抗iLRP单克隆抗体的制备和鉴定

为了研究抗iLRP (Immature laminin receptor protein)单克隆抗体在免疫治疗中的应用,本研究采用重组iLRP对6~8周龄BALB/c雌性小鼠进行免疫,取免疫后小鼠脾细胞与骨髓瘤细胞系SP2/0进行细胞融合,在HAT (Hypoxanthine aminopterin thymidine)半固体培养基上挑选分离明显的细胞克隆于96孔板培养后,取上清液通过ELISA法进行克隆筛选,初次筛选得到23株阳性克隆,复筛后得到13株稳定分泌抗体的细胞株。抗体类别鉴定显示,其中9株为IgM,3株为Ig G1,1株为IgG2a,轻链类型都为κ型。其中1株IgG1和IgG2a用于制备小鼠腹水,经Protein-G柱纯化,SDS-PAGE检测抗体纯度,Western blotting验证抗体特异性。结果表明,抗体纯度均大于90%,并与i LRP有明显的特异性结合。此结果为后续的深入研究提供了一套完整有效的抗iLRP单克隆抗体生产和鉴定的程序,对以iLRP为治疗靶点的单克隆药物研究以及最近兴起的CAR-T (Chimeric antigen receptor modified T cell)免疫治疗具有积极的意义。

Preparation and Characterization of Anti-i LRP Monoclonal Antibodies

To study the possible application of monoclonal antibodies against immature laminin receptor protein(iLRP) in tumor immunotherapy, 6~8 weeks old mice were immunized with recombinant iLRP. Splenocytes from the immunized mice were used to perform cell-fusion with mouse myeloma cell line SP2/0. The fused cells were cultured in semisolid media with Hypoxanthine/Aminopterin/Thymidine(HAT). The well isolated cell clones were picked up into 96-well plates for screening positive clones by a method of ELISA. We obtained 23 iLRP-positive hybridomas at the first round of screening, and thirteen of them were confirmed to be able to secrete anti-iLRP antibodies stably following the second round of screening. Then, an analysis of subclasses was carried out for them. Results showed that nine of them belonged to IgM class, three of them were IgG1, and one of them was IgG2a, and the types of light chain for all of them were κ. Afterwards, large amount of antibodies were produced in mice by planting the hybridoma into peritoneal cavity for 1 hybridoma of IgG1 and the hybridoma of IgG2a and then purified by Protein-G column. The purity was up to 90% determined by SDS-PAGE and the binding to iLRP was obviously specific confirmed by Western Blotting. This study provided us with an effective procedure of de novo making iLRP-specific monoclonal antibodies, which will help us to explore their therapeutic applications,such as, in developing of antibody drugs and CAR-T(Chimeric antigen receptor modified T cell) immunotherapyby using iLRP as a target protein.