Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells.     

VH Subgroup Restriction in Human Erythrocyte Antibodies: Studies of Anti-A, Anti-B, and Anti-Duffy Antibodies

IgG from 13 anti-A, 26 anti-B, and 10 anti-Duffy antisera were used to coat human erythrocytes. With antisera specific for each of the three VH subgroups, VHI, VHII, and VHIII, a clear VH subgroup restriction was shown in these antibody preparations. Of the 26 IgG anti-Bcoated cell preparations, 21 were agglutinated exclusively by the anti-VHIII antiserum, and 5 were agglutinated mainly by the anti-VHIII antiserum but showed also some reactions using anti-VHI or anti-VHII antiserum, or both. Similarly, 11 out of the 13 IgG anti-A antibodies belonged to VHI, and only 2 to VHIII. The 6 Igg anti-Fy(a)antibodies were restricted to subgroups VHI and VHII, and of the 4 IgG anti-Fy(b) antibodies, 3 belonged to subgroup VHIII and one to VHII. Additional experiments indicated that IgM anti-A and IgM anti-B antibodies showed the same type of restriction to VH subgroups as the corresponding IgG antibody preparations.     

Biochemical characterization of a monoclonal antibody to the H type-2 antigen: comparison to other ABH antibodies

Monoclonal and polyclonal antibodies to the ABH blood group antigens were tested for their specificity to glycoproteins with ABH activity on immunoblots of solubilized erythrocyte membranes. Immunoblots were stained with monoclonal antibody G10 to the H type-2 carbohydrate structure or with commercially prepared monoclonal and polyclonal antibodies to A, B, and H blood group antigens. G10 antibody specifically stained antigens in the regions that contain the erythrocyte membrane bands 3 and 4.5; the staining was proportional to the expected H content of the erythrocytes (O greater than A2 greater than B greater than A2B greater than A1 greater than A1B). No specific staining was observed with membranes derived from Oh (Bombay) erythrocytes which lack the H type-2 structure. A commercially prepared monoclonal anti-H did not specifically stain erythrocyte membrane antigens. Monoclonal and polyclonal anti-A specifically stained bands from A and AB but not O, B, or Oh erythrocytes (A1 greater than A1B greater than A2 greater than A2B). Polyclonal anti-B serum specifically stained bands from B and AB but not O, A, or Oh erythrocytes (B greater than A2B greater than A1B). However, no specific staining was observed in tests with monoclonal anti-B. Monoclonal antibodies G10 and anti-A and polyclonal anti-A and -B blood typing sera will be useful in the further characterization of the molecular nature of the ABH antigens.     

Monoclonal antibodies to cholera toxin with special reference to cross-reactions with Escherichia coli heat-labile enterotoxin.

Seventy monoclonal antibodies to cholera toxin were prepared and characterized. All were of immunoglobulin G (IgG) isotypes (39 IgG1, 29 IgG2, and 2 IgG3). A total of 61 clones produced antibody directed against the B subunit, and 9 clones produced antibodies with specificity for the cholera toxin A subunit. Among both the anti-B and anti-A antibodies, there were representatives which showed full cross-reactivity with the heat-labile enterotoxin of Escherichia coli (14 clones), others which gave partial cross-reactions (12), and still others (44) which did not cross-react. Although 24 of 25 tested anti-B monoclonal antibodies could neutralize cholera toxin, none of the 9 anti-A clones had any detectable neutralizing ability. Among the anti-B antibodies, those which cross-reacted completely with E. coli heat-labile enterotoxin all had strong cholera toxin-neutralizing capacity, whereas those with lesser or no degree of cross-reactivity varied more in their neutralizing potency. The isolation of monoclonal antibodies that distinguish between enterotoxins of different bacterial origin suggests the possibility of developing immunodiagnostic methods allowing species-specific enterotoxin detection in stools of patients with diarrheal disease.     

Interference in immunoenzymometric assays caused by IgM anti-mouse IgG antibodies

Serum samples from a female patient gave falsely elevated results in nine of ten immunoenzymometric assays (IEMAs) that used mouse monoclonal antibodies specific for human chorionic gonadotropin (n = 8), thyrotropin, or creatine kinase-MB isozyme. In contrast, normal results were obtained in five radioimmunoassays that used either mouse monoclonal antibodies or antisera specific for human chorionic gonadotropin (n = 3) or thyrotropin (n = 2). Incubation of her serum with IgG from different species or with F(ab)'2 from mouse IgG prior to IEMA showed that the interference was markedly inhibited by mouse IgG, indicating an antibody specific for the Fc portion of mouse IgG. The interfering activity was bound to and eluted from a column containing Protein A-Sepharose CL-4B. Fractionation of the eluted protein over another column containing Sephacryl S300 showed the activity was enriched in the first protein peak, which contained predominantly IgM. A model is proposed to explain how IgM anti-mouse IgG antibody selectively interferes in IEMAs that use mouse monoclonal antibodies.     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Enzyme-linked immunosorbent assay for IgG and IgM antibodies against human parvovirus B19: use of monoclonal antibodies and viral antigen propagated in vitro

Enzyme-linked immunosorbent assay (ELISA) systems for serum IgG and IgM antibodies to human parvovirus B19 were established by utilizing anti-B19 monoclonal antibodies (mAbs) and human plasma B19 antigen. The specificities of IgG and IgM ELISA were confirmed by indirect immunofluorescence staining and Western blot immunoassay with panel sera. The series of serum specimens obtained from two B19-infected patients were examined with ELISA. The IgM antibody titers increased quickly after the onset of the symptoms and returned to a negative range after five months. The IgG antibody titers also increased just after the increasing of IgM titers and the elevated levels continued for more than a year. We also established the same ELISA systems by utilizing in vitro propagated B19 antigen and similar results were obtained.     

Diagnosis of toxoplasmosis by joint detection of immunoglobulin A and immunoglobulin M.

An indirect immunofluorescence test with total anti-human immunoglobulin conjugate (IgG,A,M-IIF) can be used for joint detection of immunoglobulin A (IgA) and IgM antibodies, provided serum IgG is previously absorbed with anti-human IgG. To determine the validity of the IgG,A,M-IIF assay with absorbed sera, the results obtained were compared with those obtained by methods routinely used for the detection of acute-phase markers, IgA and IgM IIF and enzyme immunoassay. Accordingly, 114 serum samples were selected from patients showing titers of > or = 1:1,024 by IgG,A,M-IIF. (i) In 90 of the samples, neither IgA nor IgM was detected by any of the methods employed; (ii) the remaining 24 samples showed IgA and/or IgM. In all cases, the IgG,A,M-IIF assay with absorbed sera was positive. These comparative data support the use of IgG,A,M-IIF, performed with absorbed and unabsorbed sera simultaneously, for determining the presence of specific IgG, IgA, and IgM by employing a single technique (IIF), one conjugate (anti-IgG,A,M), and only one sample (with and without previous absorption), thus providing a useful initial tool for the diagnosis of toxoplasmosis.     

Monoclonal antibodies against group- and type-specific lipopolysaccharide antigens of Vibrio cholerae O:1

Hybrid cell lines producing monoclonal antibodies against the O-antigenic determinants of Vibrio cholerae O:1 have been established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay inhibition experiments by using lipopolysaccharides from V. cholerae O:1 strains and type strains of groups O:2 and O:21. The anti-A antibody was of the immunoglobulin M (IgM) class, whereas the anti-B and -C antibodies were IgG3. The antibodies had a good agglutinating capacity when tested against V. cholerae O:1 strains in the slide agglutination test.     

Significant ABO hemolytic disease of the newborn in a group B infant with a group A2 mother

ABO hemolytic disease of the newborn (HDN) occurs almost exclusively in infants of blood group A or B who are born to group O mothers because IgG anti-A or -B occurs more commonly in group O than in group A or B individuals. We report a case in which clinically significant ABO-HDN occurred in a group B neonate from anti-B of a group A2 mother. The IgG anti-B titer was much higher (256) than that found in a group A1 mother/infant control group (相似文献   

Analysis of the hepatobiliary transport of IgA with monoclonal anti-idiotype and anti-allotype antibodies

The processing and fate of mixed immune complexes is influenced by the antibody isotypes present. The hepatobiliary transport of mixed immune complexes containing the mouse IgA myeloma protein J558 and corresponding monoclonal IgG or IgM anti-J558 idiotype or monoclonal IgG anti-mouse IgA allotype antibodies has been studied. The anti-idiotype or anti-allotype antibodies were radiolabeled and injected into mice with or without mouse polymeric IgA (J558). IgG anti-idiotype antibodies to J558 IgA were selectively transported into bile by J558 IgA. This process occurred with a radiolabeled Fab preparation of the IgG anti-idiotype and was inhibitable with IgA of an irrelevant antigenic specificity. Thus, polymeric IgA influenced the fate of IgA-IgG idiotype-anti-idiotype serum immune complexes. A monoclonal anti-idiotype antibody of the IgM isotype (D8-3) was not selectively transported into bile by itself or as an IgA-IgM complex. A monoclonal IgG antibody (CB5-6) to a mouse allotype determinant in the Fc portion of IgA was not selectively transported into bile. This anti-allotype monoclonal antibody inhibited the hepatobiliary transport of 125I-polymeric J558 IgA and therefore appeared to directly or indirectly block the site in the Fc region of IgA recognized by the hepatic receptor.     

Molecular characterization of a monoclonal murine anti-blood group A antibody

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.     

Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections.

Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.     

Protein A vectorized toxins--II. Preparation and "in vitro" cytotoxic effect of protein A-ricin A chain conjugate on antibody coated human tumour cells

Protein A of Staphylococcus aureus was covalently bound to reduced ricin A chain toxin by N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate consisting mainly of one molecule of protein A bound to two molecules of A chains (Mr 107,000) was purified by tandem affinity chromatography on ConA-Sepharose 4B and IgG-Sepharose 4B. The purified protein A-A chain conjugate was able to bind and kill human lymphoma cells coated either with monoclonal mouse IgG2a anti-kappa antibody or with polyclonal rabbit anti-kappa antibody. The cytotoxic activity of protein A-A chain conjugate in conjunction with either mouse or rabbit anti-kappa antibodies was 10 times higher than that of rabbit IgG anti-mouse IgG coupled with A chain on Daudi cells coated with mouse anti-kappa antibody and 100 times higher than that of rabbit anti-kappa antibody coupled with A chain on non-coated Daudi cells. The cytotoxic effect of protein A-A chain conjugate on antibody-coated Daudi cells (9 x 10(-12) M) was comparable with that of ricin toxin on non-coated Daudi cells (2 x 10(-12) M). The results recommend the use of protein A-ricin A chain toxin conjugate as a unique specific toxin for the "in vitro" killing of antibody-coated target cells.     

Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases

The level of serum IgM, IgG and IgA antibodies including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 subclass-specific antibodies to Bacteroides (Porphyromonas) gingivalis fimbriae and to lipopolysaccharide (LPS) were analysed in patients with different forms of periodontal disease (PD) and control subjects by ELISA. Among PD subjects, sera obtained from adult periodontitis (AP), rapidly progressive periodontitis (RPP) and gingivitis contained high titres of fimbriae-specific IgG antibodies (7500-15,000 ELISA units) followed by IgA (90-700 units) and IgM (30-90 units). In contrast, sera from localized juvenile periodontitis (LJP) subjects exhibited much lower titres of fimbriae-specific IgG (89 +/- 11 units), IgA (31 +/- 5 units) and IgM (17 +/- 3 units) antibodies. A similar response pattern was also seen in sera from normal subjects aged 35-41 years who practice normal oral hygiene, while sera of younger adults (aged 18-24) with superior hygiene did not have any antigen-specific antibodies. Analysis of IgG subclass anti-fimbriae responses revealed that the major response was IgG3 followed by IgG1, IgG2 and IgG4 in AP, RPP and gingivitis. Although lower, a similar pattern of IgG subclass titre was seen in LJP and normal subjects aged 35-41 years. When IgA subclass responses were measured in AP and RPP, higher titres of the fimbriae-specific response were noted with IgA1 when compared with IgA2. However, lower but approximately equal levels of fimbriae-specific IgA1 and IgA2 titres were seen in other PD groups. When anti-B. gingivalis LPS-specific responses were measured, the sera of AP patients contained high levels of IgG antibodies (2265 +/- 224 units) followed by IgA (411 +/- 90 units) and IgM (214 +/- 56 units). Further, IgG anti-LPS responses were mainly IgG2 followed by IgG4, IgG3 and IgG1. For IgA subclass responses, higher titres of anti-LPS-specific antibodies were noted in IgA2 subclass over IgA1. These results showed that higher anti-B. gingivalis antibody responses occur in PD when compared with healthy individuals and protein and lipid-carbohydrate antigens of B. gingivalis induce distinct patterns of antigen-specific IgG and IgA subclass responses.     

Severe hemolytic disease of the newborn in a group B African-American infant delivered by a group O mother

Maternal-fetal ABO incompatibility is a common hematological problem affecting the newborn. In general, hemolysis is minimal and the clinical course is relatively benign, rarely causing the escalating levels of hyperbilirubinemia and significant anemia commonly associated with Rh hemolytic disease of the newborn (HDN). The incidence of HDN ranges from one in 150 births to 1:3000 births, depending on the degree of anemia and level of serum bilirubin. The etiology of ABO hemolytic disease of the newborn (ABO-HDN) is complex because anti-A and anti-B antibodies are composed mainly of IgM. Since only IgG antibodies cross the placenta, those pregnant women with high levels of IgG anti-A,B, anti-A, or anti-B with an ABO incompatible fetus will be the ones to give birth to an infant with ABO-HDN. We describe a case of a B/Rh positive term newborn born to an O/Rh negative African-American mother demonstrating aggressive hemolysis and a robust response of the bone marrow. This case was successfully managed with phototherapy and simple RBC transfusion without the need for exchange transfusion.     

The specificity of the cross-reacting antibodies in blood group O sera which produce mixed agglutination

γM cross-reacting antibodies in group O sera produce mixed agglutination with secretor buccal cells, but not with non-secretor cells. The mixed agglutination with sera which also contain immune anti-B is produced only with A buccal cells and B indicator red cells, and not with B buccal cells, and is inhibited by B secretor saliva or galactose, but not by A secretor saliva. Mixed agglutination with sera containing immune anti-A is produced only with B buccal cells and A indicator red cells, and is inhibited by A secretor saliva or 2-acetamido-2-deoxygalactose (N-acetyl-D-galactosamine), but not by B secretor saliva. If two sera, one containing immune anti-A and the other containing immune anti-B, are mixed together in equal volumes, mixed agglutination is no longer produced with either A buccal cells (and B indicator red cells) or B buccal cells (and A indicator red cells). These observations are thought to be most readily explicable by the hypothesis that the combining site on the cross-reacting antibody is smaller than on specific anti-A or anti-B, and that it reacts with that part of the antigenic determinant which is common to both A and B antigens. As a consequence of the restricted size of the combining site, it is suggested that cross-reacting antibody will have a lower affinity for A or B antigens than specific anti-A or anti-B does, and competes unsuccessfully with specific anti-A or anti-B for combination with antigen on buccal cells.     

A、B血型单克隆抗体临床应用的研究

为了探索人血型分型试剂工业化生产的可能性，应用人Ａ、Ｂ血型单克隆抗体，进行了抗体特性及临床血型分型的研究。经血凝特性试验，测得Ｄ２８６－Ｅ１２单克隆抗体仅对Ａ血型特异，６－１－Ｇ１１单克隆抗体仅对Ｂ血型特异。由盐水试管法测得它们的血凝滴度均为１：５１２。以血库常规人抗Ａ、抗Ｂ血清为标准对照，采用盐水试管法，对３７０４例血液样品进行了临床血型分型试验。结果显示，Ａ、Ｂ血型单克隆抗体分型试剂与标准人血清１００％相符。证明：Ｄ２８６－Ｅ１２和６－１－Ｇ１１两株杂交瘤分泌的Ａ、Ｂ血型单克隆抗体可作为非常优越的血型分型试剂。具有工业化生产血型单克隆抗体的潜力。     

Differences in ABO antibody levels among blood donors: a comparison between past and present Japanese, Laotian, and Thai populations

Passively transfused blood group antibodies cause clinical problems. High titers of anti-A and anti-B seem to be one reason for hemolytic transfusion reactions and for ABO HDN. In Japan, anti-A and anti-B titers notably decreased in the 15 years between 1986 and 2001. At present, titers of more than 100,as measured using the saline method, are rare. Differences in the level of anti-A and anti-B among ethnic populations have been reported; these differences were found to be the result of environmental factors rather than hereditary factors. In the present study, the anti-A and anti-B titers of random donors in three Asian populations are compared. In Thailand, the IgM anti-A and anti-B titers are low and are similar to the Japanese titers reported in 2001, but the IgG anti-A and anti-B titers are high and are similar to the Japanese titers reported in 1986. In the Lao People's Democratic Republic, both the IgM and the IgG anti-A and anti-B titers are high and are similar to those reported in Japan in 1986. In addition, anti-A and anti-B titers of different sex donors and of various age groups were also compared. High titers were found in 8.8 percent of the female donors in the younger than 30 age group and in 36.7 percent of the female donors in the older than 50 age group.