Human monoclonal antibodies specific for blood group antigens demonstrate multispecific properties characteristic of natural autoantibodies.

A panel of 72 human monoclonal antibodies with specificities for blood group antigens, A, Rh D, Rh C, Rh c, Rh E, Rh e, Rh G, Jka and Jkb, has been established from the peripheral blood of deliberately immunized donors. Previous work has established that the antibodies are highly specific for their respective blood group antigens, and a number of them are in routine clinical use as blood grouping reagents. This panel was screened for reactivity against six unrelated foreign and autoantigens by ELISA, for rheumatoid factor activity by ELISA and agglutination techniques, and for reactivity with a number of different tissues by immunofluorescence. Binding of the monoclonal antibodies to unrelated exo- and autoantigens was commonly seen amongst the antibodies of the IgM class, and to a lesser degree amongst the IgG class, with reaction patterns similar to those given by natural autoantibodies. Only five of the IgM antibodies failed to demonstrate any unexpected cross-reactivities and these included 1/13 anti-D, 2/7 anti-E, 1/13 anti-c and 1/2 anti-A. We propose that rather than natural autoantibodies representing a distinct population of immunoglobulins, multispecificity (polyspecificity, or polyreactivity) may be a feature of antibodies produced in response to exogenous antigens. The implications of this for the study of autoantibodies are discussed.     

Identification of blood group A and B antigens in human glycophorin

Glycophorin A (GPA), the major sialoglycoprotein of human erythrocyte membranes, was isolated separately from blood group A and B erythrocytes using phenol-water extraction. After purification, performed as gel filtration in the presence of SDS, two glycophorin samples GPA-A and GPA-B were run, in duplicate, in SDS-PAGE and electroblotted onto Immobilon P. After staining with 1) anti-glycophorin antibody and 2) with relevant anti-blood group (A or B) antibody it was shown that the band pattern of the samples in each duplicate was the same. GPA-A and GPA-B samples were also degraded using Carlson degradation (beta-elimination in mild alkaline/strong reducing conditions) and from reaction products the fractions of O-glycans and N-glycans were isolated; they were used in hemagglutination inhibition test. It was shown that both sugar fractions derived from GPA-A did inhibit agglutination of blood group A erythrocytes by anti-A antibody, whereas oligosaccharide fractions derived from GPA-B inhibited agglutination of blood group B erythrocytes by anti-B antibody. These results, obtained using immunochemical methods, confirm the presence of blood group A and B determinants in the carbohydrate moiety of human glycophorin, derived from the blood group A or B erythrocytes, respectively.     

Universal red blood cells--enzymatic conversion of blood group A and B antigens.

Accidental transfusion of ABO-incompatible red blood cells (RBCs) is a leading cause of fatal transfusion reactions. To prevent this and to create a universal blood supply, the idea of converting blood group A and B antigens to H using specific exo-glycosidases capable of removing the immunodominant sugar residues was pioneered by Goldstein and colleagues at the New York Blood Center in the early 1980s. Conversion of group B RBCs to O was initially carried out with alpha-galactosidase extracted from coffee beans. These enzyme-converted O (ECO) RBCs appeared to survive normally in all recipients independent of blood group. The clinical trials moved from small infusions to single RBC units and finally multiple and repeated transfusions. A successful phase II trial utilizing recombinant enzyme was reported by Kruskall and colleagues in 2000. Enzymatic conversion of group A RBCs has lagged behind due to lack of appropriate glycosidases and the more complex nature of A antigens. Identification of novel bacterial glycosidases with improved kinetic properties and specificities for the A and B antigens has greatly advanced the field. Conversion of group A RBCs can be achieved with improved glycosidases and the conversion conditions for both A and B antigens optimized to use more cost-efficient quantities of enzymes and gentler conditions including neutral pH and short incubation times at room temperature. Of the different strategies envisioned to create a universal blood supply, the ECO concept is the only one, for which human clinical trials have been performed. This paper discusses some biochemical and clinical aspects of this developing technology.     

Rh血型抗体的检测及结果分析

目的:调查Rh血型抗体的检出率及其特异性分布特点.分析Rh血型抗体的临床意义及产生规律.方法:采用微柱凝胶抗球蛋白技术筛查和鉴定红细胞血型不规则抗体,对鉴定为Rh血型抗体者,采用单克隆抗-D、抗-C、抗-c、抗-E、抗-e鉴定红细胞Rh血型抗原,以确认抗体的准确性;检测抗体的效价、Ig类型及37℃反应性,以明确其临床意义;询问孕产史、输血史,如果为新生儿检测其母亲血浆中是否有相同特异性的抗体,以分析抗体产生的原因.结果:就诊者54000例,共检出Rh血型抗体47例,检出率为0.087%,其中有妊娠史者27例,有输血史者13例,既有妊娠史又有输血史者1例,抗体来自母体的新生儿6例;抗体的特异性为:抗-E 29例(61.70%)、抗-D 8例(17.02%)、抗-cE5例(10.64%)、抗-c 4例(8.51%)、抗-C 1例(2.13%);47例Rh血型抗体均为IgG或IgG IgM类,37℃均可与具有相应抗原的红细胞反应,抗体效价介于1～4096.结论:被检就诊者Rh血型抗体的检出率低于白种人;在检出的Rh血型抗体中,抗-E占绝对多数,而抗-D的检出率呈逐步减少的趋势;妊娠和输血引起的同种免疫是Rh血型抗体产生的原因,新生儿自母体被动获得的Rh血型抗体是Non-ABO-HDN最主要的致病抗体.     

ABO blood group and related antigens, natural antibodies and transplantation

The current success rate of transplant surgery and immunosuppression has led to a demand for organs that has outstripped the supply. This has required investigation of alternate strategies. Therefore, allotransplantation across the ABO blood group barrier has commenced, and pig-to-human xenotransplantation is under consideration. The first immunological barrier to both these types of transplantation is the prevention of the antibody-mediated rejection. This rejection is a result of natural preformed antibodies circulating in the serum of the recipient binding to either ABO (for allo) or alpha-galactose (alpha-Gal) (for xeno) antigens expressed on the donor tissue. These antibodies recognise antigens that are, in both cases, carbohydrate molecules with the characteristic feature that the nonreducing terminal carbohydrate is either a Gal or N-acetlygalactosamine residue in an alpha1,3 linkage. These epitopes are synthesised by closely related members of a single family of glycosyltransferases. This review discusses the carbohydrate antigens, the enzymes involved in their synthesis and the consequences of natural antibodies binding these antigens.     

Detection of Epstein-Barr virus antigens and antibodies by peroxidase-labeled specific immunoglobulins.

Detection of the Epstein-Barr (EBV) antigens, early antigen (EA), viral capsid antigen (VCA), and nuclear antigen (EBNA) by the indirect immunoperoxidase technique was highly sensitive. Antibody titers to EBNA, EA, and VCA were determined in more than 25 sera of patients with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), or normal persons. A good correlation between the titers of these antigens was obtained by the immunoperoxidase and immunofluorescence methods. The indirect (anti-IgG) immunoperoxidase technique for the detection of EBNA is, in contrast to the indirect immunofluorescence method, highly sensitive. EBNA was associated with the chromosomes in cells arrested in the metaphase with colchicine.     

Monoclonal antibodies to blood group antigens on ruminant red cells

Hybridomas were made between NSI/1-Ag 4-1 mouse myeloma cells and spleen cells from Balb/c mice immunized with red cells from sheep (SRBC), goats (GRBC) and cattle (CRBC). The monoclonal antibodies thus produced were tested against a panel of RBC from 80 sheep, 20 goats and 100 cattle of known blood type. One antibody reacted with all RBC from all 3 species, two with all GRBC and SRBC but not CRBC, one was specific for SRBC and two others for CRBC. In addition two monoclonal antibodies were reactive in the B blood group system and two in the FV system of cattle. IgM and IgG concentration in ascite fluids reached up to 8 mg/ml. It is concluded that the hybridoma technique has considerable potential for the production of blood-typing reagents.     

Monoclonal antibodies specific for Neisseria meningitidis group B polysaccharide and their peptide mimotopes

From five mice immunized with Escherichia coli K1 bacteria, we produced 12 immunoglobulin M hybridomas secreting monoclonal antibodies (MAbs) that bind to Neisseria meningitidis group B (NMGB). The 12 MAbs also bound the capsular polysaccharide (PS) of E. coli K1 [which, like NMGB, is alpha(2-8)-linked polysialic acid (PSA)] and bound to EV36, a nonpathogenic E. coli K-12 strain producing alpha(2-8) PSA. Except for HmenB5, which cross-reacted with N. meningitidis group C, none of the MAbs bound to N. meningitidis groups A, C, and Y. Of the 12 MAbs, 6 were autoantibodies as defined by binding to CHP-134, a neuroblastoma cell line expressing short-chain alpha(2-8) PSA; five of these MAbs killed NMGB in the presence of rabbit complement, and two also killed NMGB with human complement. The other six MAbs, however, were nonautoreactive; all killed NMGB with rabbit complement, and five killed NMGB with human complement. To obtain peptide mimotopes of NMGB PS, four of the nonautoreactive MAbs (HmenB2, HmenB3, HmenB13, and HmenB14) were used to screen two types of phage libraries, one with a linear peptide of 7 amino acids and the other with a circular peptide of 7 amino acids inserted between two linked cysteines. We obtained 86 phage clones that bound to the screening MAb in the absence but not in the presence of E. coli K1 PSA in solution. The clones contained 31 linear and 4 circular mimotopes expressing unique sequences. These mimotopes nonrandomly expressed amino acids and were different from previously described mimotopes for NMGB PS. The new mimotopes may be useful in producing a vaccine(s) capable of eliciting anti-NMGB antibodies not reactive with neuronal tissue.     

The gel test: sensitivity and specificity for unexpected antibodies to blood group antigens

The recently FDA-licensed anti-IgG gel test for pretransfusion antibody detection requires crossover validation before implementation. Six hundred coded samples sent for routine pretransfusion tests were used to compare GEL (ID-MTS, Ortho Diagnostic Systems Inc., Raritan, NJ) with L?w and Messeter's low-ionic-strength saline (LISS). There were 456 GEL-LISS-, 97 GEL+LISS+, 45 GEL-LISS+, and 2 GEL+LISS- tests. The 144 positive tests involved 157 antibodies; 67 of these (cold auto, anti-M, -Le, etc.) were considered harmless with respect to transfusion management. GEL-LISS+ tests included seven samples containing potentially significant antibodies (assumed from specificity): anti-K(4), -Jka, -Fyb, and -S. Two potentially significant antibodies (anti- C and -D) were GEL+LISS-. Sensitivity and specificity for potentially significant antibodies were 92% and 96% for GEL, and 98% and 90% for LISS, respectively. The seven GEL-LISS+ samples associated with potentially significant antibodies were from six patients. Five of these antibodies, all detected in immune-suppressed patients, reacted predominantly as agglutinins in LISS. None of these seven antibodies were detected reliably by polyethylene glycol and LISS-additive tube methods. In light of the immune status of the patients with GEL-LISS+ agglutinins with specificity normally considered potentially significant, and because other valid methods did not detect these antibodies, their clinical importance is questionable. Excluding these questionable antibodies, GEL has the same sensitivity and better specificity than LISS. GEL is a valid method for pretransfusion antibody detection.     

A sensitive and specific ELISA using monoclonal capture antibodies for the detection of HLA antigens in blood stains

A double antibody ELISA technique is described to detect HLA antigens in extracts of blood stains. The assay involves capture of free HLA determinants using an immobilized monoclonal antibody directed against monomorphic regions of HLA class I and HLA class II antigens. The captured antigens are then detected using alloantisera directed against the polymorphic regions of the captured HLA entities. The technique is able to detect specific HLA-A, B, and DR antigens in extracts prepared from blood smears as well as from dried and freshly thawed lymphocytes. The assay may be of potential use in forensic medicine, particularly in instances where extraction of nucleic acids for fingerprinting is not feasible.     

Loss of epithelial blood group antigens A and B in oral premalignant lesions.

Tissue from 40 oral premalignant lesions were investigated for the presence of blood group antigens A and B. The material included 18 leukoplakias, 1 erythroplakia, and 3 lichen planus, all with varying degrees of epithelial dysplasia, and 18 leukoplakias without histological evidence of impending malignancy. Thirty-eight benign keratotic oral muscosal lesions were included as a control group. The antigens were demonstrated by a double layer immunofluorescence technique, and the reactivity was compared, by titration, to the reactivity of adjacent normal epithelium from the same patient. All 22 lesions with dysplsia showed decreased reactivity for blood group antigen. Among the 18 leukoplakias without any signs of impending malignancy 4 cases demonstrated loss of antigen reactivity. None of 38 benign control lesions showed any change in antigen reactivity as compared to normal adjacent epithelium.     

Identification and isolation of Lewis blood group antigens from human saliva using monoclonal antibodies

Solid-phase radioimmunoassay, polyacrylamide gel electrophoresis and thin-layer chromatography were used to compare, identify, and characterize the Lewis antigens from human salivas, using monoclonal antibodies directed to the Lea and Leb determinants. Sialylated Lea glycolipid was detected in saliva from individuals with Le(a+ b+) and Le(a+ b-) phenotypes. Immunoaffinity chromatography of the saliva from individuals with different phenotypes revealed a glycoprotein of molecular weight greater than 200 kD bearing the Lewis antigenic determinants.     

Mouse monoclonal IgE antibodies specific for ragweed pollen antigens

In order to obtain monoclonal IgE antibody specific for the major allergens in ragweed pollen extracts, hybridomas were constructed from spleens of mice immunized with ragweed antigen E (AgE). Two hybridomas were selected for thorough study, both secreting antibodies of the IgE class. Large quantities of IgE antibodies were isolated by affinity purification using Sepharose 4B conjugated with ragweed pollen proteins (Fraction A). Both MAbs were found to bind to a high molecular weight and heterogeneous population of proteins, but not to monomeric AgE as demonstrated by protein blot analysis. It is suspected that both MAbs react with low affinity with AgE determinants and binding could be demonstrated only with aggregated forms of AgE. Although the specific antigens with which they react are unknown, these monoclonal IgE antibodies should be useful reagents, complementing the previously obtained monoclonal anti-dinitrophenyl IgE antibody, for studying various aspects of the mouse and human IgE antibody systems.     

Human humoral antibodies specific for primate C-type viral antigens

Medical Microbiology and Immunology -     

The case for measuring antibodies to specific citrullinated antigens

Anti-citrullinated protein/peptide antibodies (ACPA) are the principal autoantibody system associated with rheumatoid arthritis (RA), with diagnostic sensitivity of 70% and specificity of 95%. Current testing for ACPA uses the anti-cyclic citrullinated peptide assay (anti-CCP) which measures a generalized reactivity with citrulline-containing peptides, thus giving no insight into reactivity to specific RA antigens. Of these, the best characterized are, α-enolase, fibrinogen/fibrin, vimentin, Type 2 collagen and filaggrin, antibodies to each of which are found in approximately 30–60% of RA cases. Given reports of cross-reactivity between citrullinated antigens, we discuss whether or not measuring these specific antibodies could aid: clinical diagnosis, identification of clinical subsets and drug responses, or provide insight into pathogenic mechanisms or etiology of RA.