A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA–HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30–1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08–4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

中文翻译：

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用于透明质酸检测的竞争性alpha筛选测定

描述了一种使用AlphaScreen®（扩增发光邻近均相测定）技术对乙酰透明质酸（HA）浓度进行特异性定量的方法。使用两种类型的水凝胶包被的和发色团负载的胶乳纳米珠。通过激发供体珠导致单线态氧的产生以及在暴露于单线态氧时受体珠的化学发光来检测溶液中珠的接近性。在HA分析中，供体珠被链霉亲和素修饰，并结合生物素标记的HA。受体珠用Ni（II）修饰，并用于结合带有His标签的特定重组HA结合蛋白（例如HABP；聚集蛋白聚糖G1-IGD-G2）。溶液中游离的未标记HA对HA–HABP相互作用的竞争性抑制作用用于定量。该测定法是针对HA的，并且不依赖于十糖以上的HA分子量。可以使用2.5μL样品在大约30–1600 ng / mL的浓度范围内对HA进行定量，可检测的质量范围约为0.08–4 ng HA。由于体积小，AlphaScreen测定的灵敏度比现有的类似ELISA的方法更高。在从HA中去除结合蛋白并稀释或去除其他干扰蛋白和脂质之后，可以使用AlphaScreen测定法在生物体液中检测到HA。由于体积较小的要求。在从HA中去除结合蛋白并稀释或去除其他干扰蛋白和脂质之后，可以使用AlphaScreen测定法在生物体液中检测到HA。由于体积较小的要求。在从HA中去除结合蛋白并稀释或去除其他干扰蛋白和脂质之后，可以使用AlphaScreen测定法在生物体液中检测到HA。