AN IMPROVED SEMIQUANTITATIVE ENZYME IMMUNOSTAINING PROCEDURE FOR GLYCOSPHINGOLIPID ANTIGENS ON HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAMS
AN IMPROVED SEMIQUANTITATIVE ENZYME IMMUNOSTAINING PROCEDURE FOR GLYCOSPHINGOLIPID ANTIGENS ON HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAMS
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DOI：
10.1016/0022-1759(86)90038-4
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发表时间：
1986-01-01
影响因子：
2.2
通讯作者：
MUHLRADT, PF
中科院分区：
文献类型：
--
作者：
BETHKE, U;MUTHING, J;MUHLRADT, PF
描述了一种免疫测定法，它允许在高性能薄层色谱 (HPTLC) 上检测鞘糖脂 (GSL) 抗原。该方法包括：（1）在HPTLC上分离GSL； (2) 与针对 GSL 碳水化合物结构的特异性抗体一起孵育，以及 (3) 使用碱性磷酸酶缀合的第二抗体和 5-溴-4-氯-3-吲哚基磷酸 (BCIP) 作为底物检测特异性结合的抗体。使用针对 Forssman GSL 的单克隆大鼠 IgG2c 抗体、针对 asialo GM2 的 BALB/c 单克隆抗体和针对 asialo GM1 的多克隆兔抗体，结果表明，只需 4 小时即可检测到 3 ng GSL 抗原。该测定应可用于筛选对 GSL 抗原具有潜在特异性的单克隆和多克隆抗体、组织提取物中 GSL 抗原的检测和定量以及确定抗 GSL 抗体的特异性。
An immunoassay is described which allows the detection of glycosphingolipid (GSL) antigens on high performance thin layer chromatograms (HPTLC). The method involves: (1) the separation of GSL on HPTLCs; (2) incubation with specific antibodies against carbohydrate structures of GSL, and (3) the detection of specifically bound antibodies with alkaline phosphatase-conjugated second antibodies and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as substrate. Using a monoclonal rat IgG2c antibody against Forssman GSL, a BALB/c monoclonal antibody against asialo GM2, and polyclonal rabbit antibodies against asialo GM1, it was shown that as little as 3 ng GSL antigen could be detected in a procedure taking only 4 h to perform. The assay should be useful for screening mono- and polyclonal antibodies with potential specificity for GSL antigens, for the detection and quantification of GSL-antigens in tissue extracts, and for defining the specificity of anti-GSL antibodies.