Endoplasmic reticulum chaperone-specific monoclonal antibodies for flow cytometry and immunohistochemical staining
Endoplasmic reticulum chaperone-specific monoclonal antibodies for flow cytometry and immunohistochemical staining
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DOI：
10.1034/j.1399-0039.2003.00114.x
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发表时间：
2003-11-01
期刊：
影响因子：
--
通讯作者：
Ferrone, S
中科院分区：
文献类型：
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作者：
Ogino, T;Wang, X;Ferrone, S
抗原加工机制的内质网伴侣在HLA I类抗原复合物的组装和抗原递呈中起着至关重要的作用。由于缺乏或有限的适合免疫组织化学染色的ER伴侣特异性单克隆抗体（mAb），这些伴侣在正常组织和恶性病变中表达的表征受到阻碍。为了克服这一限制，我们用多肽和重组蛋白免疫的BALB/c小鼠产生了人calnexin、ERp57、calreticulin和tapasin特异性单克隆抗体分泌杂杂瘤。单抗to -5、to -2、to -11和to -3分别对calnexin、ERp57、calreticulin和tapasin具有特异性，因为它们在ELISA中与相应的免疫肽发生特异性反应，在Western blotting中与人淋巴样细胞裂解物检测时与相应的蛋白发生特异性反应。此外，用福尔马林固定、微波处理和皂素渗透的细胞进行间接免疫荧光检测，用福尔马林固定、石蜡包埋的组织切片进行免疫过氧化物酶反应，检测四种单抗与相应细胞内抗原的反应性。这些结果表明，我们开发的ER伴侣特异性单抗是表征正常和病理细胞抗原加工机制中ER伴侣表达的有用探针。这些信息将有助于确定其表达异常对HLA I类抗原表达和功能的影响，以及对靶细胞与宿主免疫系统相互作用的影响。
Endoplasmic reticulum (ER) chaperones of the antigen processing machinery play a crucial role in HLA class I antigen complex assembly and antigen presentation. The characterization of the expression of these chaperones in normal tissues and malignant lesions has been hampered by the lack or limited availability of ER chaperone-specific monoclonal antibodies (mAb) that are suitable for immunohistochemical staining. To overcome this limitation, we have generated human calnexin, ERp57, calreticulin and tapasin-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant proteins. The mAb TO-5, TO-2, TO-11 and TO-3 were shown to be specific for calnexin, ERp57, calreticulin and tapasin, respectively, as they react specifically with the corresponding immunizing peptides in ELISA and with the corresponding proteins when tested with human lymphoid cell lysates in Western blotting. Furthermore, the reactivity of the four mAb with the corresponding intracellular antigens yielded intracellular staining when the mAb were tested with formalin-fixed, microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in the immunoperoxidase reaction. These results suggest that the ER chaperone-specific mAb we have developed are useful probes for characterizing the expression of ER chaperones of the antigen processing machinery in normal and pathological cells. This information will contribute to defining the effects of abnormalities in their expression on HLA class I antigen expression and function and on the interactions of target cells with the host's immune system.