Douwes J, Doekes G, Montijn R, Heederik D, Brunekreef B
Department of Epidemiology and Public Health, Agricultural University Wageningen, The Netherlands.
Appl Environ Microbiol. 1996 Sep;62(9):3176-82. doi: 10.1128/aem.62.9.3176-3182.1996.
beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.
β(1→3)-葡聚糖以其诱导非特异性炎症反应的强大能力而闻名,并且被认为在生物气溶胶诱发的呼吸道症状中起作用。开发了一种抑制酶免疫测定法(EIA),用于定量职业和居住环境灰尘样本中的β(1→3)-葡聚糖。通过用牛血清白蛋白偶联的海带多糖[β(1→3)-葡聚糖]免疫并在环氧-琼脂糖偶联的β(1→3)-葡聚糖上进行亲和层析来制备免疫特异性兔抗体。抑制EIA中基于海带多糖的校准曲线范围约为40至3000 ng/ml(抑制率为15至85%)。另一种β(1→3)-葡聚糖(凝胶多糖)显示出类似的抑制曲线,但按重量计反应性低三至五倍。假定为β(1→6)-葡聚糖的普鲁兰多糖在浓度比海带多糖高10倍时显示出平行的剂量反应曲线。分别用高碘酸钠和β(1→3)-葡聚糖酶处理以破坏β(1→6)-和β(1→3)-葡聚糖结构的对照实验表明,该测定中普鲁兰多糖的免疫反应性是由于β(1→3)-葡聚糖而不是β(1→6)-葡聚糖结构。其他多糖,如甘露聚糖和α(1→6)-葡聚糖,在抑制EIA中不发生反应。通过热处理(120℃)对灰尘样本进行β(1→3)-葡聚糖在水中(加温和洗涤剂)的提取,因为在室温下获得的水提取物中未检测到可检测水平的β(1→3)-葡聚糖。该测定法显示可检测在各种职业和环境环境中收集的灰尘样本中的热可提取β(1→3)-葡聚糖。基于对灰尘样本的重复分析,计算出变异系数约为25%。得出的结论是新的抑制EIA为室内β(1→3)-葡聚糖暴露评估提供了一种有用的方法。