Chicken A blood groups system antigens: molecular characteristics and lack of expression on lymphocytes

The molecular structure of two antigens (A2 and A4) of the chicken A blood group system was determined by using an A4-specific monoclonal antibody (ISU-cA) and several alloantisera specific for chicken A blood group antigens. Molecules immunoprecipitated from erythrocytes were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under either reducing or non-reducing conditions. Molecules of relative molecular weights 53.0 and 54.5 Kd were identified under reducing conditions for A2 and A4 antigens, respectively, and non-reduced molecules had a relative molecular weight of 44.5 Kd for both antigens. Two-dimensional electrophoresis showed a similar, single, diffuse band near pH 6.5 for each antigen. The data are consistent with a glycosylated molecule with one or more intrachain disulphide bonds. Allelic differences between A2 and A4 antigens seem to be due to an additional moiety on A4 antigen with a net neutral charge. Binding to chicken lymphocytes of antibody specific for A antigens was not detected by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitations of radiolabelled peripheral blood lymphocyte-surface molecules using ISU-cA and A-specific alloantisera also did not detect A blood group antigens. Thus, chicken A blood group antigens are not indicated to be present on lymphocytes.     

Ontogeny and expression of chicken A blood group antigens

Expression of chicken red blood cell (RBC) surface antigens was studied by using a monoclonal antibody (ISU-cA) specific for chicken A blood group antigens. Erythrocytes were examined from embryos of 3-18 days of incubation and from chicks at hatch up to 21 weeks of age. Specific antigens were detected on embryonic RBC surfaces by immunofluorescence as early as 3 days of incubation. Antigenic expression was examined by both haemagglutination and immunofluorescence and found to increase with age from embryos to mature birds. The antigen concentration on the cell surface was found to be affected by genotype; heterozygotes had an intermediate level of antigen between that of the two parental genotypes. These data confirm the co-dominance that is observed with most blood group antigens. Flow cytometric analysis allowed confirmation that the entire erythrocyte population gradually increased in antigenic expression over time, rather than having an antigen-negative subpopulation being replaced by a positive subpopulation.     

Expression of a subclass of human blood group A antigenicity in A+ rabbit jejunum: specific glycosylation of the glycocalyx and a 140 K brush border glycoprotein

A monoclonal antibody (14 A4) raised against human A erythrocytes has been produced. It specifically reacts with a subclass of human blood group A determinants. Whereas all the major secreted and membrane-bound glycoproteins of A+ rabbit jejunum epithelium bear human blood group A-like determinants recognized by anti-A polyclonal serum or a monoclonal antibody with broad specificity (Cl 3.3), expression of the A-subclass recognized by 14 A4 is very restricted. The contents of secretory granules of Paneth cells but not the mucins of goblet cells were labeled by 14 A4. In the enterocytes, glycans recognized by 14 A4 were present in the glycocalyx, on an early expressed 140 K glycoprotein of brush border membranes and also on a glycoconjugate of the basolateral membrane of immature crypt cells. In the jejunal brush border membrane of blood group A secretor humans, only one glycoprotein of molecular weight 140 K bears the A-subclass recognized by 14 A4.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Immunochemical studies on blood groups. The combining site specificities of mouse monoclonal hybridoma anti-A and anti-B

Mouse monoclonal hybridomas, five anti-blood group A, three anti-B, and one anti-AB, produced by various methods of immunization, have been characterized by quantitative precipitin tests and the fine structures of their combining sites have been mapped by oligosaccharide inhibition assays. The combining sites of antibodies of each specificity differed among themselves. Three of the five monoclonals were specific for difucosyl and two for monofucosyl A determinants. All but the anti-AB were strictly specific for blood group A or blood group B erythrocytes; all of the anti-A monoclonals gave essentially equivalent titers in hemagglutination tests with A1 and A2 erythrocytes except for a monoclonal anti-A prepared by immunization with a human gastric cancer cell line. The data provide additional evidence for the heterogeneity of the antibody response to the different antigenic determinants present on blood A and B substances and emphasize the importance of difucosyl determinants which comprise most of the determinants on the water-soluble blood group substances.     

Mapping the antigenic epitope for a monoclonal antibody against lysozyme

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of K?hler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.     

Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.     

Evidence for an IgD homologue on chicken lymphocytes

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.     

An antigen expressed in proliferating cells at late G1-S phase

A monoclonal antibody Pr-28 was prepared, which recognized an antigen present only in proliferating cells. Immunofluorescence analysis of Pr-28 antigen showed that the antigen was localized mainly in perinuclear cytoplasm. Although Pr-28 antibody was produced against a chicken cell antigen, it reacts not only with chicken cells but also other cells of murine origin, such as L-cells and NIH 3T3 cells. The molecular weight (Mr) of the antigen recognized by Pr-28 antibody was 45,000 D as determined by SDS-PAGE run under reducing conditions. The antigen disappeared in NIH 3T3 quiescent cells, reappearing in quiescent cells stimulated by fetal calf serum (FCS). The synthesis of Mr 45,000 protein occurred at late G1 phase, just before DNA synthesis in serum-stimulated quiescent NIH 3T3 cells and ceased in S phase.     

Difference in the ability of blood group-specific lectins and monoclonal antibodies to recognize the ABH antigens in human tissues

Summary Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A,-B and-H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues.Helix asparsa agglutinin (HAA),Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereasDolichos biflorus agglutinin (DBA).Griffonia simplicifolia agglutinin-I (GSA-I),Sophora japonica agglutinin (SJA) andVicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucous-like cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels.Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, whileAnguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed withLotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands

Summary 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. However, difference was also observed between monoclonal antibody and lectin staining, that is, monoclonal anti-A antibody reacted weakly but consistently with granules from blood group A nonsecretors but DBA (HPA) did not; staining with UEA-I was observed in granules from the secretor individuals of any blood groups whereas monoclonal anti-H antibody reacted with granules from blood group O and some A secretor individuals but not from B and AB secretor individuals; GSAI-B₄ reacted uniformly with granules throughout the cells whereas monoclonal anti-B antibody bound to limited number of granules in the same cells. This was confirmed by the double labeling experiments with the lectin and the antibody. These results suggest that the different types of antigens as to the binding ability for monoclonal antibodies and lectins are expressed on different granules in the same cell.     

Coexistence of fast-type and slow-type C-proteins in neonatal chicken breast muscle

Two different C-protein variants which selectively react with either monoclonal anti-fast C-protein antibody (MF-1) or monoclonal anti-slow C-protein antibody (ALD-66) were separated from neonatal chicken pectoralis muscle by hydroxylapatite column chromatography. Myofibrils isolated from the neonatal chicken muscle reacted with both monoclonal antibodies as examined by an indirect immunofluorescence method. These observations strongly indicate that both fast-type and slow-type C-proteins are expressed in the neonatal chicken skeletal muscle. Both of them are intermingled and assembled in the same myofibrils.     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Blood groups in the species survival plan®, European endangered species program,and managed in situ populations of bonobo (Pan paniscus), common chimpanzee (Pan troglodytes), gorilla (Gorilla ssp.), and orangutan (Pongo pygmaeus ssp.)

Blood groups of humans and great apes have long been considered similar, although they are not interchangeable between species. In this study, human monoclonal antibody technology was used to assign human ABO blood groups to whole blood samples from great apes housed in North American and European zoos and in situ managed populations, as a practical means to assist blood transfusion situations for these species. From a subset of each of the species (bonobo, common chimpanzee, gorilla, and orangutans), DNA sequence analysis was performed to determine blood group genotype. Bonobo and common chimpanzee populations were predominantly group A, which concurred with historic literature and was confirmed by genotyping. In agreement with historic literature, a smaller number of the common chimpanzees sampled were group O, although this O blood group was more often present in wild‐origin animals as compared with zoo‐born animals. Gorilla blood groups were inconclusive by monoclonal antibody techniques, and genetic studies were inconsistent with any known human blood group. As the genus and, specifically, the Bornean species, orangutans were identified with all human blood groups, including O, which had not been reported previously. Following this study, it was concluded that blood groups of bonobo, common chimpanzees, and some orangutans can be reliably assessed by human monoclonal antibody technology. However, this technique was not reliable for gorilla or orangutans other than those with blood group A. Even in those species with reliable blood group detection, blood transfusion preparation must include cross‐matching to minimize adverse reactions for the patient. Zoo Biol 30:427–444, 2011. © 2010 Wiley‐Liss, Inc.     

On the clinical usefulness of a few sugar antigens and a galactosyl-transferase

Using human cultured cell lines or lymphocytes, two kinds of murine- and one human-monoclonal antibodies were produced, respectively and their clinical usefulness were investigated, and the possibility of galactosyl-transferase as a new tumor maker was also discussed. (1) A murine monoclonal antibody MSN-1, which was raised against human endometrial cancer cell line and recognized blood type sugar chain Leb, reacted with about 85% of endometrial cancer tissues, indicating that useful clinical information may be obtained by applying MSN-1 to immunohistochemistry and flow cytometry. (2) A new assay system using two murine monoclonal antibodies MA54 and MA61, which were raised against human lung cancer cell line and reacted with mucin sugar residues, revealed 76% positive rate in ovarian cancer patients, especially 82% in mucinous cystadenocarcinoma, indicating the clinical effectiveness as a new tumor maker compensating for the drawbacks of CA-125. (3) Galactosyl-transferase isozyme GT-2 was analyzed by the assay system using a newly produced monoclonal antibody. GT-2 was positive in 74% of ovarian cancers, especially in 89% of meso-nephroid cancer, indicating that GT-2 could be a useful tumor maker in ovarian tumors. (4) Human monoclonal antibody, which recognized "type 1 sugar chain" or iso-paragloboside, reacted about one half of endometrial cancer tissues. The production of human monoclonal antibody may contribute to the cancer imaging and the missile therapy.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

Structural differences around hormonogenic sites on thyroglobulins from different species detected by monoclonal antibodies

Thyroxine remains attached to its synthetic site in thyroglobulin until it is released by proteolysis. Strong homology in the primary sequence surrounding thyroxine-forming residues in thyroglobulins from various species suggests a unique three-dimensional structure at hormonogenic sites. To examine this, two thyroxine-binding mouse anti-(chicken thyroglobulin) monoclonal antibodies, 1A10 and 5F6, were used as probes for this region in an enzyme-linked immunosorbent inhibition assay. The thyroxine content of thyroglobulins had a marked positive influence on the monoclonal antibody binding: when the thyroxine content of human thyroglobulin rose by 6.6-fold, cross-reactivities rose 25-fold for the 1A10 monoclonal antibody and 17.6-fold for the 5F6 monoclonal antibody. However, interspecies comparison of thyroglobulin preparations with similar thyroxine content showed lower than expected cross-reactivities for human, pig and sheep thyroglobulins when compared with chicken thyroglobulin. Only when the thyroxine content of heterologous thyroglobulin preparations was two or three times higher did the cross-reactivities equal or surpass that of chicken thyroglobulin. It is concluded that in thyroglobulin there are structural differences in the different animal species near the thyroxine-forming sites bound by these monoclonal antibodies. The known primary sequence similarity does not seem to result, therefore, in identical three-dimensional structures about this site. These differences may reflect species-specific variations in distant regions brought close as a result of chain folding to form the hormonogenic site, such as those around the donor diiodotyrosine residue or in polysaccharide structures. These monoclonal antibodies provide information about the structure of thyroglobulin, which cannot be obtained from knowledge of the amino acid sequence alone.     

Differing reactions of monoclonal anti-A antibodies with oligosaccharides related to blood group A

Inhibition radioimmunoassays with blood group A-related oligosaccharides have been used to investigate the specificities of six monoclonal anti-A antibodies, three of which had been intentionally generated by immunization of mice with blood group A erythrocytes and A-active blood group substance, and three were incidentally produced following immunization of mice with human tonsil cell membranes or a human colon cancer cell line. By hemagglutination, these antibodies are highly specific for human blood group A erythrocytes. However, they differ from one another in their reaction patterns with mono- and difucosyl A antigen structures and the corresponding afucosyl sequences on Type 1 and Type 2 backbone structures. The six antibodies, together with four previously characterized anti-A monoclonal antibodies (originally raised against the receptor for epidermal growth factor) have been classified into five groups. The first two groups consist of antibodies with broad specificities for A-related structures. There are five antibodies in the first group (TL5, 29.1, A17/3D1, MH2/6D4, and MH1/5D1) reacting to varying degrees with the mono- and difucosyl A antigen structures on either type of backbone sequence. In the second group are two antibodies (A15/3D4 and A15/3D3) which are difficult to inhibit with the oligosaccharides tested, but they reacted best with monofucosyl A structure on either type of backbone. Each of the remaining three antibodies had a distinct and more restricted reaction pattern, with a specificity for the difucosyl A antigen on both types of backbone (antibody EGR/G49) or the Type 1-based mono- and difucosyl A antigen structures (antibody MAS 016c) or the Type 2-based monofucosyl A antigen structure (antibody 455). The reactions of four of the antibodies with N-acetylgalactosamine or with oligosaccharides containing the afucosyl sequence GalNAc alpha 1-3Gal suggest that they may react with certain glycoconjugates with alpha-N-acetylgalactosaminyl termini ("A-like" structures) that are unrelated to the products of the blood group A gene-specified alpha-N-acetylgalactosaminyl-transferase. Knowledge of the differing reactions of these monoclonal antibodies is important for interpreting their reactions with glycoproteins and glycolipids of diverse origins.