Monoclonal antibodies against human erythrocyte membrane antigens and the antigens recognized by these antibodies

Six monoclonal antibodies (KOR-E1-E6) were raised against human erythrocyte membrane antigens. Aggregating reactions of normal human erythrocytes with or without enzyme treatment and specific antigen deficient (null type) erythrocytes were used for detection of the antigens. SDS-polyacrylamide gel electrophoresis with Western blotting and immunoperoxidase methods were also used to confirm the results. The antigen recognized by KOR-E1 and KOR-E5, which was sensitive to protease, trypsin, and neuraminidase, and only expressed on human erythrocytes, was identified as Pr1h. The antigen recognized by KOR-E2 and KOR-E6 was identified as the EnaTS portion of glycophorin A, because the antigen was sensitive to protease and trypsin, but resistant to neuraminidase, and was not present on En(a-) erythrocytes. The antigen recognized by KOR-E3 that was protease-, trypsin-, and neuraminidase resistant, and absent on En (a-) erythrocytes, was identified as Wrb antigen. As KOR-E4 reacted with all erythrocytes examined, the antigen it recognizes could not be determined.     

An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.     

The quantification of erythrocyte antigen sites with monoclonal antibodies.

The application of monoclonal antibodies to the quantification of blood group antigen sites on erythrocytes was examined. A second antibody technique using labelled anti-mouse IgG could not be used as it was not possible to predict the binding ratio between this and the monoclonal antibody. A series of monoclonal antibodies (R10, R18, BRIC 13, BRIC 14) to the erythrocyte sialoglycoprotein alpha (syn: glycophorin A) showed the number of antigen sites to be from 0.3 X 10(6) to 1.2 X 10(6) per erythrocyte and supported the conclusion that the Wrb antigen is located on this protein. An antibody with a specificity related to the Rh blood group system (R6A) showed 4.6 - 10.4 X 10(4) binding sites to be present on cells of phenotype cCDEe. On cells of phenotype -D- 1.24 X 10(4) binding sites were present but protease treatment increased the number of available sites to 1.3 X 10(5). An antibody to a Kell-related antigen (BRIC 18) recognized 2.5 - 5.9 X 10(3) sites per erythrocyte on cells of phenotype Kk. However, a similar number also appeared to be present on cells of the McLeod and Ko phenotypes although the affinity for the antigen on these cells was very much reduced. The potential of using monoclonal antibodies for this purpose and the value of this in the study of blood group systems has been demonstrated.     

Analysis of human antibodies to erythrocyte binding antigen 175 of Plasmodium falciparum

Invasion of human erythrocytes by Plasmodium falciparum merozoites is a multistep process. For many strains of the parasite, part of this process requires that the erythrocyte binding antigen 175 (EBA-175) of the merozoite binds to sialic acid residues of glycophorin A on the erythrocyte surface, a receptor-ligand interaction which represents a potential target for inhibition by antibodies. This study characterizes the reactivity of naturally acquired human antibodies with four recombinant proteins representing parts of EBA-175 (region II, regions III to V, and the dimorphic C and F segment region) in populations in which the organism is endemic. Serum immunoglobulin G (IgG) recognizing the recombinant proteins is predominantly of the IgG1 and IgG3 subclasses, and its prevalence increases with age. In a large population study in The Gambia, serum positivity for IgG or IgG1 and IgG3 subclass antibodies to each of the EBA-175 recombinant antigens was not significantly associated with subsequent protection from clinical malaria. However, there was a trend indicating that individuals with high levels of IgG to region II may have some protection.     

Use of horseradish peroxidase-labelled antiglobulin for the colorimetric quantitation of erythrocyte antibodies

Horseradish peroxidase (HRP)-labelled antiglobulin has been used extensively as a histochemical marker. In the method described the stable reaction product formed by using 3,3′-diaminobenzidine (DAB) as the hydrogen donor in the peroxidase reaction was dissolved in a fluoro alcohol, 1,1,1,3,3,3-hexafluoroisopropanol (HPF). Reproducible standardisation curves resulted with HRP. Based on these observations a colorimetric assay procedure was developed to quantitate the amount of antibody coating red cells. This was achieved by treating the coated red blood cells with HRP-labelled rabbit anti-human IgG followed by hypotonic lysis, separating the ghosts for colour development with the DAB reagent, solubilisation in HFP, and reading at 450 nm. Preliminary estimates of the number of anti-D molecules on Rhesus positive red cells were found to approximate the results reported using radioiodinated antibodies. The smallest number of anti-D molecules detected was 350 per red cell. Qualitative studies indicate that this procedure can be applied to other blood group systems.     

Clinical consequences of enzyme deficiencies in the erythrocyte

The anucleate mature erythrocyte also lacks ribosomes and mitochondria and thus cannot synthesize enzymes or derive energy from the Krebs citric acid cycle. Nevertheless, the red blood cell is metabolically active and contains numerous residual enzymes and their products which are essential for its survival and normal functioning. Enzyme deficiencies in the Embden-Myerhoff glycolytic pathway can result in nonspherocytic hemolytic anemia (NSHA), and some are also associated with neuromuscular or neurologic disorders. Glucose-6-phosphate dehydrogenase deficiency in the hexose monophosphate shunt also results in hemolytic anemia, especially following exposure to various drugs. Defects in glutathione synthesis and pyrimidine 5'-nucleotidase deficiency also cause NSHA, as does increased adenosine deaminase activity. Gluthathione synthetase deficiency which is not limited to the red cell also presents as oxoprolinuria with neurologic signs. All red cell enzyme defects appear as single gene errors, in most cases recessive in inheritance, either autosomal of X-linked.     

Clinical features of anti-chromo antibodies associated with anti-centromere antibodies

Anti-chromo antibodies (AChA) are autoantibodies accompanying anti-centromere antibodies (ACA). We determined the frequency and clinical significance of AChA in autoimmune rheumatic diseases. Serum samples from 252 patients with rheumatic diseases were examined by immunoblotting with HeLa nuclear extract and with recombinant N-terminus of 25-kD chromo protein (p25). AChA were detected in 28 (36%) of 77 sera with ACA. AChA were found only in ACA-positive sera. Twenty-two (79%) of 28 recognized a recombinant N-terminal portion of p25, including the chromo domain which is conserved among species. AChA were related to leucopenia, thrombocytopenia, elevated erythrocyte sedimentation rate, and existence of Sjögren*s syndrome (SS). In ACA-positive patients, AChA might be a serologic indicator of systemic sclerosis (SSc), having features of systemic lupus erythematosus and/or SS or diseases other than SSc.     

Clinical relevance of antiprothrombin antibodies

Antiprotrombin antibodies belong to the family of antiphospholipid antibodies (aPLs). The clinical relevance of antiprothrombin antibodies has not been established and it depends on the applied detection method. Antibodies against phosphatidylserine-prothrombin complex (aPS/PT) are closely associated with clinical features of antiphospholipid syndrome (APS) and lupus anticoagulant rather than antibodies against prothrombin alone. The determination of aPS/PT in routine clinical practice should be done in conjunction with other aPLs detection to improve the likelihood of recognising the APS, which would ultimately facilitate the management of the disease.     

Clinical applications of monoclonal antibodies

This review highlights the properties, problems, and potentials of monoclonal antibodies as diagnostic and therapeutic agents. The most extensive experience has been obtained with antibodies specific for functionally distinct subsets of lymphocytes. They have been used to monitor the immunosuppression of organ-graft recipients and to attempt to elucidate the disturbance of immunologie function in a variety of conditions. Current therapeutic applications under trial include immunosuppression to treat organ-graft rejection and to eliminate cells responsible for graft-vs-host disease from the bone marrow. Applications to cancer diagnosis and treatment have been hampered by the difficulty of identifying truly tumor-specific antigens. Successes have, however, been obtained in the location of metastases and in the extracorporeal treatment of autologous marrow to purge it of malignant cells, and, more rarely, with the direct administration of monoclonal antibodies in vivo. The conjugation of cell-type specific monoclonal antibodies with cytotoxic agents should overcome a number of limitations.     

Production of erythrocyte autoantibodies in NZB mice is inhibited by CD4 antibodies.

NZB mice spontaneously develop haemolytic anaemia as the result of production of erythrocyte autoantibodies. The mechanisms leading to breakdown in tolerance to erythrocyte autoantigens are unknown. Antibodies to CD4 have been successfully used to treat several murine models of autoimmune disease. In this study we injected NZB mice with non-depleting CD4 antibodies and were able to prevent and abrogate erythrocyte autoantibody production in young (Coombs' negative) and old (Coombs' positive) mice, respectively. Our data indicate the dependency of autoantibody production on CD4+ T cells. However, withdrawal of anti-CD4 antibodies resulted in the appearance of erythrocyte autoantibodies, showing that under these conditions we were unable to re-establish tolerance to autoantigens on erythrocytes using anti-CD4 treatment.     

Monoclonal antibodies against human erythrocyte membrane antigens and their reactivities with hematopoietic cells

Four monoclonal antibodies against human erythrocyte membrane antigens were established. The antigenic determinants of KOR-E1, E3, E6 were Pr1h antigen, Wrb antigen, and the trypsin sensitive portion of glycophorin A (EnaTS) respectively. The antigen recognized by KOR-E4 could not be determined. The reactivities of these antibodies with normal hematopoietic cells, malignant hematopoietic cell lines (N = 31), and fresh leukemic cells obtained from 128 patients with various types of leukemias were studied. All antibodies reacted only with erythrocytes among peripheral blood cells, and also KOR-E6 reacted only with erythroid cells among bone marrow cells. KOR-E3 had no reactivity with any cell lines examined, and KOR-E1 and KOR-E4 were reactive with some lymphoid cell lines. However, KOR-E6 had specific reactivities with erythroid (HEL, K562), megakaryocytic (CMK-1), multiphenotypic (KOPM-28), and basophilic (KU-812) cell lines. The antigen (glycophorin A) recognized by KOR-E6 was expressed on a small population of mononuclear cells separated from acute lymphoblastic leukemia (3/70), acute myelogenous leukemia (2/12), monosomy 7-myeloproliferative disorder (1/1), juvenile CML (1/1), and transient myeloproliferative disorder with Down's syndrome (4/12), although it could not be determined whether these cells were leukemic cells or not. KOR-E6 was reactive with a large population of leukemic blasts in erythroleukemia (2/2) and acute megakaryoblastic leukemia (3/6). Thus, KOR-E6 appears to be an erythroid marker of leukemic cells.     

Clinical significance of anti-Ro52 (TRIM21) antibodies non-associated with anti-SSA 60kDa antibodies: results of a multicentric study

Ro52 antigen has recently been identified as TRIM21 protein, but the clinical significance of anti-Ro52/TRIM21 antibodies remains controversial. The aim of this multicentric study was to investigate the significance of anti-Ro52 antibodies without anti-SSA/Ro60 antibodies in various connective diseases. Sera were selected by each laboratory using its own method (ELISA, immunodot or Luminex technology), and then performed with ANA Screen BioPlex? reagent (BIO-RAD). Among the 247 screened sera, 155/247 (63%) were confirmed as anti-Ro52 positive and anti-SSA/Ro60 negative. These sera were analyzed for the detection of other antibodies in relation with clinical settings. Isolated anti-Ro52 antibodies were detected in 89/155 (57%) sera. For the remaining sera (66/155), the main antibodies associations were Sm/SmRNP or Chromatin (n=38; 57%), Jo1 (n=17; 26%) and CenpB (n=9; 14%). Clinical data from the 155 patients showed high prevalence in autoimmune diseases (73%) including myositis or dermatomyositis (n=30), lupus (n=23); Sj?gren and/or sicca syndrome (n=27); CREST or Systemic sclerosis (n=11) and autoimmune hepatitis (n=11). We found that pulmonary manifestations were often associated with the presence of anti-Ro52 antibodies (n=34, 22%), in addition with anti-tRNA synthetases, anti-SRP or anti-Ku antibodies (18/34) or isolated in half of cases (16/34). Separate detection of anti-Ro52 antibodies might be useful in related antisynthetase syndrome diagnosis. The presence of anti-Ro52 antibodies should probably precede development of autoimmune disease and must induce sequential follow-up of positive patients, particularly in interstitial lung disease progression.     

脐血瘦素水平与胎儿出生体重的关系

目的探讨脐血瘦素水平与胎儿出生体重的关系.方法应用特异放射免疫分析法测定91例正常分娩或剖宫产的新生儿脐血清瘦素水平.根据新生儿出生体重与胎龄的关系将研究对象分成对照组44例,大于胎龄儿(LGA)组28例和小于胎龄儿(SGA)组19例.结果91例新生儿脐血清瘦素范围为1.8-40.5ng/ml,平均为9.9±7.4.男性为1.8-35.5ng/ml,平均为5.3±5.6.女性为2-42.5ng/ml,平均为15.0±8.0.两性新生儿出生体重、体重指数比较无显著差异.脐血清瘦素男性显著低于女性(P=0.011).LGA组、SGA组脐血清瘦素水平、出生体重、体重指数分别与对照组比较差异均有显著性(P＜0.01).新生儿脐血清瘦素水平与出生体重、体重指数均呈正相关.相关系数分别为r=0.59、r=0.37.结论瘦素是胎儿生长调节系统的重要因子,与胎儿出生大小有密切相关性.     

孕妇外周血中胎儿细胞对3种抗体表达的研究

目的：利用流式细胞仪研究孕妇血中胎儿细胞对三种抗体表达的情况。希望能更准确的标记孕妇血中的胎儿细胞。以助提高胎儿细胞的分离纯度。方法：用CD71、CD45、GPA（糖蛋白A）分别标记抗凝的22例孕妇血、28例脐血、30例正常未孕妇女血各100ul。在流式细胞仪上检测。同时对已标记2种抗体的2例20ml母血在流式细胞仪上进行胎儿细胞分离,并经PCR技术鉴定男性SRY基因。结果：1、正常未孕妇女血中几乎无CD71 细胞存在。2、在脐血和孕妇血中CD71 细胞主要有两群。高表达群和低表达群。有核红细胞（表达CD71 GPA /CD45-）主要在高表达群中,脐血是58.73%,孕妇血36.43%。3、无论是高表达群 低表达群,CD45 细胞占多数。在脐血中低表达群是90.86%,高表达群占22.26%。孕妇血CD71 低表达群中CD45 细胞占82.11%。高表达群CD45 细胞占79.29%。4、利用流式细胞仪分离已标记三种抗体的母血中胎儿细胞,经PCR技术鉴定分离后的标本中存在男性SRY基因的胎儿细胞。结论：1、正常未孕妇女血中几乎不存在有核红细胞。2=在脐血和孕妇血中有核红细胞主要有两群。高表达群和低表达群。有核红细胞主要在高表达群中。3、无论高表达群或低表达群。CD45 细胞（增殖的白细胞）仍占多数,说明依然缺乏识别胎儿细胞的特异性标记物。主张多参数标记胎儿细胞有利于提高分防细胞纯度。     

Molecular mechanisms resulting in pathogenic anti-mouse erythrocyte antibodies in New Zealand black mice.

The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9- and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgG1 and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss- or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None of the eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5- B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five of the eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) of the IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.     

Naturally acquired and vaccine-elicited antibodies block erythrocyte cytoadherence of the Plasmodium vivax Duffy binding protein

Malaria merozoites require the presence of specific surface receptors on the red blood cell for invasion. Plasmodium vivax, requires the Duffy blood group antigen as an obligate receptor for invasion. The parasite Duffy binding protein (DBP) is the ligand involved in this process, making the DBP a potential vaccine candidate. A preliminary objective was to study whether people exposed to vivax malaria acquire antibodies that have the ability to block erythrocyte cytoadherence to the PvDBP. In comparison, we studied the immunogenicity of various recombinant DBP vaccines and investigated their potential to induct antifunctional antibodies. In order to do so, recombinant proteins to different regions of the putative ectodomain of the DBP and a DNA vaccine were used to immunize laboratory animals. An in vitro cytoadherence assay was used to investigate the presence of antifunctional antibodies in plasmas from people naturally exposed to vivax malaria, as well as in antisera obtained by animal vaccination. Our results showed that human plasma from populations naturally exposed to vivax malaria, as well as antisera obtained by vaccination using recombinant proteins, a DNA vaccine, and a synthetic peptide to DBP, inhibited in vitro binding of human erythrocytes to the DBP ligand domain (DBP(II)) in correlation to their previously measured antibody titer. Our results provide further evidence for the vaccine potential of this essential parasite adhesion molecule.