Direct Antiglobulin Reaction in ABO-Haemolytic Disease of the Newborn

The minimum number of IgG anti-A (or anti-B) molecules detectable on A or B red cells by the antiglobulin reaction was found to be the same—that is, about 150 molecules per red cell—with newborn as with adult cells. Furthermore, the ratio of anti-IgG bound to IgG anti-A (or anti-B) molecules was the same whether the anti-A (or anti-B) molecules were present on newborn or on adult cells and was similar to that found for anti-IgG bound to IgG anti-Rh.In 15 infants (11 group A, 4 group B) with haemolytic disease of the newborn due to ABO-incompatibility the amount of anti-A or anti-B on the red cells ranged from 0·25 to 3·5 μg antibody per ml red cells, corresponding to 90-1,320 antibody molecules per cell; only five infants had more than 0·55 μg antibody per ml of red cells. These amounts are far smaller than those found in most moderate or severe cases of Rh-haemolytic disease.It is concluded that the weak direct antiglobulin reactions observed in ABO-haemolytic disease are due simply to the fact that the number of anti-A (or anti-B) molecules on the infant''s red cells is at the lower limit of sensitivity of the test. Since ABO-haemolytic disease can be quite a severe process it seems probable that IgG anti-A and anti-B molecules are more effective than anti-Rh molecules in bringing about red cell destruction.     

Blood groups of apes and monkeys. VII. The human-type A-B-H factors in gelada monkeys (Therapithecus gelada)

A series of 21 gelada monkeys (Theropithecus gelada) all showed strong reactivity of their saliva for H substance, but no reactivity for either A or B. Tests on their sera in no case showed the simultaneous presence of both the agglutinins anti-A and anti-B; instead some animals had only anti-A, others had only anti-B, while the remainder had neither anti-A nor anti-B. These findings distinguish gelada monkeys from all other species of Old World monkeys tested to date. They also provide further evidence supporting the genetic independence of the H substance and the A-B-O blood groups.     

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes

Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I:A-active ganglioside II:The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.     

Effect of specific antisera upon Streptococcus mutans adherence to saliva-coated hydroxylapatite

Abstract IgG fractions of antisera against Streptococcus mutans cell-surface protein antigens A and B were used to examine the role of these molecules in adherence to saliva-coated hydroxylapatite. Anti-B antibody inhibited S. mutans adherence by 20–50% depending upon the strain used, while anti-A antibody was without effect. Some IgG-mediated agglutination of cells occurred in the course of these experiments which was overcome by using Fab fragments prepared from the anti-A and anti-B IgG's. Anti-B Fab inhibited S. mutans adherence by 50% but anti-A Fab had no effect. These observations suggest that antigen B is an important factor in the adherence of S. mutans to saliva-coated hydroxylapatite.     

Do Toxocara canis larval antigens used in enzyme-linked immunosorbent assay for visceral larva migrans cross-react with AB isohemagglutinins and give false positive results?

A variety of helminth parasites have A and B blood group antigens on their surface. These antigens may cross-react with elevated concentrations of A and B isohemagglutinins in some patients and give false-positive results in the serologic diagnosis of visceral larva migrans caused by T. canis. To clarify this point, serum from patients with visceral larva migrans and elevated T. canis antibody titers as determined by ELISA were absorbed with AB blood cells and retested by ELISA without a demonstrable decline in T. canis antibody titers. Similarly, absorption with T. canis embryonated egg antigens of serum containing elevated levels of anti-A or anti-B isohemagglutinins failed to decrease the isohemagglutinin titer. This indicates that the ELISA using T. canis embryonated egg antigen does not give false positive results with sera containing high concentrations of anti-A or anti-B isohemagglutinins.     

Acquired B antigen: further studies using synthetic oligosaccharides

The reactivity of acquired-B red cells with various antisera has been investigated by agglutination inhibition assays using four trisaccharides obtained by chemical synthesis. Two of these had the structure GalNAc alpha 1-3 (LFuc alpha 1-2). Gal and Gal alpha 1-3 (LFuc alpha 1-2) Gal which are characteristic of the A and B determinants respectively and were indeed strong inhibitors of human anti-A and -B antibodies. The other two sugars denoted B-OAc and B-NH2 are derivatives of the B-trisaccharide by substitution of the hydroxyl group on carbon-2 of the alpha-galactose residue with the O-acetyl group (B-OAc) or the amino group (B-NH2) respectively. We have shown that both B and B-NH2 trisaccharides inhibited strongly the agglutination of acquired B red cells by the anti-B reagents (crude or affinity purified) whereas sera containing "anti-acquired B" agglutinins were specifically inhibited by B-NH2 but not by the A, B or B-OAc structures. We have also shown that the agglutination of Tk-activated erythrocytes by the BS-II lectin is specifically inhibited by N-acetylglucosamine but not by B, B-OAc or B-NH2 structures. These results and the observation that anti-acquired B agglutinins cannot be adsorbed on Tk red cells suggest that (i) the "B-like" and the "acquired-B" determinants share a common structure best represented by B-NH2 and (ii) Tk and "acquired-B" antigens are not identical.     

An auto-anti-b in an a1b person. Serological studies.

The serum of a patient (Mr. Lat) with the regular blood group A1 B contains an anti-B reacting with all cells having a B antigen except Bx and cis AB. The anti-B reacts at 4 degrees C and occasionally at room temperature as shown by agglutination, absorption-eluction and by thermo-dynamic assays. The antibody is regarded as an irregular autoantibody belonging to the group of the so called "suppressed" or "latent" antibodies.     

Biochemical characterization of the feline AB blood group system

The biochemical nature of the feline AB blood group system was characterized by analysing red blood cells from homozygous (genotype A/A) and heterozygous (A/B) type A, type B (B/B), and type AB cats. High performance thin layer chromatography (HPTLC) of red cell gly-colipids revealed that specific neuraminic acids (NA) on gangliosides, containing ceramide dihexoside (CDH) as a backbone, correlated with the feline AB blood group antigens. Although disialogangliosides predominated, mono- and trisialogangliosides were also isolated. B cats expressed solely N-acetyl-NA (NeuNAc) on these gangliosides. In addition to expressing N-glycolyl-NA (NeuNGc) containing gangliosides, A red cells have gangliosides with only NeuNAc or mixtures of both NA. HPTLC profiles of disialogangliosides from homozygous and heterozygous A cats differed slightly in the quantity of disialogangliosides. Equal amounts of NeuNAc and NeuNGc containing disialogangliosides, as well as two intermediary forms, were recovered from AB erythrocytes. Analysing disialogangliosides from red cells belonging to 17 genetically related cats, we consistently obtained the expected disialoganglio-side profile, based on blood typing and pedigree information. SDS-PAGE of red cell membrane proteins and blotting with Triticum vulgaris, a lectin recognizing NeuNAc, revealed glycoproteins of approximately 51, 53, and 80 kD in B and AB cats but only a faint band of approximately 53 kD in A cats. By haemagglutination, Triticum vulgaris could also distinguish different blood types by specifically binding to B and AB cells. Flow cytometry showed that more anti-B bound to B cells than anti-A bound to A cells. Although AB cells had a broad range of fluorescence when compared to the profiles seen with A and B cells, the mean fluorescence with AB cells was half of that seen with A or B. These results further characterize the antigens determining the feline AB blood group system illustrating differences between A, B and AB cats, and between homozygous and heterozygous A cats.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Maternal IgG anti-A and anti-B titres predict outcome in ABO incompatibility in the neonate

Hemolytic disease of newborn (HDN) is an alloimmune hemolytic disease which occurs due to red blood type incompatibility between mother and fetus. An AB blood type neonate was admitted to Shengjing hospital with severe anemia. Major crossmatch incompatibility was found with some random donors. Serological tests were administered to the neonate and his parents. The mother was B blood type, while the father was AB blood type. The neonate's direct antiglobulin test (DAT) was negative, but the elution test was positive with A₁ cell and negative with A₂ cell. Titers 64 anti-A₁ and 2 anti-A in the mother's serum were detected after treated by dithiothreitol (DTT). The mother's red cells showed a weak agglutination with anti-A under microscopy. The neonate was diagnosed with HDN. After phototherapy and A₂B red blood cell (RBC) transfusion, the neonate was discharged with a recovery of his hemoglobin and physiological index. This study describes a rare case of HDN caused by anti-A1 allo-antibodies.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.     

Galactosialidosis: a direct evidence that a 46-kilodalton protein restores deficient enzyme activities in fibroblasts

The intracellular function of a specific protein to protect lysosomal beta-galactosidase and neuraminidase activities against proteases in human fibroblasts was studied. Beta-Galactosidase was purified from human placenta to different degrees; a preparation (A) contained also two concomitant proteins, and a highly purified preparation (B) contained only the mature beta-galactosidase. The protein concentrate of the culture medium of normal fibroblasts restored the activities of the deficient enzymes, beta-galactosidase and neuraminidase, in galactosialidosis cells. This effect was inhibited only by the anti-A anti-serum, and not by the anti-B antiserum. A 46-kilodalton protein, secreted from fibroblasts cultured in the presence of ammonium chloride, was detected again only by the anti-A antiserum, and not by the anti-B antiserum. It was concluded that this protein has a function to restore their activities in fibroblasts from galactosialidosis patients after being endocytosed from the culture medium.     

The effect of pyruvate on antibody interaction with group-specific erythrocyte antigens

Using the AB0 antibody-antigen model the influence of natural metabolite pyruvate on the antibody interaction with of erythrocyte antigens, defining their group specificity has been investigated. Before agglutination reaction erythrocytes of A (II)–AB (IV) blood groups, monoclonal anti-A and anti-B antibodies were incubated with sodium pyruvate. Visualization of agglutinates was performed by means of flow cytometry and laser scanning confocal microscopy. Computer-aided prediction of the spectrum of biological activity of pyruvate by a PASS program proposed major regulatory pathways, in which pyruvate may be involved. It has been demonstrated that pyruvate can regulate the intensity of antigen-antibody interaction. These results suggest the possibility of using small molecules, for example pyruvate, as molecular probes and prospects of the use of erythrocytes with antigenic determinants of the ABO system expressed on their membranes for studies of protein-protein interactions due to convenient visualization and possibility of quantitative evaluation of this process.     

血型检测用反向定型试剂的研制

将正常的红细胞在特定条件下用甲醛处理,使红细胞膜固定但不影响膜表面糖蛋白血型抗原的活性。采用与正向定型相同的平板凝集试验方法,４０６０份血样正向和反向定型结果完全一致。经稳定性观察９０天,处理后的红细胞与相应抗体的凝集性能未见明显改变。实验结果表明本文介绍的红细胞试剂可用于ＡＢＯ血型鉴定的反向定型试验。     

A new symmetry: A anti-B is anti-(B anti-A), and reverse enhancement

Immune system network theory leads to a new symmetry, namely that the antibodies produced in an allogeneic A anti-B immune response (where A and B are, say, two different mouse strains), should have complementary shapes to the antibodies in a B anti-A response. That is, A anti-B is anti-(B anti-A). This symmetry is due to the existence of two readily separable populations of antibodies that are present in alloantisera: anti-foreign and anti-anti-self antibodies. The theoretical basis for the symmetry is described, and results indicating the presence of anti-anti-self antibodies in each of 12 alloantisera (six made in B10-congenic strains, and six made with the unrelated chains CBA, SJL, and C57BL/6) are reported. The finding that hyperimmune alloantisera routinely contain anti-anti-self antibodies suggests that network regulation plays an important role in maintaining self-tolerance during responses to allogeneic cells. We further show that A anti-B serum absorbed against B can specifically prolong the survival of A grafts in a B strain animal. We suggest that this result can be interpreted as being due to A anti-(B anti-A) antibodies preventing B anti-A cells from rejecting the A grafts. We call this phenomenon "reverse enhancement" because it involves the converse antiserum to that used in conventional enhancement of graft survival by specific antibodies.     

Quantitative and thermodynamic study of B antigen in a family including individuals with phenotypes B 3 and AB 3

Four B3 and one A1B3 erythrocyte samples belonging to the same family were studied using several series of quantitative measurements: percentage of agglutination, agglutination kinetics and thermodynamic methods. For this last assay the erythrocytes were used fresh and after treatment by formalin. From the obtained results evidence was given that while B antigen density was lower in A1B3 than in B3O cells, the reactive structure is qualitatively the same in these two kinds of cells. The enthalpy change of the reaction of this B3 antigen with a non stimulated anti-B from A1O individual was -- 5,000 cal/mole, i.e. weaker than when normal B cells were concerned (-- 16,000 cal/mole).     

Haptoglobin type and isoantibodies (anti-A and anti-B)

In 582 sera of blood donors of the groups A1, A2, B and 0 the Hp type as well as the anti-A or anti-B isoantibody titres respectively were determined. The frequency distribution of isoantibody titres in serum samples with different Hp-type were compared. As far as the numerical difference of distribution was concerned there was only one significant observation in the group of B alpha--titres of test persons with a different Hp-type. On the basis of these findings the following Hp-type/isoantibody relation can be established: low anti-A or anti-B titres respectively (approximately 1:8) will occur more frequently in persons with the Hp-type 1-1, higher titres (approximately 1:64) are to be found predominantly in persons with Hp2 genes. These findings are in accordance with other results, on the basis of which is was suggested that persons with Hp2 gene product have a higher immunogenic reactivity in comparison to type Hp 1-1.     

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.