Determination of the glucosidase-stimulating proteins by competitive enzyme-linked immunoassay.
Determination of the glucosidase-stimulating proteins by competitive enzyme-linked immunoassay.
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通过竞争性酶联免疫测定测定葡萄糖苷酶刺激蛋白。
DOI：
10.1016/0003-2697(84)90537-2
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发表时间：
1984
影响因子：
2.9
通讯作者：
Radin,NS
中科院分区：
文献类型：
--
作者：
Datta,SC;Radin,NS
描述了小鼠组织中共水解酶鞘脂-I 的免疫测定程序。这种共水解酶（实际上是至少四种相关蛋白质的混合物）刺激或激活β-葡萄糖苷酶，该酶可水解神经酰胺葡萄糖苷（一种广泛存在的鞘糖脂）。该方法包括用盐缓冲溶液从组织匀浆中提取共水解酶，通过调节pH至6去除蛋白质，通过加热进一步去除蛋白质，并用小尺寸排除柱去除干扰物质。在兔体内针对牛共水解酶产生抗体，并用共水解酶制成的亲和柱进行纯化。免疫测定涉及共水解酶样品 (20-200 pg) 与抗体的结合，与已与辣根过氧化物酶化学连接的共水解酶竞争。将混合物用颗粒连接的二抗处理并离心；然后用荧光法测定沉淀的过氧化物酶含量。该方法的初步应用表明，辅水解酶存在于所有研究的小鼠组织中，并且其浓度与葡萄糖脑苷脂酶的浓度相对接近。两种物质随年龄（14 天和 92 天）的变化以类似的方式发生。
A procedure for the immunoassay of cohydrolase sphingolipid-I in mouse tissue is described. This cohydrolase (actually a mixture of at least four related proteins) stimulates or activates the β-glucosidase which hydrolyzes ceramide glucoside, a widely occurring glycosphingolipid. The method involves extraction of cohydrolase from tissue homogenate with a salt-buffer solution, removal of proteins by adjustment to pH 6, further removal of proteins by heating, and removal of interfering materials with a small size exclusion column. Antibodies were raised to bovine cohydrolase in rabbits and purified with an affinity column made from cohydrolase. The immunoassay involves binding of antibody by the cohydrolase sample (20–200 pg) in competition with cohydrolase that has been chemically linked to horseradish peroxidase. The mixture is treated with particle-linked second antibody and centrifuged; the pellet is then assayed fluorometrically for peroxidase content. Initial application of the method showed that cohydrolase was present in all mouse tissues studied and that its concentration paralleled that of glucocerebrosidase relatively closely. Changes with age (14 and 92 days) occurred in a similar fashion for the two substances.