Quantitative Two-Dimensional Ultrastructural Distribution of Rh _o (D) Antigenic Sites on Human Erythrocyte Membranes ^*

Abstract

A method is described for determining the two-dimensional distribution of specific antigens on cell surfaces, and is applied to the D antigen of the Rh antigenic system. Rh-positive human erythrocytes are allowed to react with purified 1251-labeled human anti-Rh0(D) -yglobulin antibodies, and the sensitized cells are then lysed at an air-water interface. The residual cell membranes are spread flat by surface forces, and are picked up on a carbon-strengthened collodion-coated electron microscope grid. The membranes are then stained with ferritinconjugated goat -antibodies directed against human -yglobulins. Only Rh-positive cells sensitized with antiRho(D) antibodies bind the ferritin-conjugated antihuman -y-globulins. The

Specific Leukocyte Receptors for Small Endogenous Hormones. DETECTION BY CELL BINDING TO INSOLUBILIZED HORMONE PREPARATIONS

“…One method involves insolubilization of the hormone, antigen, or hapten, preventing its entrance into the cell and allowing study of the function of the cell membrane or the intact cell when it makes contact with the chemical. Thus either the pharmacologic effect of the insoluble hormone agonist (1)(2)(3), the detection of a receptor to antigen on selected cells of a suspension (4,5), or the distribution of the contact points of an antigen for its receptor on the cell can be studied (6). A second and complementary approach has been to interact the radio-labeled substance with a cell containing the receptor and to determine the influence of [1349][1350][1351][1352][1353][1354][1355][1356][1357][1358][1359][1360][1361] labeled stubstances on the binding property of the labeled eheiical.…”

Ultrastructural mapping of methyldopa and anti-D IgG erythrocyte antigen receptors.

“…1 and which has been described by Voak and Williams (24), may be due to technical limitations of the method or may represent true red cell heterogeneity with respect to IgG binding. This variation in staining may be due to differences associated with in vivo red cell age heterogeneity or may in part be due to antigen mobility (13). Except for EMI and autoradiograph of individual red cells, such cell-to-cell differences would not be detectable with standard serological techniques, in which the IgG bound per cell represents an average value calculated from the quantity of IgG bound to a large number of cells.…”

“…Another potential problem is the unconjugated anti-IgG in the chromatographed immunoferritin conjugate, which would lead to an underestimation of the cell-bound IgG. This source of error was controlled by comparing the ferritin sites with the number expected from the cell-bound radioactivity when ['«I]anti-D IgG was used alone (13). In this previous study the estimate of red cell D-antigen sites with immunoferritin conjugates was in good agreement with the number of D sites determined independently with [1I]anti-D binding over a range of antigen site density from 10,000 to 20,000/red cell.…”

“…The ferritin counting data, as presented in the accompanying tables, indicate the total number of ferritin clusters counted for each experiment. The value of 145 ,um2 was used for the total red cell surface area (13) (Table I). Although the D-negative cell (rr) consistently bound less a-AID IgG (from 4 to 40% less), the difference was significant at the P < 0.01 level in only two of the three experiments.…”

Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes.

“…Microsome sections having one to eight conjugates were classified into eight classes (classes [1][2][3][4][5][6][7][8] according to the number of conjugates. Sections in each class were further classified in the following way according to the number of "unit areas" in which the conjugates were distributed.…”