The Method of Microassay system of Sulfuric Glycosaminoglycans in saliva

龈沟液（GCF）中硫酸化糖胺聚糖（S-GAGs）的测定在寻找疾病生物标志物中具有重要意义。为了快速、准确地分析GCF中S-GAGs的含量，我们改进了以前用于尿S-GAGs的微孔板法，以在不干扰GCF中其他组分的情况下估计GCF中S-GAGs的含量。收集6颗上颌前牙的龈沟液样本，将稀释后的龈沟液样本分为两组（各20 μl），有或无软骨素酶ABC处理，与二甲基亚甲基蓝（DMB）染料溶液（180 μl）混合以检测S-GAG，使用酶标仪在540 nm处测量吸光度，并根据标准S-GAG的校准曲线计算S-GAG的量。在浓度范围（0-3 μg/100 μl）内存在线性关系的GAG。其他GCF成分如糖蛋白和血清成分不影响S-GAG的测定。尽管在健康牙周组织的对照中发现了痕量的S-GAG（0.05 μg/100 μl±0.04，平均值±S.E.，晚期牙周病组中S-GAG含量为0.42 ± 0.11，n=6，P<0.05。该方法简便、快速、准确，可用于牙周病的筛查。

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.