Scheffold A, Miltenyi S, Radbruch A
Institute for Genetics, University of Cologne, Germany.
Immunotechnology. 1995 Aug;1(2):127-37. doi: 10.1016/1380-2933(95)00014-3.
Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cells according to specific marker molecules. Due to the limited sensitivity at least several thousands of antigens per cell are required for optical detection. Molecules expressed at low copy numbers cannot be analysed, although they may be of considerable functional importance.
Development of a magnetic and fluorescent staining reagent for analysis and sorting of cells according to antigens expressed at low number. To this end, uniformly sized, antibody-conjugated liposomes loaded with large amounts of dye molecules and small magnetic particles were generated.
A method for the preparation of homogeneously sized large unilamellar liposomes which contain carboxyfluorescein, magnetic particles and surface-bound antibodies had to be developed. These liposomes were then tested for their ability to enhance immunofluorescence compared to conventional staining in a model system and by staining of CD25 on resting B and T cells.
Large unilamellar liposomes, homogeneous in size and loaded with fluorescein and magnetic beads can be prepared by combining membrane extrusion and magnetic filtration. Hapten-specific antibodies conjugated to their surface make them a universal tool for immunofluorescence. With such liposomes, intensity of fluorescent staining can be increased 100-1000-fold without increased background fluorescence, compared to conventional fluorochrome-conjugated antibodies. Due to the simultaneous magnetic labelling, stained cells can easily be isolated by MACS. The magnetofluorescent liposomes proved to be useful for improvement of sensitivity of detection and physical separation in general and to visualize and sort cells according to antigens expressed at low levels. The high affinity IL2 receptor CD25 is expressed in low copy number on a significant fraction of resting B and T lymphocytes in human peripheral blood, as can be shown exclusively by the magnetofluorescent liposomes.
免疫荧光和免疫磁珠技术是根据特定标记分子对细胞进行分析和分选的重要技术。由于光学检测灵敏度有限，每个细胞至少需要数千个抗原才能被检测到。低拷贝数表达的分子无法被分析，尽管它们可能具有相当重要的功能意义。
开发一种磁性和荧光染色试剂，用于根据低拷贝数表达的抗原对细胞进行分析和分选。为此，制备了负载大量染料分子和小磁性颗粒的尺寸均匀、偶联抗体的脂质体。
必须开发一种制备尺寸均匀的大单层脂质体的方法，该脂质体包含羧基荧光素、磁性颗粒和表面结合抗体。然后在模型系统中以及通过对静息B细胞和T细胞上的CD25进行染色，测试这些脂质体与传统染色相比增强免疫荧光的能力。
通过结合膜挤压和磁过滤，可以制备尺寸均匀、负载荧光素和磁珠的大单层脂质体。其表面偶联的半抗原特异性抗体使其成为免疫荧光的通用工具。与传统的荧光染料偶联抗体相比，使用这种脂质体，荧光染色强度可提高100 - 1000倍，且背景荧光不增加。由于同时进行磁性标记，染色细胞可通过磁珠细胞分选轻松分离。磁荧光脂质体被证明总体上可用于提高检测灵敏度和物理分离，并根据低水平表达的抗原对细胞进行可视化和分选。人外周血中相当一部分静息B淋巴细胞和T淋巴细胞上高亲和力的白细胞介素2受体CD25以低拷贝数表达，这仅通过磁荧光脂质体才能显示出来。
