An enzyme-linked lectin assay for α1,3-galactosyltransferase
An enzyme-linked lectin assay for α1,3-galactosyltransferase
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DOI：
10.1006/abio.2000.4504
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发表时间：
2000-05-01
影响因子：
2.9
通讯作者：
Bovin, NV
中科院分区：
文献类型：
--
作者：
Khraltsova, LS;Sablina, MA;Bovin, NV
UDP-Gal：Gal β 1-4GlcNAc α 1，3-半乳糖基转移酶（α 3GalT）负责碳水化合物异种抗原Gal α 1-3Gal β 1-4GlcNAc的合成。在这项工作中，一个方便和灵敏的测定系统定量的α 3GalT活性的酶联凝集素测定（ELLA）与比色检测。将表面已用二糖Gal β 1-4GlcNAc：（受体）的聚丙烯酰胺缀合物包被的微量滴定板威尔斯孔与α 3 GalT在作为糖基供体的“冷”UDP-Gal存在下孵育。通过生物素化的植物凝集素槲寄生凝集素检测聚糖链的酶促延伸形成的产物。在没有（背景）或已知量的酶活性的情况下运行标准测定后，完成用于正确定量α 3GalT活性的标准曲线。以这种方式检测到的产物形成与酶活性以及受体和糖基供体UDP-Gal的浓度成比例。根据α 3GalT的已知特异性，使用该测定法未观察到Le（x）酶促转化为Gal α Le（x）。使用暴露于二甲双胍的亲和吸附剂分离人α Gal抗体，并研究其特异性。相对于这些天然免疫球蛋白作为产物检测工具的应用，ELLA被证明是更敏感的。(C)北京大学出版社.
UDP-Gal:Gal beta 1-4GlcNAc alpha 1,3-galactosyltransferase (alpha 3GalT) is responsible for the synthesis of carbohydrate xenoantigen Gal alpha 1-3Gal beta 1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha 3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Gal beta 1-4GlcNAc:(acceptor) are incubated with alpha 3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin, The standard curve for correct quantification of alpha 3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl donor UDP-Gal, In accordance with the known specificity of alpha 3GalT, no enzymatic conversion of Le(x) into Gal alpha Le(x) was observed using this assay, Human alpha Gal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive. (C) 2000 Academic Press.