Environmental health 2000.

Quantification of oestradiol binding at the surface of human lymphocytes by flow cytofluorimetry

Steroid binding to human lymphocytes was previously detected by a fluorescence assay (Tubiana et al., 1984). In the present study fluorescence assays were carried out with a macromolecular complex constituted by covalently binding steroid to bovine serum albumin (BSA). In this way it was possible to eliminate transmembrane diffusion and confine the study to that of external binding only. The percentage of positive cells observed microscopically was recorded. Based on the findings, it can be safely assumed not only that binding takes place on the plasma membrane but also that this binding is saturable, rapid and stereospecific. It was also observed that binding was partially temperature dependent and that it could be inhibited by proteolysis and reversed by incubation for 18 h in culture medium. Binding was demonstrated on healthy and diseased donor B lymphocytes (Tubiana et al., 1983) as well on B cell lines, but not on T lymphocytes. Calibrated flow cytometry has two great advantages. The first is that the intensity of the fluorescence on the labelled cells can be compared to the same calibrated standard throughout the experiment. The second is that it allows Scatchard analysis by plotting the results obtained with decreasing amounts of the fluorescent compound.
The F:P ratio of the compound determined after correction for quenchling was 1. Before use the compound was centrifuged for 50 min at 100,000g.
Calibration was achieved with an Epics V cell sorter and the relationship between the number of fluorochrome molecules and the intensity of fluorescence was established, as already described (Le Bouteiller et al., 1983), using aminoethyl-Sephadex G-25 beads labelled with different amounts of FITC as microfluorometric standards. The lower threshold of detection for this method is 3,400 fluorochrome molecules per bead (Le Bouteiller et al., 1983). Each test sample contained 10,000 viable cells; dead cells, debris and clumps of cells were eliminated by light scatter gating.
The fluorescence curves encompassed channels 0-255. At gain 50, cells included in channels 17-255 were considered positive. Five different intervals (channel numbers) of fluorescence intensity corresponding to increased levels of bound FITC molecules were slected as follows: 17-26, 27-84, 85 Figure 1 shows the difference in the percent of positive staining and in the intensity of fluorescence between ER' and ERcells.
Interesting data were obtained with increasing concentrations of E2-BSA-FITC and Scatchard 256 256 analysis was performed. Specific binding, (B), for each concentration was determined directly using the Epics V cell sorter. Controls were carried out with the same concentrations of E2-BSA alone. The amount of free steroid (F) was calculated by substracting the amount of compound bound from the total concentration used. To determine the dissociation constant (Kd) and the number of specific sites per cell, the concentration of bound steroid was plotted versus bound/free (B/F) ratio. A linear regression analysis was performed on chosen points of the Scatchard plot and the characteristics of the binding are calculated as described (Faden et al., 1976).
As shown in Figure 2, the binding data obtained with Raji cells, were best fitted by two straight lines. This would suggest that E2-BSA-FITC bound to two classes of receptor sites with different affinities. Kd was calculated from the slope of the lines. For component A, it was 6.25 x 10-9 with a correlation coefficient of -0.92. For component B, it was 2 x 10-7 with a correlation coefficient of 0.92. The total number of binding sites per cell was read as the intersection of the high affinity component with the X axis and was 120,000 per cell. To quantify each binding component, the contribution to total binding of one component must be substracted from that of the other. Kd calculated by this method was 1.72 x 10-9. The same findings were obtained with peripheral

ER-cells with Kd =0
.4 x 10-9 for specific binding and Kd =10-for non specific binding. For ER+ cells, only non specific binding was detected. Flow cytofluorimetry confirms the binding of E2-BSA-FITC on. human lymphocytes previously reported with fluorescence microscopy. This binding is different for T and B lymphocytes.
The experiments described herein also provide valuable insight into the extent of this binding and its biochemical characteristics. In particular, the dissociation constant and number of binding molecules were determined. As in epithelial cells (Berthoix, 1983) two binding components with different affinities were found.
As shown by these findings, flow cytofluorimetry is not only more sensitive than microscopy but also enabled simple and reliable quantification of estradiol binding by fluorescence assay.