Assay of mucins in human tear fluid
Assay of mucins in human tear fluid
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DOI：
10.1016/j.exer.2007.01.018
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发表时间：
2007-05-01
影响因子：
3.4
通讯作者：
Gipson, Ilene
中科院分区：
文献类型：
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作者：
Spurr-Michaud, Sandra;Argueso, Pablo;Gipson, Ilene
据报道，分泌型（MUC2、MUC5AC、MUC5B、MUC7）和膜相关型（MUC1、MUC4、MUC16）粘蛋白基因均由眼表上皮细胞表达。本研究的目的是使用针对每种粘蛋白的多种抗体全面检测人泪液中的粘蛋白含量，并开发一种灵敏的、半定量的泪液粘蛋白检测方法。通过向眼表滴注生理盐水，然后从下穹窿收集泪液冲洗液。泪液蛋白在1%琼脂糖凝胶中分离，通过真空印迹转移到硝酸纤维素膜上，并用识别MUC1、MUC2、MUC4、MUC5AC、MUC5B、MUC7和MUC16的多种抗体进行检测。使用化学发光法检测结合情况，并通过光密度测定法确定量。对正常个体的混合泪液进行系列稀释检测，以确定可检测性的线性范围。在人泪液中始终能检测到MUC1、MUC4、MUC16、MUC5AC以及低水平的MUC2，而未检测到MUC5B和MUC7。使用识别同一粘蛋白上不同表位的几种抗体证实了这些发现。粘蛋白抗体与泪液系列稀释液呈线性结合（r²>0.9），表明半定量的可行性。与从结膜组织中分离的MUC5AC相比，泪液中的MUC5AC具有更高的电泳迁移率。本研究提供了明确的证据，表明泪液中的粘蛋白成分是分泌型和脱落的膜相关型粘蛋白的混合物，并首次在泪液中检测到MUC16。使用琼脂糖凝胶电泳和化学发光检测的泪液免疫印迹为粘蛋白提供了一种半定量检测方法，这对于与患病眼睛的泪液或药物干预后的泪液进行比较将是有用的。（C）2007爱思唯尔有限公司。保留所有权利。
Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of delectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r(2) > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention. (C) 2007 Elsevier Ltd. All rights reserved.