New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting,Western blotting, and ELISA
New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting,Western blotting, and ELISA
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DOI：
10.1158/1078-0432.ccr-05-0578
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发表时间：
2005-08-15
影响因子：
11.5
通讯作者：
Pastan, I
中科院分区：
文献类型：
--
作者：
Onda, M;Willingham, M;Pastan, I
目的：间皮素是一种细胞表面蛋白，在一些恶性肿瘤中高表达，是免疫治疗的一个有前景的靶点。最近的数据表明间皮素是一种粘合剂。蛋白质，可能在卵巢癌的转移中发挥作用。尽管已经生产了一些针对间皮素的单克隆抗体（MAb），但由于它们的亲和力低或仅与变性间皮素蛋白具有反应性，因此它们对于研究天然间皮素的表达具有局限性。我们生产了针对间皮素的新型单克隆抗体，以帮助研究间皮素功能并开发改进的间皮素表达肿瘤的诊断和免疫疗法。实验设计：用编码间皮素的质粒 cDNA 免疫间皮素缺陷小鼠，并在细胞融合前用间皮素-兔 IgG Fc 融合蛋白进行加强。使用涂有间皮素-Fc蛋白的板通过ELISA筛选杂交瘤。结果：建立了17个产生抗间皮素抗体的杂交瘤，并显示与间皮素上的两个表位反应。一组与最初用于识别间皮素的低亲和力抗体 K1 反应相同的表位。另一个是与新表位反应的新基团。每组中选择一种抗体进行进一步研究，并在 ELISA、免疫组织化学和活细胞荧光激活细胞分选中显示出强烈反应。结论：我们的两种新建立的 MAb（MN 和 MB）与目前用于通过免疫组织化学、荧光激活细胞分选、ELISA 和蛋白质印迹检测间皮素的抗体相比，具有不同且有用的特性。
Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive. protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors.Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein.Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low-affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells.Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.