Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
Anal Biochem. 2011 Sep 1;416(1):18-26. doi: 10.1016/j.ab.2011.05.006. Epub 2011 May 10.
Microtiter plate colorimetric assays are widely used for analysis of carbohydrates and glycoconjugates. However, mucins are often not easily detected, as they have low neutral sugar content. We have adapted and optimised the periodic acid-Schiff's reagent (PAS) staining for microtiter plate assay by examining five factors: concentration and volume of periodic acid, oxidation time, volume of Schiff's reagent, and color development time. This assay requires just 25 μl of sample, utilises standardised Schiff's reagent, and has decreased assay time (140 min to completion). Seventeen monosaccharides (acidic, neutral, basic, phosphorylated, and deoxy) and four disaccharides were assessed. PAS-positive carbohydrates (amino, N-acetylamino, deoxy, and certain neutral monosaccharides, and sialic acids) responded linearly within a 10-100 nmol range approximately, which varied for each carbohydrate. The assay response for fetuin and porcine gastric mucin (PGM) was linear up to 150 μg (highest concentration tested), with no response from nonglycosylated protein. A lower response for asialofetuin was observed, but desialylated PGM preparations were similar or higher in response than their sialylated counterparts. The simplicity and low sample consumption of this method make it an excellent choice for screening or quantitation of chromatographic fractions containing carbohydrates and glycoconjugates, especially in the case of mucins.
微量滴定板比色分析广泛用于碳水化合物和糖缀合物的分析。然而，由于粘蛋白的中性糖含量低，因此通常不易检测到。我们通过检查五个因素（过碘酸的浓度和体积、氧化时间、希夫试剂的体积和显色时间），对微量滴定板检测中的过碘酸-Schiff 试剂（PAS）染色法进行了改编和优化。该测定法仅需 25 μl 样品，使用标准化的 Schiff 试剂，并且缩短了测定时间（140 分钟即可完成）。我们评估了 17 种单糖（酸性、中性、碱性、磷酸化和脱氧）和 4 种二糖。PAS 阳性碳水化合物（氨基、N-乙酰氨基、脱氧和某些中性单糖以及唾液酸）在约 10-100 nmol 范围内呈线性响应，每种碳水化合物的响应范围都不同。胎球蛋白和猪胃粘蛋白（PGM）的测定响应在 150 μg（测试的最高浓度）范围内呈线性，而无糖基化蛋白的响应。脱唾液酸胎球蛋白的响应较低，但脱唾液酸 PGM 制剂的响应与它们的唾液酸化对应物相似或更高。该方法简单，样品消耗低，是筛选或定量含有碳水化合物和糖缀合物的色谱馏分的绝佳选择，尤其是在粘蛋白的情况下。
