在Balb/c小鼠中抗人CD20单克隆抗体的研制与特性分析

Development and characterization of monoclonal antibodies against human CD20 in Balb/c mice.

作者信息

Sepehr Koushan Sineh, Baradaran Behzad, Majidi Jafar, Abdolalizadeh Jalal, Aghebati Leili, Shahneh Fatemeh Zare

机构信息

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

出版信息

Hum Antibodies. 2012;21(3-4):57-64. doi: 10.3233/HAB-130263.

DOI:10.3233/HAB-130263

PMID:23549022

Abstract

BACKGROUND

Targeting CD20 antigen on B-lymphocytes provides good opportunity for management of the target cells in patients with B-cell malignancies. By the advent of hybridoma technology, monoclonal antibodies applications exert extensive changes in medical fields such as diagnosis, treatment and purification.

OBJECTIVE

The prim aim of this study was to produce monoclonal antibody against CD20 for exploitation in diagnosis.

METHODS

In this study, Balb/c mice were immunized with two peptides from extracellular domain of CD20. Poly Ethylene Glycol (PEG) fused spleen cells of the most immune mouse with SP2/0 (myeloma cells). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The desired clones were selected for limiting dilution (L.D). Afterward, specificity and cross reactivity of these antibodies were determined by immunological assay such as ELISA and western blot analysis (WB) and Immunofluorescence. Large scale of monoclonal antibodies was produced by ascetic fluid method. Monoclonal antibody was purified by chromatography then confirmed by SDS-PAGE.

RESULTS

In this study, between five positive clone wells, 3 clones were chosen for limiting dilution. Limiting dilution product was one monoclone with absorbance about 2.

CONCLUSIONS

These results indicate that such monoclonal antibodies against CD20 can be used in diagnosis of CD20 in the cells surface.

摘要

背景

靶向B淋巴细胞上的CD20抗原为治疗B细胞恶性肿瘤患者的靶细胞提供了良好契机。随着杂交瘤技术的出现，单克隆抗体在诊断、治疗及纯化等医学领域引发了广泛变革。

目的

本研究的主要目的是制备抗CD20单克隆抗体用于诊断。

方法

本研究中，用来自CD20胞外域的两种肽免疫Balb/c小鼠。将免疫反应最强的小鼠的脾细胞与SP2/0（骨髓瘤细胞）用聚乙二醇（PEG）融合。通过酶联免疫吸附测定（ELISA）筛选杂交瘤细胞的上清液以检测抗体。选择所需克隆进行有限稀释（L.D）。之后，通过ELISA、蛋白质免疫印迹分析（WB）及免疫荧光等免疫测定方法确定这些抗体的特异性和交叉反应性。通过腹水法大量制备单克隆抗体。通过层析法纯化单克隆抗体，然后用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。