IMMUNOREACTIVE ALPHA A CRYSTALLIN IN RAT NONLENTICULAR TISSUES DETECTED WITH A SENSITIVE IMMUNOASSAY METHOD
IMMUNOREACTIVE ALPHA A CRYSTALLIN IN RAT NONLENTICULAR TISSUES DETECTED WITH A SENSITIVE IMMUNOASSAY METHOD
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DOI：
10.1016/0167-4838(91)90146-q
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发表时间：
1991-10-25
期刊：
影响因子：
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通讯作者：
OHSHIMA, K
中科院分区：
文献类型：
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作者：
KATO, K;SHINOHARA, H;OHSHIMA, K
为了定量分析透镜中α-晶状体蛋白A亚基（alpha-A）的含量，并探讨非晶状体组织中α-晶状体蛋白的可能存在，我们建立了一种高灵敏度、高特异性的α-晶状体蛋白A免疫分析方法。 用从牛透镜纯化的α-A或α-A的C-末端十肽（EEKPSSAPSS）（α-A（pep））在兔中产生抗α-A的抗血清。 通过使用α-A偶联的Sepharose 4 B柱纯化α-A和α-A（pep）的抗体。 将纯化的抗α-A IgG的F（ab '）2片段固定在聚苯乙烯球上，并用来自大肠杆菌的β-D-半乳糖苷酶标记纯化的抗α-A（pep）IgG的Fab'片段。 使用两种抗体制剂的夹心型免疫测定的最低检测限为< 10 pg alpha-A，与alpha-B没有任何交叉反应性。 通过采用本方法，发现在大鼠脾脏和胸腺中存在显著量的免疫反应性α-A。 在直肠、盲肠、肝脏、肾脏、肾上腺、小脑和脑干中检测到极低水平的免疫反应性α-A。 脾提取物中的免疫反应性α-A通过使用抗α-A（pep）偶联的琼脂糖凝胶部分纯化（约50%纯度）。 在3周龄之前，脾脏中的α-A浓度< 1 ng / mg蛋白质。 然而，5周龄后，它线性增加，到18周龄达到约20 ng / mg蛋白质。 免疫组化结果显示，α-A定位于脾脏和胸腺的网状细胞中。
For the quantitative analysis of the A subunit of alpha-crystallin (alpha-A) in the lens and for the survey of possible existence of alpha-A in the non-lenticular tissues, we have established a highly sensitive and specific immunoassay method for alpha-A. Antisera to alpha-A were raised in rabbits with alpha-A purified from bovine lens, or the C-terminal decapeptide (EEKPSSAPSS) of alpha-A (alpha-A(pep)). The antibodies to alpha-A and alpha-A(pep) were purified by the use of an alpha-A-coupled Sepharose 4B column. The F(ab')2 fragments of purified anti-alpha-A IgG were immobilized on polystyrene balls and the Fab' fragments of purified anti-alpha-A(pep) IgG were labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the sandwich-type immunoassay using the two antibody preparations was < 10 pg alpha-A without any cross-reactivity with alpha-B. By employing the present methods, it was found that a significant amount of immunoreactive alpha-A was present in rat spleen and thymus. Very low levels of immunoreactive alpha-A were detected in the rectum, caecum, liver, kidney, adrenal, cerebellum and brainstem. The immunoreactive alpha-A in the spleen extract was purified partially (about 50% purity) by the use of anti-alpha-A(pep)-coupled Sepharose. The concentration of alpha-A in the spleen was < 1 ng / mg protein before 3 weeks of age. After 5 weeks of age, however, it increased lineally reaching about 20 ng / mg protein by 18 weeks of age. Immunohistochemically, the alpha-A was localized in the reticular cells in the spleen and thymus.