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首页> 外文期刊>Biotechnology Progress >Towards an Indirect Screening Technique Facilitating Detection of Cellular Populations Bearing Specific Cell Surface Markers
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Towards an Indirect Screening Technique Facilitating Detection of Cellular Populations Bearing Specific Cell Surface Markers

机译：致力于促进筛选具有特定细胞表面标记的细胞群体的间接筛选技术

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摘要

We describe and demonstrate a technique for detection of cell surface antigens, with potential use in tissue antigen research. Briefly, a small volume of wetted chromatographic beads (specifically, 100 μL of Nickel-nitriloacetic acid (NTA) agarose) was bound to small quantities (specifically, ~0.1-0.2 μg) of a single-chain Fv antibody recognizing the Class I MHC heavy chain antigen. The beads were then used to capture and detect cells bearing this antigen, through SDS polyacrylamide gel electrophoresis of boiled bead-cell complexes. A key feature of this method is its use of "signal amplification." Although the antibody-mediated binding and immobilization of intact cells involves relatively small numbers of antibodies, and cells, what is being detected is simply the presence of cells. Each bound cell contains a number of abundant proteins that are present in millions of copies per cell, and the abundance of these proteins ensures that they are detectable on SDS-PAGE, signaling cell-binding. As few as 10~(10)-10~(12) scFv antibodies immobilized on 100 μL of Ni-NTA beads are thus enough for the trapping of enough cells to allow visualization of their abundant proteins. Conceptually, this method could be easily developed and applied to detection of cells bearing any other cell specific antigen.
机译：我们描述并演示了一种用于检测细胞表面抗原的技术，具有在组织抗原研究中的潜在用途。简而言之，将少量润湿的色谱珠（具体而言，100μL的镍-硝酸镍（NTA）琼脂糖）与少量（具体而言，约0.1-0.2μg）的识别I类MHC的单链Fv抗体结合重链抗原。然后，通过煮沸的珠-细胞复合物的SDS聚丙烯酰胺凝胶电泳，将珠用于捕获和检测带有此抗原的细胞。该方法的关键特征是其使用“信号放大”。尽管抗体介导的完整细胞的结合和固定涉及相对少量的抗体和细胞，但检测到的仅仅是细胞的存在。每个结合的细胞都包含大量丰富的蛋白质，每个细胞以百万个拷贝的形式存在，这些蛋白质的丰富性确保了它们在SDS-PAGE上可检测到，从而发出细胞结合信号。因此，固定在100μLNi-NTA磁珠上的scFv抗体只有10〜（10）-10〜（12）个，足以捕获足够的细胞，从而可视化其丰富的蛋白质。从概念上讲，此方法可以轻松开发并应用于检测带有任何其他细胞特异性抗原的细胞。

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