Preparing Monoclonal Antibody against human Retinoblastoma by solid tumor cell and cloning its Light Chain Variable Gene

Abstract

Abstract: : Purpose: To prepare anti–human retinoblastoma(RB) monoclonal antibody (McAb) and to clone its variable gene of light chain (VL). Methods: With hybridoma technology , anti–human RB McAb was prepared using RB solid tumor cells acquired from the patient with RB as immune antigen . Then, using RT–PCR method , the VL was amplified from the total RNA of identified hybridoma cells with mouse VΚ general primers, and sequenced by sanger's method. Homologous analysis was done by NCBI BLAST . Results: We established 3 hybridoma cell lines secreting anti–human RB McAb ,which belongs to subgroup IgG1 . Both immunofluorescence and immunohistochemistry demonstrated that corresponding antigen of the McAb was specifically expressed in RB tumor cell membrane and cytoplasm .Western Blot indicated that the McAb could bind to 25 kD protein band of RB antigen ,which is different from P^110Rb. The VL sequence consisted of 321 bp encoding 107 amino acid residues , which was most homological to a member of the Vk9 gene family , and its chain utilized the Jk1 gene segment. Conclusions: We have prepared the McAb against human RB . The antigen molecular weight bounded to this antibody were 25 kD or so ,which suggested that it was possible for a new unidentified gene to cause the tumor formation of RB . The VL gene of anti–human RB McAb was amplified successfully , which belonged to the Vk9 gene family and utilized Vk–Jk1 gene rearrangement .This study lays a good basis for constructing a recombinant humanized antibody and doing tumor immune targeting therapy .

Keywords: retinoblastoma