Shimizu M, Sekine K, Iwaguchi T
Department of Cancer Therapeutics, Tokyo Metropolitan Institute of Medical Science, Japan.
J Biochem Biophys Methods. 1990 Apr-May;20(4):335-43. doi: 10.1016/0165-022x(90)90095-t.
A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.
本文描述了一种用于检测针对绵羊红细胞（SRBC）的体液免疫的简单流式细胞术方法。将SRBC与来自经SRBC免疫的小鼠的血清、抗SRBC单克隆抗体或用SRBC进行脾细胞体外原代培养获得的上清液一起孵育。通过免疫荧光和流式细胞术评估与SRBC结合的抗体。当将流式细胞术直方图中峰值的通道数作为SRBC荧光强度的指标进行测量时，该数值与抗SRBC的IgM和IgG类别的浓度显著相关。在SRBC免疫血清和抗SRBC单克隆抗体中，将流式细胞术方法与作为经典方法的血凝试验（HA）进行了比较。流式细胞术测定的灵敏度远高于HA。通过流式细胞术发现抗SRBC抗体的最低可检测浓度为3.4 ng/ml。通过流式细胞术而非HA检测SRBC在体外原代培养中的剂量反应，且反应随SRBC剂量增加。使用该方法，在体外培养系统中检测了体外和体内刀豆蛋白A（Con A）诱导的脾细胞对针对SRBC的体液反应的影响。发现体外Con A诱导的淋巴细胞抑制抗SRBC反应（IgM和IgG），而体内Con A诱导的淋巴细胞增强该反应。因此，这种新方法被发现是检测体外原代体液抗体反应的一种好方法，已知该反应具有较低的反应性。
