEL-recognition site in V_H, linked to gp130, and transduced the resultant vector into LE-expressing cells. After HEL selection, the mutant V_H showed comparable binding affinity to the wild-type V_H. Furthermore, V_H library was amplified by PCR from splenocyte of mice immunized with superoxide dismutase (SOD), and fused with gp130 intracellular domain. When cells were transduced with this V_H library, some growing clones were obtained, indicating the possibility of library selection by using the chimeric receptor.In addition, we constructed a vector in which chimeric receptor gene is flanked with two loxP sequences. By using this vector, we successfully developed a system in which a chimeric receptor-expressing cell can be sequentially converted into an antibody producer cell, which was attained by expressing Cre recombinase after antigen selection in the cell. Furthermore, we developed a technique to make 324 homogeneous spots of antibody at 150 μm diameter on a glass plate (9mm x 9mm) by an electrospray deposition method. Sandwich ELISA using this antibody microarray reproducibly detected antigen concentration at 0.1 to 1 ng/ml of detection limit.