Cerrone M C, Kuhn R E
Department of Biology, Wake Forest University, Winston-Salem, NC 27109.
J Immunol Methods. 1991 Apr 8;138(1):65-75. doi: 10.1016/0022-1759(91)90065-n.
Limiting dilution analysis has been a valuable approach for both determining the frequency of cell subpopulations elicited during immune responses, as well as for the analysis of immunoregulatory circuits. We describe a simple, visually scored spot test for evaluating the response of Mishell-Dutton microcultures used in limiting dilution analysis. This spot test is based on a microELISA using immunoreagents conjugated to the enzyme, urease, as an alternative to the hemolytic spot test. The assay as performed in Terasaki trays requires minute quantities (less than 10 microliters) of culture supernatant, yet the ELISA yields a distinct color difference between tray wells containing culture supernatants derived from responding (purple) and nonresponding (yellow) microcultures. Although designed to be scored rapidly by visual inspection, the assay can be quantified by manual alignment of the Terasaki tray wells on commercially available ELISA plate readers with an accuracy and reproducibility comparable to assays performed in 96-well ELISA plates. Determination of anti-sheep RBC responses in limiting dilution Mishell-Dutton microcultures with both the hemolytic spot test and the urease-microELISA spot test showed a very close correlation between the results of the two assays. However, the urease-microELISA should be amenable for use with antigens not readily conjugated to an indicator RBC, and should be useful in those situations where determination of the antibody subclass(es) produced by responding microcultures is desired.
有限稀释分析一直是一种有价值的方法，可用于确定免疫反应过程中引发的细胞亚群频率，以及分析免疫调节回路。我们描述了一种简单的、通过视觉评分的斑点试验，用于评估有限稀释分析中使用的米谢尔-达顿微量培养物的反应。这种斑点试验基于一种微量酶联免疫吸附测定（ELISA），使用与脲酶结合的免疫试剂替代溶血斑点试验。在寺崎培养板中进行的该测定法需要微量（少于10微升）的培养上清液，但ELISA在含有来自反应性（紫色）和非反应性（黄色）微量培养物的培养上清液的培养板孔之间产生明显的颜色差异。尽管设计用于通过目视检查快速评分，但该测定法可通过将寺崎培养板孔手动对准市售ELISA读板仪进行定量，其准确性和重现性与在96孔ELISA板中进行的测定相当。用溶血斑点试验和脲酶-微量ELISA斑点试验测定有限稀释米谢尔-达顿微量培养物中的抗绵羊红细胞反应，结果表明两种测定法的结果之间具有非常密切的相关性。然而，脲酶-微量ELISA应该适用于与指示性红细胞不易结合的抗原，并且在需要确定反应性微量培养物产生的抗体亚类的情况下应该是有用的。
