HYDROCOATING - A NEW METHOD FOR COUPLING BIOMOLECULES TO SOLID-PHASES
HYDROCOATING - A NEW METHOD FOR COUPLING BIOMOLECULES TO SOLID-PHASES
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DOI：
10.1016/0022-1759(94)00330-y
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发表时间：
1995-04-12
影响因子：
2.2
通讯作者：
ELSNER, HI
中科院分区：
文献类型：
--
作者：
GREGORIUS, K;MOURITSEN, S;ELSNER, HI
固相免疫测定，如酶联免疫吸附测定，需要固定其中一种测定组分。这通常是通过抗原或抗体被动吸附到疏水聚合物表面来实现的，例如聚苯乙烯。或者，生物分子可以通过被动吸附的载体蛋白间接结合，或者使用交联剂直接通过固相上的官能团结合。在这里，我们描述了一种新的技术-水包膜-共价固定生物分子，如肽，在高度亲水性的环境。多肽通过与活化的亲水性聚合物的共价键固定在微滴板上。可溶性葡聚糖被2,2,2-三氟乙烷磺酰氯（三酰氯）激活，导致葡聚糖聚合物上羟基的活化。这种活化的葡聚糖分子被固定在含有氨基的表面上，留下足够数量的活性基团用于其他生物分子的二次结合。在聚苯乙烯上吸附时无法检测到或难以识别的多肽，在用氢涂层技术固定时很容易识别。此外，与以共价固定在含有仲氨基的表面上的相同肽相比，用这种方法固定的肽的识别能力提高了5-10倍。该技术似乎为生物分子在固相上的被动吸附提供了一种替代方法，并可能在未来免疫测定的发展中发挥作用。
Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique - hydrocoating - for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays.