Dellaratta D V, Hicklin D J, Kishore R, Kageshita T, Ferrone S
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595, USA.
Tissue Antigens. 1997 Dec;50(6):567-75. doi: 10.1111/j.1399-0039.1997.tb02914.x.
Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C-terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.
通过用与每个亚基C末端区域相对应的LMP2和LMP7特异性肽或TrxLMP2和TrxLMP7重组蛋白免疫兔子，制备了效价至少为1:10,000的抗人LMP2和抗人LMP7血清。用LMP特异性肽或重组蛋白免疫诱导产生的IgG抗体在ELISA中与其各自的免疫原表现出反应性。此外，用两种免疫原诱导产生的抗体在蛋白质印迹中识别天然和重组的LMP2和LMP7亚基，并且能够从淋巴细胞裂解物中免疫沉淀作为20S蛋白酶体组分的LMP2和LMP7。在ELISA中，用与一个LMP亚基相对应的LMP肽和重组蛋白产生的抗体亚群与另一个亚基具有交叉反应性。在用抗血清与相应免疫原孵育分离得到的亲和纯化抗体群体中未检测到该抗体亚群。抗LMP2和抗LMP7血清均未与同源蛋白酶体亚基Delta和MB1表现出交叉反应性。在免疫组织化学反应中，亲和纯化的抗LMP2和抗LMP抗体在正常皮肤的冰冻和福尔马林固定组织切片中均能对细胞进行染色。这些结果表明，用肽和重组蛋白诱导产生的抗LMP2和抗LMP7血清都是用于LMP2和LMP7生化特性鉴定以及分析它们在正常细胞和转化细胞中表达的有用试剂。
