Edward J. Victoria

Antigen Site Densities and Ultrastructural Distribution Patterns of Red Cell Rh Antigens

The ultrastructural distribution pattern and antigen site density of the major Rh antigens (C, c, E, e, and D) on red blood cell ghosts sensitized with IgG Rh antibodies were determined using electron microscopy and ferritin conjugated rabbit anti‐human IgG. The ferritin particle distribution pattern of all the Rh antigens studied was random and ranged from relatively isolated monodis‐persed clusters containing less than eight ferritin particles to large localized aggregates or masses of 20 or more ferritin particles. Evidence is presented to indicate that most of the antigen clustering was due to the conjugate during staining of the cell bound IgG on the stroma preparations. The conjugate‐induced antigen clustering was influenced by both the titer of the conjugate and the staining time. Whether a similar degree of antigen mobility exists in the native membrane remains to be determined. Estimates of the number of cell bound IgG molecules (number of antigenic determinants) based on ferritin particle scoring of IgG Rh antibody sensitized red blood cells under nonsaturating conditions were in the range of 20,000 to 32,000 for each of the Rh antigens. These findings are consistent with the view that the Rh antigen complex is associated with a single membrane component.

Synthesis of a cyclic-thioether peptide which binds anti-cardiolipin antibodies

Abstract Thioether cyclized peptide analogue, 1 , was synthesized using solid phase FMOC chemistry on HMPB-MBHA resin. α-Methylproline was introduced as an FMOC-alanyl-α-methylproline dipeptide, and the thioether was incorporated as FMOC-protected thioether tetrapeptide intermediate, 8 . Compound 1 has been shown to bind anti-cardiolipin antibodies.

In vivo characterization of bioconjugate B cell toleragens with specificity for autoantibodies in antiphospholipid syndrome.

This study investigated the use of well-defined bioconjugate molecules to suppress antigen-specific B cell responses to domain I (DI) of human beta(2)-glycoprotein I (beta(2)GPI) in rats. DI is the dominant target of pathogenic autoimmune antibodies in patients with antiphospholipid syndrome (APS), a disease characterized by antibody-mediated thromboembolic events. Rats primed with DI conjugated to keyhole limpet hemocyanin (DI-KLH) were rendered tolerant to subsequent antigen challenge by treatment with multivalent conjugates of DI. Antibodies to DI were suppressed 89-96% with intravenous doses of 500 micro g, and reductions were paralleled by decreases in splenic antigen-specific antibody-forming cells (AFC). Suppression was achieved with a variety of conjugates having two to four copies of DI and circulating half-lives of 2.6-8.7 h. Antibodies to KLH were not suppressed, indicating the specificity of the approach. These results establish the basis for further development of therapeutic B cell toleragens to suppress pathogenic antibodies in APS and other autoimmune diseases.

Immunoreactivity of the Rho(D) antigen in cytoskeleton‐free vesicles

Skeleton‐free microvesicles derived from D‐positive red cells immunospecifically bind anti‐D. Anti‐D saturation binding yielded an equilibrium constant value (K = 1–2 × 108 I mole−1) similar to intact red cells. The vesicles contained band 3, the major sialoglycoproteins and, at high protein loads, low‐molecular‐weight polypeptides. Anti‐D binding was referenced to total protein, phospholipid, and band 3 content to determine whether there was nonrandom segregation of the D antigen into vesicles. Selective segregation of the D antigen into vesicles could not be demonstrated unequivocally based on the methods and the assumptions of this study. The results, however, indicate that D reactivity does not require a membrane skeletal association.

Rh antigen immunoreactivity after histidine modification

125I-anti-D IgG and unlabeled blood group allo-antisera in combination with 125I-protein A were employed in assessing antibody binding to red cells (RBC) treated with histidine reagents. The acylating reagent diethylpyrocarbonate (DEP), the alkylating reagent p-bromophenacyl bromide (pBPB) and the photosensitizer dye Rose Bengal (RB) were used under conditions that usually result in the selective modification of histidine in isolated proteins. Progressive apparent inactivation of the D antigen in ghost membranes occurred with increasing DEP concns, which was not demonstrably reversible by hydroxylamine since this reagent itself inactivated the D antigen. Exposure of red cells to 5 mM p BPB resulted in a 50% decrease in binding of 125I-anti-D IgG. Photo-oxidation of RBC in the presence of Rose Bengal apparently inactivated all the major Rh antigens as detected either by labeled anti-D IgG binding, IgG agglutinating serological reagents, or the binding of 125I-labeled protein A following the sensitization of cells with unlabeled antisera. Under conditions of RB treatment, where hemolysis was absent or minimal, 125I anti-D IgG binding decreased to 38-49% of the level seen in controls. Rose Bengal treatment of R1r RBC revealed varying inactivation of all the Rh antigens, i.e. D 15%, C 89%, c 73%, e 54% inactivated, whereas antibody binding activity of the Fya and Fyb antigens present in the same cell was unaffected. Previous reports as well as the pH profile of anti-D binding have implicated the participation of histidine in Rh antigen expression. Our results are consistent with histidine involvement in Rh activity. Whether Rh antigens have essential histidine(s) involved directly in epitope structure, or instead depend on a critical histidine(s) at the lipid-protein interface that modulates antigen expression remains to be determined.

The IgG binding function of the normal red cell plasma membrane: identification of integral polypeptides that bind IgG

Summary. Non‐immune IgG binds to red cell integral membrane proteins obtained by mild alkaline extraction of ghosts. Detergent gel chromatography and electrophoretic analyses of IgG‐membrane protein complexes obtained by nonionic detergent solubilization and affinity binding to protein A indicate that three polypeptides participate in the binding of IgG. These have apparent molecular weights of 90 000, 44 000 and 22 000 and are present in a 1:3:0.9 stoichiometry. Evidence obtained indicates that the major sialoglycoproteins are not involved in this type of binding.

Labeling of Rh antibodies on solid-phase protein A

Rh0(D) antibodies which retain immune specificity after radiolabeling were prepared by a procedure which does not require IgG isolation from serum, requires 10-fold less isotope than conventional techniques and yields antibody solutions of defined composition. The method involves radioiodination of IgG on immobilized protein A, depends on employing human red cells reduced in surface cytophilic IgG, and exploits the inability of goat IgG to interact with Staphylococcus aureus protein A. The technique concentrates IgG by affinity adsorption and should prove useful in preparing radiolabeled alloantibodies from dilute human antisera and for red cell autoantibodies.

Some properties of alkali-extracted red cell ghost membranes

The properties of integral membrane proteins obtained by dilute alkali extraction of red cell ghosts were examined. A variety of conditions promoted the disulfide-mediated aggregation of integral membrane proteins, principally band 3. Procedural modifications which minimized aggregation were the use of EDTA and S-alkylation. Integral membrane proteins were solubilized under non-denaturing conditions using Brij 36T, a lauryl polyoxyethylene ether with an NMR-determined average chain length of 8.2 (oxyethylene) units. Detergent gel filtration revealed a chromatographic shoulder due to aggregated band 3 when membrane proteins were not alkylated. Analyses of the column profile also revealed a discrete peak for sialoglycoproteins and two phosphate peaks, an early one due to phospholipid and a later one not identified, but probably due to phosphoinositide.

Estimation of anti-D IgG in red blood cell eluates using the specific radioactivity of 125I-labeled IgG: effect of unlabeled, cytophilic IgG

The specific radioactivity of conventionally prepared 125I IgG anti‐D eluates is significantly less (from 1/5 to 1/20) than that of the 125I IgG fraction used to prepare the eluate. This discrepancy is due to the release of unlabeled, cytophilic IgG from normal red blood cells during eluate preparation and does not represent an underestimation of the eluate anti‐D IgG content. Cytophilic IgG content of eluates plays an important role in reducing the nonimmunologic binding of labeled antibody IgG. The results justify the assumption used in numerous studies that the specific radioactivity of 125I IgG fractions can be used to provide a valid estimate of the anti‐D IgG content of eluates.

Effects on blood group antigens from storage at low ionic strength in the presence of neomycin.

Abstract. Red blood cells (RBC) stored without plasma in a neomycin, low ionic strength medium at 4°C in excess of 24 h show alterations in antigen reactivity. There is a loss of protease‐sensitive RBC antigens and a protease‐type increased IgG saline agglutinability of Rh antigens that is associated with increased binding of 125I anti‐D. Both the serological findings and the alteration in RBC membrane polypeptides are consistent with protease modification of the membrane due to contamination of the RBC by leukocytes. Neomycin, low ionic strength or leukocytes alone or in dual combination do not produce the observed changes in antigen reactivity. The role of neomycin and low ionic strength in this phenomenon and implication for quality control of reagent RBC used for antibody detection and identification are discussed.