Hicklin D J, Kageshita T, Ferrone S
Department of Microbiology and Immunology, New York Medical College, Valhalla, USA.
Tissue Antigens. 1996 Jul;48(1):38-46. doi: 10.1111/j.1399-0039.1996.tb02603.x.
The limited availability of sera to human MHC-linked transporters associated with antigen processing (TAP) has hampered the analysis of the role of these molecules in the reduced HLA Class I antigen expression by normal and transformed cells. To overcome these limitations, anti-human TAP1 and anti-human TAP2 xenoantisera have been generated and characterized. To this end rabbits have been immunized with TAP1-specific or TAP2-specific peptides which correspond to nonhomologous, hydrophilic regions of each transporter subunit. The immunized rabbits developed high titer IgG antibodies which displayed specific reactivity with the immunizing peptides in ELISA. Both anti-TAP1 and anti-TAP2 antisera immunoprecipitated the 70-76 kDa TAP complex from TAP(+)-TAP2+ cell lines WALK and Colo 38, but precipitated no component from TAP(-)-TAP2- cell lines T2 and SK-MEL-19. Furthermore, in immunodepletion experiments anti-TAP1 and anti-TAP2 antisera removed the molecules recognized by each of them in a lymphoid cell extract. Lastly, in Western blotting assays anti-TAP1 and anti-TAP2 antisera reacted specifically with isolated TAP1 and TAP2, respectively. The latter results in conjunction with those of the immunodepletion experiments indicate that TAP1 and TAP2 are not detectable as isolated subunits in a cell extract and that TAP heterocomplex is the major, if not the only detectable molecular species in cells. Anti-TAP1 and anti-TAP2 antisera were evaluated in immunohistochemical staining of both frozen and formalin fixed sections of skin and primary malignant melanoma lesions. Both antisera stained the cytoplasm of keratinocytes in normal skin and of melanoma cells in malignant lesions. The antisera we have elicited with TAP1- and TAP2-specific peptides appear to be useful reagents to characterize the role of TAP in abnormalities of HLA Class I antigen expression.
用于分析与抗原加工相关的人类主要组织相容性复合体（MHC）转运体（TAP）的血清有限，这阻碍了对这些分子在正常细胞和转化细胞中HLA I类抗原表达降低过程中所起作用的研究。为克服这些限制，已制备并鉴定了抗人TAP1和抗人TAP2异种抗血清。为此，用与每个转运体亚基的非同源亲水区相对应的TAP1特异性或TAP2特异性肽免疫兔子。免疫后的兔子产生了高效价IgG抗体，这些抗体在酶联免疫吸附测定（ELISA）中与免疫肽表现出特异性反应。抗TAP1和抗TAP2抗血清均能从TAP（+）-TAP2+细胞系WALK和Colo 38中免疫沉淀出70 - 76 kDa的TAP复合体，但从TAP（-）-TAP2-细胞系T2和SK-MEL-19中未沉淀出任何成分。此外，在免疫去除实验中，抗TAP1和抗TAP2抗血清在淋巴细胞提取物中去除了各自识别的分子。最后，在蛋白质印迹分析中，抗TAP1和抗TAP2抗血清分别与分离的TAP1和TAP2发生特异性反应。后一结果与免疫去除实验结果表明，在细胞提取物中无法检测到分离的TAP1和TAP2亚基，并且TAP异源复合体是细胞中主要的（如果不是唯一可检测到的）分子形式。在皮肤和原发性恶性黑色素瘤病变的冰冻切片和福尔马林固定切片的免疫组织化学染色中评估了抗TAP1和抗TAP2抗血清。两种抗血清均能对正常皮肤中的角质形成细胞和恶性病变中的黑色素瘤细胞的细胞质进行染色。我们用TAP1和TAP2特异性肽诱导产生的抗血清似乎是用于表征TAP在HLA I类抗原表达异常中作用的有用试剂。
