Studies of Bovine Blood Cell Surfaces

Citation

Rosenberg, Suzanne Thelma Ostrand (1975) Studies of Bovine Blood Cell Surfaces. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/SCPF-E841. https://resolver.caltech.edu/CaltechETD:etd-09222004-140527

Abstract

The cell surface expression of genetically defined bovine red cell antigens has been studied by electron microscopic and serological techniques. Electron microscopic cell surface localization of specific antigens on bovine red cells has been achieved with the use of an indirect labeling reagent: hemocyanin glutaraldehyde coupled to rabbit-antibovineimmunoglobulin (Hcy-RABI). When the specific antigenic sites on the cell surface are combined with their corresponding antibodies, the secondary application of Hcy-RABI serves to visualize these sites for electron microscopy. Serological dosage reagents for the Z antigen are known to differentiate Z homozygotes (Z/Z) from heterozygotes (Z/-) in terms of the kinetics of complement mediated hemolysis. In the present study, cells homozygous for the Z antigen and saturated with anti-Z antibody were found to take up approximately twice as much Hcy-RABI as cells heterozygous for Z; cells negative for Z showed only background labeling values. Cells possessing the J antigen, a soluble serum substance secondarily absorbed to the red cell surface, were also examined for their quantitative uptake of hemocyanin. Many intergrades of J positive cells exist, ranging from cells which require large amounts of anti-J antibody for complement mediated lysis to cells which are lysed by minute quantities of specific antibody. The quantity of label taken up by a sampling of cells was found to be inversely related to the amount of antibody necessary to lyse those cells. Sequential double labeling studies were conducted to characterize the cell surface steric configurations of antigens whose genes reside in 1) the same blood group system; 2) different blood group systems; and 3) cis versus trans conformations within a system. In no case was steric hindrance found. This result indicates that each antigenic determinant examined is spacially distant from others; it suggests that the determinants may be coded for by distinct genes, and that the antigens labeled are not a series of determinants on a common backbone macromolecule. Sequential double labeling of one set of antigens gave a value which was twice the sum of the two single label values. This phenomenon was noted only for one particular pair of antigens, and only on cells treated initially with one of the antisera. The increased uptake of the second antibody was highly specific. This observation suggests that new antigenic sites are revealed in the presence of bound antibody directed against another specificity, on cells labeled for this particular pair of antigens. Concanavalin A (Con A) binding experiments on trypsinized and nontrypsinized cells strongly indicate that the Con A receptor and the A antigen are molecular unique cell surface entities. Trypsinization of A positive cells caused increased binding of anti-A, accompanied by clustering of the A antigen sites and of the intramembranous particles seen in freeze-fracture experiments. These phenomena were accompanied by cell agglutination. Using bovine red cell blood typing reagents in a leukocyte microcytotoxicity system, bovine leukocytes were found to have specific surface antigens. In this preliminary study there is no obvious association between leukocyte antigens and red cell antigens of any individual animal. The leukocyte and erythrocyte antigenic systems appear to be distinct from each other.

Thesis Files