Distribution and Mobility of the A, D and c Antigens on Human Red Cell Membranes: Studies with a Gold‐labelled Antiglobulin Reagent

Abstract

Studies are presented in which a gold-labelled anti-IgG reagent has been used to map the distribution of A, D and c antigen sites on the human red cell membrane. Red cells were combined with IgG antibodies; ghosts were produced which were then labelled with gold-anti-IgG. D antigen sites were found to be single entities dispersed on the red cell membrane of lysed but otherwise untreated Rh positive cells. Following papain treatment prior to lysis, a clustered distribution was observed. Clustering of D sites was also observed on non-enzyme-treated red cells of the -D- phenotype. Further studies indicated that D and c antigen sites are closely associated on ccDEE red cells. The distribution of A antigen sites was found to be clustered in proportion to the amount of IgG anti-A combined with the cells. Reasons are given for considering such distribution as indirect evidence of mobility of A sites. There was an association between agglutinability of red cells by IgG antibodies and the extent of the clustering of the antigen sites.

Scanning Electron Microscopy Shows that both Antigen Mobility and Membrane Deformation Occur in the Hemagglutination Reaction

“…Distribution of the antigen was found to be random. These observations were in agree ment with previous observations that glutaraldehyde fixation results in retention of an Previous studies of membrane mobility [9] of red cell antigens have labeled ghosts with ferritin [4,11] or gold [7] and examined the reaction with the transmission electron microscope. Freeze-etching procedures have also been used [5].…”

“…The results with the anti-NOVA antiserum, which attaches to the same number of sites as the anti-D serum, were of interest because they show that surface antigen density was not the major factor influencing the aggluti nation reaction [2]. These experiments were consistent with the hypothesis that cluster ing of sites was important for agglutination [7,11,12], no clumping or agglutination of the red cells. Distribution of the antigen was found to be random.…”

“…[1]. Previous works have shown the D antigen to be present on about 10,000-25,000 sites [4,7] while the EM antigen was present on about 500,000 sites [3]. The secondary antiserum was either goat anti-rabbit or goat anti-human con jugated to alkaline phosphatase.…”

“…Though its endpoint, the aggregation of antibodycoated red cells, is easily visualized, the bio physical events are complex [10], Although antigenic mobility has been studied in detail by a number of investigators using techniques such as immunoelectron microscopy [4,12] and varying physiochemical methods of manipulating the agglutination reaction [7], these approaches have not satisfactorily de scribed these events, since in part they yield a two-dimensional picture of a three-dimen sional process. Several developments have made it possible to study the cell surface re action by scanning electron microscopy.…”

A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress

“…The main limitation of such techniques is usually the inability to combine particulate immunochemical marking with good preservation of antigenicity and ultrastructural morphology. Gold probes measuring 5-20 nm have been used to label ultrathin frozen sections in investigations involving postembedding electron microscopic immunocytochemistry (Newman et al 1983) and in studies of cell surface proteins (Romano et al 1975). However, with pre-embedding techniques, the relatively large size of the gold probes has made it difficult to visualize intracellular antigens and at the same time to achieve reasonable morphological preservation.…”