Demonstration of Ferritin‐Labelled Antibodies Bound to Human Erythrocytes Fixed with Glutaraldehyde¹

Abstract

Summary In this immuno‐electron microscopic study, the capability of the combining antibody and the agglutinability was examined in glutaraldehyde‐fixed erythrocytes. Glutaraldehyde‐fixed human red cells preserved a strong ability to bind specific antibodies as unfixed cells, even though they decrease in agglutinability. As the fixed cells were separated from one another, even when were agglutinating, the localization of antibodies on a cell in the fixed cells was very clear. The number of A antigen sites per red ceil of different phenotypes was estimated individually as each values of single cell but not as mean value of many cells. It was confirmed that the ability of the combining antibody varied greatly from cell to cell in the same blood of umbilical cord.

The Heterogeneity of Erythrocyte Antigen Distribution in Human Normal Phenotypes: an Immunoelectron Microscopy Study

“…Labelling pattern. The surface staining always appeared as a continuous layer of dense reaction, and differed from that obtained by others with ferritin-conjugated reagents (Lee & Feldman, 1964;Matsukura, 1972;Voak & Williams, 1971;Williams& Voak, 1972). In these latter experiments, ferritin molecules are distributed in a discontinuous pattern, suggesting a periodic distribution of antigen sites which lends to quantitative estimations.…”

“…In addition such variations have already been reported by several workers using the immunofluorescence method (Cohen & Zuelzer, 1965;Fischer & Stege, 1967;Poskitt et ul, 1974;Reed, 1964) or sophisticated immunoadherence techniques (Poskitt & Fortwengler, 1974). Some differences in surface labelling were also noted without comment in recent immunoelectron microscopic experiments using ferritin-conjugated antibodies (Voak & Williams, 1971;Matsukura, 1972).…”

“…The present investigations were carried out on cell suspensions which were glutaraldehyde-fixed (g-f} prior to incubation with antibodies. This procedure was chosen because g-f erythrocytes retain their ability to bind specific antibodies but are much less agglutinable (Matsukura, 1972; Reyes et al, 1973). This coupled with the resuspension of cells by pipetting after each sensitization, resulted in the virtual absence of cell clumps and allowed a comparison of the surface staining of separated single cells.…”

“…W e first undertook therefore a study of the cellular distribution of A and A, antigens in various normal A subgroups, as a basis for further investigations of abnormal acquired phenotypes. In previous studies of normal phenotypes devoted to the cellular detection of these antigens, emphasis has been given to quantitative estimations of antigen sites at the cell surface, either by immunoelectron microscopy using ferritin-conjugated reagents (Matsukura, 1972;Voak & Williams, 1971 ;Williams & Voak, 1972) or radio-isotope techniques (Cartron et al, 1974;Economidou et a!, 1967). From these data little is known about the relative distribution among cells of a given sample.…”

Binding patterns of monoclonal anti‐B, anti‐H and anti‐(Leb+ Y) on erythrocytes, imaged in the scanning electron microscope

“…The binding of anti-A antibodies to erythrocytes has been studied previously in immunofluorescence (1, 2), transmission electron microscopy (3)(4)(5)(6) and in scanning electron microscopy (SEM) (7). Both polyclonal (1)(2)(3)(4)(5)(6) and mouse monoclonal anti-A (7) have been investigated. These studies have revealed that amount and composition of the antigen A vary between and within the various subgroups of A, between single cells, and between the convex and concave parts of the cells.…”