Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Immunol Invest. 2010 Jan;39(2):114-31. doi: 10.3109/08820130903496785.
Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.
癌抗原 125（CA 125）在卵巢癌中过表达，针对它的抗体被广泛用于诊断目的。然而，CA 125 抗原结构域的稀有性及其高度糖基化结构是一个问题，可能会阻止免疫小鼠产生针对 CA 125 的多样化抗体群体。在这项研究中，使用了一种增强免疫反应的免疫原性策略，即通过杂交瘤技术生产针对稀有和免疫原性差的决定簇的单克隆抗体（mAbs）。然后通过 ELISA、western blot、免疫沉淀、免疫组化和 OVCAR-3 细胞系的免疫荧光染色评估 mAbs 的反应性。总共产生了 10 个克隆，其中 3 个为 IgG 同种型，其余为 IgM。三分之二的克隆在固定和活细胞中识别同源抗原，并在 IHC 染色中具有强烈的免疫反应性。在 Western blot 中，我们的抗体识别 CA 125 作为高分子量抗原，主要迁移到 3%的堆积胶中。mAbs 对 OVCAR-3 细胞裂解物的免疫沉淀导致非常相似的迁移模式，这再次证实了它们的特异性。本研究中产生的 mAbs 是评估癌性卵巢组织中 CA 125 表达的诊断和研究领域中非常有价值的工具。
