Kato K, Shinohara H, Kurobe N, Goto S, Inaguma Y, Ohshima K
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Biochim Biophys Acta. 1991 Oct 25;1080(2):173-80. doi: 10.1016/0167-4838(91)90146-q.
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 Apep). The antibodies to alpha A and alpha Apep 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 Apep 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 less than 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 Apep-coupled Sepharose. The concentration of alpha A in the spleen was less than 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.
为了对晶状体中α晶状体蛋白A亚基（αA）进行定量分析，并探究αA在非晶状体组织中可能的存在情况，我们建立了一种针对αA的高度灵敏且特异的免疫测定方法。用从牛晶状体中纯化得到的αA或αA的C末端十肽（EEKPSSAPSS，αApep）免疫家兔制备抗αA血清。利用与αA偶联的琼脂糖4B柱纯化抗αA和抗αApep抗体。将纯化的抗αA 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αApep IgG的Fab'片段。使用这两种抗体制备的夹心型免疫测定的最低检测限低于10 pg αA，且与αB无任何交叉反应。通过采用本方法，发现大鼠脾脏和胸腺中存在大量免疫反应性αA。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αA。脾脏提取物中的免疫反应性αA通过使用抗αApep偶联的琼脂糖进行部分纯化（纯度约为50%）。3周龄前脾脏中αA的浓度低于1 ng/mg蛋白质。然而，5周龄后，其呈线性增加，到18周龄时达到约20 ng/mg蛋白质。免疫组织化学显示，αA定位于脾脏和胸腺的网状细胞中。
