The Method of Microassay system of Sulfuric Glycosaminoglycans in saliva

在寻找疾病的生物标志物时，测定龈沟液中的S糖胺聚糖具有重要意义。为了快速、准确地分析牙周膜中S-GAG的含量，我们改进了尿S-GAG的微量板法，使之能在不干扰其他成分的情况下测定牙周膜中S-GAG的含量。采集6颗上颌前牙牙周组织标本，稀释后随机分为2组(每组20μ)，分别加入或不加入软骨素酶ABC处理，与二甲基亚甲基蓝染料溶液(180μL)混合检测S-GAG，用540 nm微板仪测定吸光度，标准S-GAG的浓度在0~3μg/100μL范围内呈线性关系，计算出S-GAG的含量。糖蛋白和血清成分等其他成分不影响S-GAGS的测定。虽然在健康牙周组织中发现了微量的S-GAG(0.05μg/100μL±0.04，平均值±S.E.，n=5)，但在晚期牙周病患者中也发现了相当数量的S-GAG(0.42±0.11，n=6，p&lt；0.05)。该方法简便、快速，可用于牙周疾病的筛查。

The measurement of sulphated glycosaminoglycans (S-GAGs) in gingival crevicular fluid (GCF) is important in the search of disease biomarkers. To analyze the amount of S-GAGs in GCF quickly and accurately, we have modified a microplate method previously used for urinary S-GAGs to estimate the content of S-GAGs in GCF without interference of other GCF components. GCF samples were collected from six maxillary anterior teeth and the diluted GCF samples were divided into two groups (20 μl each), with or without chondroitinase ABC treatment, mixed with a dimethylmethylene blue (DMB) dye solution (180 μl) to detect S-GAG.The absorbance was measured using microplate reader of 540 nm and the amount of S-GAG was calculated from calibration curves for standard S-GAGs where a linear relationship existed in the concentration range (0-3 μg/100 μl). Other GCF components such as glycoprotein and serum constituents did not influence the measurement of S-GAGs. Although trace amounts of S-GAGs were found in control with a healthy periodontium (0.05 μg/100 μl±0.04, mean±S.E., n=5), considerable amounts of S-GAGs were found in patients with advanced periodontal disease (0.42 ± 0.11, n=6, p<0.05). The accuracy of this simple and rapid method measuring S-GAGs in GCF makes it useful for screening periodontal diseases.