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

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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.