Harriman William D, Collarini Ellen J, Sperinde Gizette V, Strandh Magnus, Fatholahi Marjan M, Dutta April, Lee Yunji, Mettler Shelley E, Keyt Bruce A, Ellsworth Stote L, Kauvar Lawrence M
Trellis Bioscience, 2-B Corporate Dr., South San Francisco, CA 94080, United States.
J Immunol Methods. 2009 Feb 28;341(1-2):135-45. doi: 10.1016/j.jim.2008.11.009. Epub 2008 Dec 16.
The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at <1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.
单个杂交瘤细胞分泌的免疫球蛋白足迹，包含约10飞克原位纯化的抗体，已通过使用由用蛋白质功能化的280纳米组合彩色荧光乳胶珠组成的检测标签同时检测了9种特性。由此在初次筛选中评估了每个杂交瘤细胞产物的特异性。通过改变珠子上抗原的密度来调节珠子与分泌的抗体足迹之间相互作用的亲和力，从而在同一初次筛选中按亲和力进行排序。随着更多标准被添加到选择过程中，阳性细胞的频率下降；在某些情况下，有利的细胞出现频率低于1/50,000。通过将细胞接种在膜顶部的粘性培养基中来实现感兴趣细胞的回收。在膜下方的捕获表面上收集抗体足迹后，将固定的细胞转移到培养箱中，同时分析足迹以定位感兴趣的杂交瘤细胞。然后通过从膜上的相应位置挑选所需细胞进行克隆。
