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.

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Whole-body hyperthermia maintains the secondary immune response of specific antitumour immune T cells.

The effect of hyperthermia on the spleen cells of Meth A-hyperimmunized BALB/c mice (Meth A-Im-SPL) was examined. Three kinds of heating were employed: (a) a cell suspension of Meth A-Im-SPL was directly heated for 1 h at 41 degrees C; (b) the whole body of Meth A-hyperimmunized mice was heated in the same manner--the antitumour activity of Meth A-Im-SPL was examined by Winn assay after heating; (c) the whole body of BALB/c mice intradermally inoculated with a mixture of Meth A-Im-SPL and Meth A cells (Winn assay) was heated in the same manner. The results showed that the antitumour activity of Meth A-Im-SPL was not affected by heating in vivo (Experiments B and C), although it was affected in vitro (Experiment A). Furthermore, slight augmentation of the antitumour activity of Meth A-Im-SPL was observed on heating in vivo. This shows the possibility of combination therapy involving adoptive transfer and whole-body hyperthermia.

Animals↗