Pike B L, Jennings G, Shortman K
J Immunol Methods. 1982;52(1):25-37. doi: 10.1016/0022-1759(82)90346-5.
A simple semi-automated method for the assay of large numbers of replicate microcultures for the presence of antibody-forming cell clones is described. The supernatant medium is removed from microcultures by a single sharp flick on inverting the tray. The cultured cells are mixed with 0.05 ml of a plaque-revealing mix containing indicator erythrocytes and complement and then transferred to new flat-bottomed 96-well microculture trays, using a multichannel pipette or 96-channel replicator. The tray is centrifuged, the indicator erythrocytes and cultured cells forming an even monolayer on the bottom surface of the well. Trays are held at 37 degrees C for 1-1 1/2 h to allow plaque development. Using a dissecting microscope, the number of plaques in each well is counted, or in the case of limiting dilution analysis, each well is simply scored as positive or negative. This assay procedure provides a simple, rapid and inexpensive means of assaying large numbers of microculture trays for the detection and enumeration of antibody-forming cell clones. There is no loss in sensitivity compared with the standard hemolytic plaque assay methods. The method is particularly useful for limiting dilution analysis which necessitates the assay of large numbers of replicate cultures for either the presence of absence of a clone of antibody-forming cells.
本文描述了一种简单的半自动方法，用于检测大量重复的微量培养物中是否存在抗体形成细胞克隆。通过倒置培养板并快速轻弹一下，即可去除微量培养物中的上清培养基。将培养的细胞与0.05 ml含有指示红细胞和补体的噬斑显示混合液混合，然后使用多通道移液器或96通道复制器转移到新的平底96孔微量培养板中。将培养板离心，使指示红细胞和培养的细胞在孔的底面上形成均匀的单层。将培养板在37℃下放置1 - 1.5小时，以使噬斑形成。使用解剖显微镜计数每个孔中的噬斑数量，或者在有限稀释分析的情况下，简单地将每个孔记为阳性或阴性。该检测程序提供了一种简单、快速且廉价的方法，用于检测大量微量培养板中的抗体形成细胞克隆并进行计数。与标准溶血空斑检测方法相比，灵敏度没有损失。该方法对于有限稀释分析特别有用，有限稀释分析需要检测大量重复培养物中是否存在抗体形成细胞克隆。
