Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Two individuals with elliptocytic red cells apparently lack three minor erythrocyte membrane sialoglycoproteins.

We have studied the erythrocytes of two individuals (P. L. and K. W.) who lack the Gerbich (Ge) blood-group antigen. The erythrocytes of P. L. and K. W. were not reactive with two monoclonal antibodies (NBTS/BRIC 4 and NBTS/BRIC 10) which reacted with normal erythrocytes. The membranes of P. L. and K. W. erythrocytes appeared to lack three minor sialoglycoproteins (beta, beta 1 and gamma). These three minor sialoglycoproteins were found to be associated with the cytoskeletons of normal erythrocytes. Approx. 10% of the erythrocytes of P. L. and K. W. were frankly elliptocytic. We suggest that one or more of the minor sialoglycoproteins may play a part in maintaining the discoid shape of the human erythrocyte.     

Evolution of glycophorin A in the hominoid primates studied with monoclonal antibodies, and description of a sialoglycoprotein analogous to human glycophorin B in chimpanzee

Comparison of human and primate erythrocyte membrane sialoglycoproteins showed that common chimpanzee, dwarf chimpanzee, gorilla, orangutan, and gibbon have major periodic acid Schiff-positive proteins resembling human glycophorin A (GPA) monomer and dimer in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Immunoperoxidase staining of Western blots with monoclonal antibodies to human GPA showed that these primate bands express some GPA antigenic determinants. A new sialoglycoprotein analogous to human glycophorin B (GPB) was detected in common chimpanzee. Although human MN blood group phenotype results from an amino acid polymorphism of GPA, Western blots showed that in chimpanzee sialoglycoprotein (GPAch) always expresses the M blood group, whereas chimpanzee sialoglycoprotein (GPBch) expresses either the N blood group or a null phenotype. This result explains the detection of M and MN, but not of N, blood group phenotypes in chimpanzee. GPBch has higher apparent m.w. than human GPB, is present in the erythrocyte membrane in greater quantity than human GPB, and contains trypsin cleavage site(s) and the 10F7 determinant (both found on human GPA but not GPB). Expression of human GPA antigenic determinants was consistent with the phylogeny of the hominoid primates; common and dwarf chimpanzee expressed most of the determinants tested, gorilla and orangutan an intermediate number, and gibbon and siamang the least. Of the GPA antigenic determinants examined, the MN blood group determinants were most consistently expressed during evolution of the hominoid primates. The results suggested that variability in expression of GPA antigenic determinants between species was due to both differences in amino acid sequence and glycosylation.     

Individuals lacking the Gerbich blood-group antigen have alterations in the human erythrocyte membrane sialoglycoproteins beta and gamma.

Membranes from erythrocytes with a new Gerbich (Ge)-negative phenotype (Leach phenotype), as well as those from two other Ge-negative phenotypes, were examined. Whereas cells of the Leach phenotype apparently lack three minor sialoglycoproteins (beta, beta 1 and gamma), the membranes of Ge- Yus- and Ge- Yus+ erythrocytes apparently lack beta- and gamma-sialoglycoproteins but contain additional diffusely migrating components of apparent Mr 30 500-34 500 and 32 500-36 500 respectively. Immunoprecipitation experiments showed that the abnormal components of both Ge- Yus- and Ge- Yus+ erythrocytes reacted with two monoclonal antibodies, BRIC 4 and BRIC 10. These antibodies have been shown to react with sialoglycoproteins beta and beta 1 in normal erythrocytes. Cytoskeletal preparations from Ge- Yus- and Ge- Yus+ erythrocyte membranes contained the abnormal components. In contrast with cells of the Leach phenotype, which are elliptocytic, Ge- Yus- and Ge- Yus+ were of normal shape, despite their apparent lack of beta- and gamma-sialoglycoproteins. It seems likely that the abnormal components in these cells contribute to their normal shape. Ovalocytic erythrocytes were shown to incorporate more radioactivity in the sialoglycoprotein-beta 1 region than normal erythrocytes after labelling by the periodate/NaB3H4 technique. It is suggested that abnormal components in Ge- Yus- and Ge- Yus+ erythrocytes result from chromosomal misalignment with unequal crossing-over at meiosis between the genes giving rise to beta-, beta 1- and gamma-sialoglycoproteins.     

Anti-human red cell monoclonal antibodies produced by macaque-mouse heterohybridomas: their reactivity with human and nonhuman primate erythrocytes.

Eighteen monoclonal antibodies (Mabs) against human red blood cells (RBCs) produced by macaque mouse heterobybridomas gave uniformly positive reactions with all human samples except for some with particular null phenotypes. Based on reactions with latter cells, the human antigenic targets of 11 antibodies could be identified: six were specific for glycophorin-related antigens (Wr(b), En(a), Ge4), and each of the live remaining antibodies showed one of the following specificities: CD55, CD44, CD59, Kell, and Rh proteins. Four Mabs recognized the Vc antigen of the chimpanzee V-A-B-D system. Six macaque Mabs detected polymorphisms in chimpanzee, gorilla, orangutan, and gibbon that did not correspond to any known blood group in these animals, while other Mabs gave monomorphic reactions with ape RBCs. The reagents produced by macaque hybridomas are useful tools not only for human blood grouping tests, but also for tracing the relationships among blood group antigens of man and anthropoid apes.     

Relic of ancient recombinations in gibbon ABO blood group genes deciphered through phylogenetic network analysis

The primate ABO blood group gene encodes a glycosyl transferase (either A or B type), and is known to have large coalescence times among the allelic lineages in human. We determined nucleotide sequences of ca. 2.2 kb of this gene for 23 individuals of three gibbon species (agile gibbon, white-handed gibbon, and siamang), and observed a total of 24 haplotypes. We found relics of five ancient intragenic recombinations, occurred during ca. 2–7 million years ago, through a phylogenetic network analysis. The coalescence time between A and B alleles estimate precede the divergence (ca. 8 MYA) of siamang and common gibbon lineages. This establishes the coexistence of divergent allelic lineages of the ABO blood group gene for a long period in the ancestral gibbon species, and strengthens the non-neutral evolution for this gene.     

抗-D抗体及其应用

Rh血型是仅次于ABO血型系统的人类红细胞抗原系统,至今已发现40多种抗原,但与临床密切相关的是D,C、c、E、e等5种抗原,其中最主要的是D抗原。相应的抗-D抗体无论是在临床输血检测,还是在Rh(D)新生儿溶血病、溶血性输血反应等的防治方面均具有非常重要的意义。传统的抗-D抗体的制备需用人的血清,来源受限。各种抗-D人源性单克隆抗体和基因工程抗体已经成为发展方向。     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Apparent expression of varicella-zoster virus proteins in latency resulting from reactivity of murine and rabbit antibodies with human blood group a determinants in sensory neurons

Analyses of varicella-zoster virus (VZV) protein expression during latency have been discordant, with rare to many positive neurons detected. We show that ascites-derived murine and rabbit antibodies specific for VZV proteins in vitro contain endogenous antibodies that react with human blood type A antigens in neurons. Apparent VZV neuronal staining and blood type A were strongly associated (by a χ2 test, α = 0.0003). Adsorption of ascites-derived monoclonal antibodies or antiserum with type A erythrocytes or the use of in vitro-derived VZV monoclonal antibodies eliminated apparent VZV staining. Animal-derived antibodies must be screened for anti-blood type A reactivity to avoid misidentification of viral proteins in the neurons of the 30 to 40% of individuals who are blood type A.     

Blood group A and B glycosyltransferases synthesize A and B determinants on different acceptor polyglycosyl peptidesin vitro

The glycosylation of polyglycosyl chains from human erythrocytes by human plasma blood group A and B glycosyltransferases was studied in order to clarify why human blood group AB erythrocyte polyglycosyl peptides carry only either A or B determinants [Eur J Biochem (1981) 113:259–65].The blood group A transferase was able to add radioactiveN-acetylgalactosamine from labeled UDP-N-acetylgalactosamine to B-type erythrocytes which had been treated with -galactosidase in order to cleave the B determinant sugar from the erythrocytes. This suggests that the enzymes specified by theA andB genes utilize the same acceptor molecules on erythrocyte membranes. Polyglycosyl peptides isolated from blood group B erythrocytes acted as acceptors for blood group A glycosyltransferase and the generation of hybrid structures containing both A and B determinants was demon-strated. When blood group O polyglycosyl peptides were used as acceptors in the simultaneous presence of both blood group A and B glycosyltransferases, however, the A and B determinant sugars were found in different polyglycosyl peptides. It is suggested that the enzyme-acceptor complex does not dissociate until the final number of determinants has been added.     

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.     

Studies on Miltenberger class III, V, Mv and Mk red cells. I. Sodium-dodecylsulfate polyacrylamide gel electrophoretic investigations

The glycoproteins in erythrocyte membrane from individuals exhibiting the rare alleles at the MNSs blood group locus Miltenberger (Mi-) III, V, Mv and Mk were studied by sodium-dodecylsulfate polyacrylamide gel electrophoretic techniques. The results suggest that the genes Mi-III and -V give rise to the formation of Ss sialoglycoproteins whose electrophoretic mobilities are altered. The Mi-V alteration is additionally associated with a decreased MN glycoprotein content. The allele Mv leads to a decreased Ss glycoprotein content. Data on ordinary Mk and Mk/Mi-III red cells suggest that the gene complex Mk does not give rise to the synthesis of Ss glycoprotein.     

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.     

Identification and immunochemical characterization of the human erythrocyte membrane glycoproteins that carry the Xga antigen.

When the membrane components of Xg(a+) erythrocytes were separated by electrophoresis and immunoblotted with an anti-Xga of human origin, two diffuse bands of approximate Mr 22,000-25,000 and 26,500-29,000 were stained. These reactive components were not evident in membranes from proteinase-treated Xg(a+) erythrocytes. Neuraminidase treatment of erythrocytes before immunoblotting resulted in one diffuse Xga-reactive band, the leading edge of which had a slightly increased mobility corresponding to a decrease in Mr of approx. 1500. These results suggest that the membrane components that carry Xga are sialoglycoproteins. A genetic relationship exists between Xga and the antigen recognized by the monoclonal antibody 12E7. An immunochemical comparison of the structures that carry the Xga and 12E7 antigens demonstrated that they differ in Mr and in the way in which they are modified by neuraminidase. This is in accord with evidence that Xga and 12E7 are the products of two separate structural loci.     

The AB blood group system of cats

Holmes (1950) and Eyquem. Podliachouk & Milot (1962) classified feline erythrocytes into two types according to their reactions with naturally occurring antibodies in cats' plasmas. Eyquem et al. (1962) designated the two antigens, A and B. and this nomenclature has been retained in the present study. The blood group system. AB. was investigated in more detail, both genetically and serologically. Frequencies of 73.3 % A and 26.3 % B were found in a survey of 1895 Brisbane cats and in addition, a new phenotype. AB. was discovered with a low incidence of 0.4 %.The results of the serological testing and limited family information suggested that the AB phenotype is inherited and not due to blood chimaerism. Preliminary genetic studies indicated that the A gene is dominant to the B in the usual situation and hypotheses to explain the occurrence of the AB phenotype are discussed.
The incidence of naturally occurring antibodies was investigated in cats, with 1895 of blood type B having anti-A and only 35 % of type A having anti-B. No subgroups of the A and B antigens were detected and no blood group substances were found in the salivas of 37 cats. There was no evidence of any serological relationship of the feline A and B antigens with the human ABO antigens.     

Anti-human red cell monoclonal antibodies produced by macaque-mouse heterohybridomas.

Eight monoclonal antibodies (Mabs) against human red cells were produced by macaque-mouse heterohybridomas. All Mabs uniformly reacted with all human red blood cells tested, but only some agglutinated the red cells of anthropoid apes occasionally detecting intraspecies polymorphisms. None was reactive with blood of Old and New World monkeys. One of the Mabs recognized the Vc antigen of the chimpanzee V-A-B-D system, the homologue of the human M-N blood group system.     

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.The results suggest that glycolipids of erythrocytes and possibly of other cell types bear the A type 3/4 determinant specific for the A1 subgroup and that A type 3/4 determinants of glycoproteins might be present in both A1 and A2 subgroups on short oligosaccharide chains which are only detectable at the level of the Golgi apparatus.     

Human-human hybridomas secreting antibodies specific to human lung carcinoma

Summary Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium. Editor's Statement Identification of monoclonal antibodies that recognize human lung adenocarcinoma cells with reasonable specificity represents a potentially important development that may prove useful in diagnosis and therapy of neoplastic disease. The selection procedures and methods for propagation of the human-human hybridomas described in this paper also represent some novel approaches that may be of general application. David W. Barnes     

A血型单克隆抗体的抗原结合部位结构多样性研究

应用杂交瘤技术,以Ａ型红细胞,Ａ１血型物质ＭＳＭ（Ａ１）和Ａ－ＲＢＣ＋ＭＳＭ（Ａ１）为免疫原,制备了一组抗人Ａ血型单克隆抗体：Ａ１２１８,Ｂ５７,ＤＥ９２３－Ｇ８,Ｄ２８６－Ｅ１２经Ｔａｋａｔｓｙ微量血细胞凝集试验证明：这组单抗仅能凝集Ａ１,Ａ２及ＡＢ型红细胞,不能凝集Ｂ,Ｏ型红细胞．采用ＥＬＩＳＡ定量抑制试验法,精确测定了它们抗原结合部位的结构,互补于Ａ活性寡糖。Ａ１２１８互补于具有双岩藻糖结构的Ａ活性五糖（Ａ－Ｐｅｎｔａ）;Ｂ５７,ＤＥ９２３－Ｇ８互补于具有单岩藻糖结构的Ａ活性六糖（Ａ－Ｈｅｘａ）;而Ｄ２８６－Ｅ１２则互补于具有单岩藻糖的Ａ活性四糖（Ａ－Ｔｅｔｒａ）．结果表明：血凝特异性相同的抗Ａ单抗,其抗原结合部位的结构可呈现多样性。即Ａ活性寡糖的糖基组成数目和含有岩藻糖数目均可不相同,各种抑制剂对不同单抗的抑制作用强弱也不相同。