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

我们构建了抗鸡蛋溶菌酶（HEL）抗体的VH和VL分别与促红细胞生成素受体的胞外D2和跨膜/胞内结构域融合的HE和LE嵌合受体。用gp 130的胞内结构域取代H_g和L_g嵌合受体。进一步构建了一系列以抗荧光素抗体单链Fv为配体识别结构域的嵌合受体。然后，我们将VH中的HEL-recognition位点的4个氨基酸随机化，连接到gp 130上，将构建的载体导入LE表达细胞中。经HEL筛选，突变体V_H与野生型V_H的结合亲和力相当。用超氧化物歧化酶（SOD）免疫小鼠，用PCR方法从脾细胞中扩增出VH文库，并与gp 130胞内区融合。用该文库转导细胞，获得了一些生长良好的克隆，表明用嵌合受体进行文库筛选是可行的。通过使用该载体，我们成功地开发了一种系统，其中嵌合受体表达细胞可以依次转化为抗体生产细胞，这是通过在细胞中抗原选择后表达Cre重组酶而实现的。此外，我们开发了一种技术，通过电喷雾沉积法在玻璃板（9mm × 9mm）上制备324个直径为150 μm的均匀抗体斑点。使用该抗体微阵列的夹心ELISA可重复地检测检测限为0.1至1 ng/ml的抗原浓度。

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.