Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands. An electron microscopic observation using lectins and monoclonal antibodies

Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B4 staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of monoclonal antibodies that recognize antigens associated with human cervical carcinoma

Six monoclonal antibodies, generated by immunization of mice with human cervical carcinoma cells maintained in tissue culture or with cells from fresh tumor tissue, reacted specifically with the malignant cells in 71% to 90% of the tumor tissue imprints and cervical smears containing neoplastic cells but not with normal cervical epithelial cells in smears from 21 to 23 healthy donors. Antibody CE 402 bound to epithelial cells associated with regeneration in 2 of the 23 normal smears tested. Considerable heterogeneity of antibody binding by malignant cells was observed. Antibody CE 400 was the most reactive, binding to more than 50% of the tumor cells in all reactive specimens. Five of these monoclonal antibodies detected protein antigens in the 80 K to 110 K molecular weight range. Our studies demonstrate the feasibility of producing monoclonal antibodies with selected specificity for cervical carcinoma. These antibodies may be of considerable diagnostic value.     

Comparison of human and siamang ABH and MN blood groups using monoclonal antibodies

Comparison of human and siamang ABHIi and MNSs blood groups using monoclonal antibodies and enzyme-modified erythrocytes showed a different organization of ABHIi in the siamang, and a greatly altered expression of the sialoglycoproteins that express MNSs. While several monoclonal antibodies to human MNSs sialoglycoproteins failed to agglutinate siamang erythrocytes, MoAb145, a monoclonal antibody that reacts with the MN sialoglycoprotein, agglutinated siamang erythrocytes to almost the same titer as human red blood cells. These studies suggest the potential usefulness of monoclonal antibodies in seroprimatology.     

Identification of capillaries in sections from skeletal muscle by use of lectins and monoclonal antibodies reacting with histo-blood group ABH antigens

This study was performed to evaluate the application of different lectins and monoclonal antibodies against ABH antigens to detect and characterize carbohydrate structures in capillaries of skeletal muscle from humans and laboratory animals. Blood group specific lectins (Griffonia simplicifolia, Griffonia simplicifolia isolectin B4,Lotus tetragonlobus, Ulex europaeus, andDolichos biflorus) and monoclonal antibodies reacting with histo-blood group carbohydrate antigens belonging to type 1 (Le^a) and type 2 (H, A and Le^y) chains were used as histological markers for capillaries in sections from skeletal muscle. The material consisted of 20 human masseter muscle biopsies from individuals with known blood types: (eight blood group O, nine blood group A, two blood group B, and one blood group AB) and masseter muscles specimens from different laboratory animals (mouse, rat, rabbit, cat, dog, pig, cow, and macaca monkey). Unfixed sections and an avidin alkaline phosphatase method were used to visualize the specific reaction.Ulex lectin stained capillaries in all human biopsies either strongly or moderately. Strong muscle capillary reaction was observed in biopsies from O, B and AB individuals while capillaries from A individuals were only moderately stained.Griffonia simplicifolia marked capillaries in A, B, and AB individuals andGriffonia simplicifolia isolectin B4 stained capillaries in muscle biopsies from B and AB donors.Dolichos biflorus was a weak marker of muscle capillaries from A individuals. Only capillaries from O individuals were stained with the antibody against H type 2. Capillary reaction was not observed with the other antibodies used.Girffonia simplicifolia was an excellent marker for capillaries in mouse muscle whileGriffonia simplicifolia isolectin B4 is recommended for rat muscles. Periodic acid treatment and subsequentLotus tetragonolobus staining is suitable to visualize capillaries in mouse, rat and pig muscle. Using a sensitive histochemical technique for staining with lectins and monoclonal antibodies reacting with blood group related antigens the microvascular density in human skeletal muscle may be estimated. Further, the carbohydrate compounds in the muscle capillaries reflect the individual blood type. A selection of lectins is suitable for demonstration of capillaries in animal skeletal muscle.     

Mouse monoclonal antibodies to swine blood group antigens

Eight mouse hybridomas with haemagglutination capacity to swine blood group antigens were obtained, three of them producing antibodies capable of being used as blood group reagents. Two detected the Ba factor and another the Fa factor. The others gave non-specific and weak reactions or cross-reaction with antigens present in more than one system. We conclude that mouse monoclonal antibodies are also suitable for use in swine as a complement of polyclonal reagents.     

ABH blood group antigens in O-glycans of human glycophorin A

The major O-linked oligosaccharide structures attached to human glycophorin A (GPA) have been extensively characterized previously. Our own recent findings, obtained by immunochemical methods, suggested the presence of blood group A and B determinants in O-glycans of human glycophorin originating from blood group A or B erythrocytes, respectively. Here, we elucidate the structure of O-glycans, isolated from GPA of blood group A, B, and O individuals by reductive beta-elimination, carrying A, B or H blood group epitopes, respectively. Structural studies based on nanoflow electrospray-ionization tandem mass spectrometry and earlier reported data on the carbohydrate moiety of GPA and ABH antigens allowed us to conclude that these blood group epitopes are elongations of the beta-GlcNAc branch attached to C-6 of the reducing GalNAc. The galactose linked to C-3 of the reducing GalNAc carries NeuAcalpha2-3 linked residue. Identified here O-glycans were found in low amounts, their content estimated at about one percent of all GPA O-glycans. These O-glycans with type-2 core, carrying the blood group A, B or H determinants, have not been identified in GPA so far. Our results demonstrate the efficacy of nanoESI MS/MS in detecting minor oligosaccharide components present in a mixture with much more abundant structures.     

The nature of ABH blood group antigens in human gastric secretion

The origin of blood group ABH activity in human gastric content was investigated. Dialyzed and lyophilized samples of ten individual gastric secretions were assayed for ABH antigen under various conditions. The native activity persisted in delipidated residue of the respective secretions, but was completely missing in the lipid extracts of the analyzed samples. The alkaline degradation of the native and delipidated samples led to total loss of blood group activity of the analyzed materials, but no effect on A-active glycosphingolipid was evolved. Purified glycolipid portion of the lipid extract was lacking ABH activity and was shown to have distinct composition. This fraction contained only glyceroglucolipids and neither sphingosine nor other carbohydrates were present. On the basis of blood group activity assays of the native, delipidated, alkaline degraded samples and also on glycolipid analysis it was established that the ABH blood group activity of stomach secretion originated entirely from the glycoprotein portion of these samples.     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Mouse monoclonal antibodies to bovine blood group antigens: additional results

Seven fusions of mouse myeloma cells with spleen cells from mice immunized with bovine red cells yielded 61 clones producing discriminant antibodies out of total of 651 secreting clones. Although antigenic factors of all known bovine blood group systems were present on the donors' cells, the antibodies identified reacted with antigenic factors from only five systems, A, B, F, S and Z. The antibody specificities produced by more than two clones were anti-A1 or -A2 (21 clones), -S (9),- Z(6),-G' (3) and -V1 (3). The absence of clones secreting antibodies to antigens of the other systems, especially the complex C system, remains unexplained. The properties of the antibodies reacting with antigens of the S system (anti-SU", anti-SUU') and of the B system (O-like antibodies) are in accordance with previous interpretations of polyclonal sera and with present knowledge of the genetic map of the B system.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Use of monoclonal antibodies to follow the phylogenic distribution of human renal antigens

Nine human differentiation antigens have been defined by monoclonal antibodies (M. Abs) developed from mice immunized with embryonic kidney cells (mesonephros or metanephros of 7 week-developmental ages). Their spatial and temporal distributions during human kidney organization were previously studied [3]. In this paper we have attempted to follow by immunofluorescence their phylogenic location, from fish to mammals. Six of them recognized the same structures as in humans: proximal convoluted tubules (PCT) (EG9.11, EG19.6, E116.1), glomerular basement membrane (GBM) (EG14.1) and extracellular matrix (EK8.1, EK17.1). However, staining was limited to certain mammals. EK17.1 has been characterized as an anti-fibronectin. These antibodies revealed the same histological structures in the human mesonephros and metanephros. The three other antibodies revealed epitopes appearing earlier in evolution and whose histological distribution varied according to species. These antibodies stained different structures in the mesonephros and metanephros. Thus, the staining particularities observed during human renal ontogenesis were found again in the phylogenetical study.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell- surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.