Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies

The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms.     

Erythrocyte morphology in genetic defects of the Rh and Kell blood group systems.

Absence of KX antigen and of normal expression of the Kell system antigens is associated with bizarre red blood cell morphology when observed by either light or scanning electron microscopy. Numerous acanthocytes and dacryocytes have been observed in the peripheral blood smear of an apparently healthy individual with McLeod-phenotype blood, in a male patient with type II chronic granulomatous disease who had a shortened 51Cr red blood cell survival time, and in a minor population of the red blood cells of his carrier mother.     

四川地区彝族ABO等8个血型的分子遗传学研究

目的了解四川地区彝族人群ABO、Duffy、Kidd、MNs、Diego、Lutheran、Dombrock、Colton血型的分子遗传背景及其基因频率的分布规律。方法采集四川省凉山彝族自治州布拖市和冕宁县共120名彝族无血缘关系健康者血样,对ABO基因第6、7外显子作测序分析;对Duffy等另外7个血型系统采用聚合酶链式反应-序列特异性引物(PCR-SSP)作基因分型。结果 120例四川凉山彝族无血缘关系标本中,共检测出5种常见ABO等位基因:A101[0.020 8(5/240)]、A102[0.166 6(40/240)]、B101[0.245 8(59/240)]、O01[0.304 2(73/240)]和O02[0.2625(63/240)]。其他7个血型的基因频率依次为M=0.683 3(164/240)、N=0.316 7(76/240)、S=0.091 7(22/240)和s=0.908 3(218/240);Lua=0(0/240)、Lub=1(240/240);Fya=0.945 8(227/240)、Fyb=0.029 2(7/240)、FyES=0.025 0(6/240);Jka=0.525 0(126/240)、Jkb=0.475 0(114/240);Dia=0.016 7(4/240)Dib=0.983 3(236/240);Doa=0.120 8(29/240)、Dob=0.879 2(211/240);Coa=1(240/240)、Cob=0(0/240)(经χ2检验,均符合Hardy-Weinberg平衡)。结论四川凉山地区彝族人的红细胞血型系统具有一定的分子遗传学特征:等位基因M频率>0.6,Dia频率<0.05,这均符合中国南方人的血型分布特点;FyES等位基因存在一定的分布(Jka>Jkb),Doa频率稍微偏高,显示出该民族一定的特异性。     

浙江畲族Kidd、Duffy血型基因型的调查

Kidd、Duffy血型系统是比较重要的血型系统之一,可引起新生儿溶血病、溶血性输血反应 等,其抗原的分布具有种族多态性[1].笔者用基因分型方法对浙江畲族人群的 Kidd、Duffy血型的基因型进行了调查,现报道如下.1 材料与方法1.1 检测对象共90例,男40例,女50例,年龄26～83 岁,皆为3代无血缘关系的健康的浙江省景宁畲族自治县境内的畲族人.     

Prenatal typing of Rh and Kell blood group system antigens: the edge of a watershed

Knowledge of the molecular basis of the blood group systems has enabled the development of assays for blood group genotyping. At this time, polymerase chain reaction (PCR)-based assays validated on fetal material obtained by invasive means (chorionic villus sampling or amniocentesis) are available for all clinically relevant fetal blood groups, However, only Rh typing (D, C, c, E, and e) and K1 genotyping assays are discussed in this review. Importantly, one must remember that results of genotyping assays will not always be concordant with serological typing. Thus, the RhD genotyping assays have to be modified in response to increased understanding of the molecular biology of this blood group system. RhD typing assays should produce negative results when tested on the black RhD-negative RHD alleles, RHDpsi and r's. PCR-based assays can be used to determine paternal zygosity. For RhD zygosity testing, the real-time quantitative PCR approach and the direct detection of the hybrid Rhesus box, which is the result of the deletion of the RHD gene are available. Recently, methods for noninvasive prenatal genotyping have been investigated. The use of fetal cells circulating in the maternal circulation has been explored; however, the scarcity of circulating fetal cells has limited the use of this approach. More promising are the results obtained with RhD typing assays with cell-free fetal DNA, which is present in the maternal circulation in a concentration of 25 genomic equivalents per milliliter of maternal blood in early pregnancy increasing to 100 copies per milliliter in the third trimester, which is cleared from the circulation within a few hours of delivery. The positive predictive value of this approach is virtually 100%, but false-negative results are (infrequently) encountered. Therefore, this assay can at present only be used for screening of RhD-negative women to make the use of antenatal prophylaxis more targeted and hence more cost-effective. For the clinical management of the pregnancies of alloimmunized women, the development of a control for the presence and the amplification of fetal DNA is needed, which is at present only available in male pregnancies. Assays for the genotyping of the other Rh antigens or Kell antigens with cell-free fetal DNA have not yet been described.     

Demonstration of gene dosage effects on antigens in the Duffy, Ss, and Rh systems using an enzyme-linked immunosorbent assay

Using a recently developed enzyme-linked immunosorbent assay, gene dosage effects were demonstrated for the Duffy, Ss, and Rh red blood cell antigen systems. We report a comparison by an antibody-binding assay of the relative amounts of Fya and Fyb antigens on FyaFya, FyaFyb, FybFyb, FyaFyx, and FyxFyx erthrocytes, and of s antigen on cells homozygous and heterozygous for s. The immunosorbent assay also was used to study Rh antigens, and data which had been obtained using radiolabelled antibodies were confirmed.     

Dosage effects in the Duffy, Ss, and Rh systems

EDITOR'S NOTE: In order to permit timely publication of correspondence, the references have not been verified as they have been for articles appearing in TRANSFUSION, and, therefore, the accuracy of cited references in Correspondence is the sole responsibility of the authors.     

Status of the MNSs antigens on human platelets

A sensitive and specific radioimmunoassay was used to determine whether human platelets possess antigens of the MNSs blood group. Mouse monoclonal IgG anti-M and anti-N were purified by Staphylococcus protein A chromatography, labeled with 125I, and incubated with platelet pellets from donors of various MN phenotypes. Human IgG anti-M, -S, and -s were purified by absorption-elution, incubated with platelet pellets from donors of different MNSs phenotypes, washed, and incubated with 125I-labeled mouse monoclonal anti-human IgG. In both assays, the platelet pellets were centrifuged through phthalate ester oils and the radioactivity in the pellets was counted. Dose-response curves and ligand bound per cell indicated no significant difference in the binding of mouse or human anti-M and anti-N to platelets from donors of the MM, MN, or NN phenotype or of human anti-S and anti-s to platelets from donors of the Ss or ss phenotype. Contrary to many previous studies, our data indicate that the MNSs antigens are not expressed on the circulating human platelet. Therefore, antibodies to these antigens probably do not play a role in refractoriness to platelet transfusion.     

Retention of HLA antigens on previously frozen human platelets

Recent technical achievements allow satisfactory cryopreservation of human platelets that, after thawing and washing, maintain functional viability for therapeutic use. We have now demonstrated that such previously frozen platelets retain full HLA antigenic activity in spite of the combination of freezing, thawing, and washing procedures and the presence of the surface-active cryopreservative DMSO. Platelets stored at 4 C without DMSO and previously frozen platelets from the same donors were tested in parallel for quantitative expression of HLA antigens. In all cases, the previously frozen platelets were quantitatively equal or superior to the platelets stored at 4 C with regard to their capacity to specifically reduce the HLA antibody activities of selected typing sera against a panel of antigen-positive lymphocytes.     

Murine monoclonal antibodies associated with Rh17, Rh29, and Rh46 antigens

After immunization with native human red cells and red cells infected with Plasmodium falciparum, mouse splenocytes were fused with a myeloma cell line to obtain hybridomas. Among the antibodies specific for blood group antigens, four antibodies (iB3C4, F12, MR432, and iB4) directed against epitopes related to the Rh antigen were selected for characterization. It is suggested that two monoclonal antibodies (iB3C4 and F12) recognized an epitope associated with the Rh29 antigen. The specificity of the monoclonal antibody iB4 seemed related to the CcEe series of antigens. MR432 did not react with red cells of people who are homozygous for the RN gene, and seemed to identify an epitope associated with the Rh46 antigen (recently numbered).     

Mapping assignment of the Rh and Duffy blood group genes to chromosome 1.

Blood group genes are inherited in a straightforward manner and their products are readily detectable. Because of this they are utilized over a wide area of scientific endeavor and provide excellent markers for use in gene mapping of the human chromosomes. Both the Rh and Duffy blood group genes have now been localized to the number 1 chromosome. Studies of a patient whose red cells exhibit mosaicism for the Rh blood group indicate that loss of the end of the short arm of chromosome number 1 is associated with loss of an Rh gene complex. The Rh gene can, therefore, be assigned with some precision to this region of the chromosome.     

罕见Rh，Kidd血型系统复合抗体引起速发性溶血性输血反应的调查研究

目的 研究罕见Rh,Kidd血型系统复合抗体引起的速发性溶血性输血反应血清学特征,调查其在输血医学中的临床意义.方法 对1例已进行5次9 U红细胞输注后发生溶血性输血反应患者的血样,分别在盐水介质、间接抗球蛋白介质中分析其抗体的特异性;并用血清学与PCR-SSP基因分型法鉴定其Rh与Kidd血型;运用微柱凝胶法(MGT)与抗球蛋白试管法(IAT)进行患者与捐血者相容性试验.结果 患者为RhE抗原阴性,Kidd血型为罕见Jk(a-b-)型,患者体内含有IgM抗E与IgG抗Jkb抗体,患者血清与28个E抗原阴性的捐血者微柱凝胶法交叉配血相合,抗球蛋白试验法只有4个捐血者相合.结论 罕见Rh,Kidd血型系统复合抗体在临床引起速发性溶血性输血反应.MGT法漏检Jkb抗体,对临床有重大意义.     

人Rh(D)血型抗原模拟表位的筛选和鉴定

目的 从噬菌体随机肽库中筛选人Rh(D)血型抗原模拟表位,并对其免疫学活性进行鉴定.方法 以抗Rh(D)单克隆抗体作为固相筛选分子,对噬菌体随机十二肽库进行3轮"吸附-洗脱-扩增"筛选过程,随机挑选35个克隆,经噬菌体ELISA和交叉反应试验,确定阳性克隆,再对这些克隆进行DNA序列测定和抗体竞争抑制试验,以获取人Rh(D)血型抗原模拟表位.然后采用蛋白质免疫印迹法(Western blot)对所获噬菌体进行鉴定和抗原性分析.结果 经过3轮淘洗后,噬菌体得到富集,获得11个阳性克隆.DNA序列测定和竞争抑制实验结果表明,这11个克隆噬菌体所展示的外源多肽中有7个克隆氨基酸序列含有相同的色氨酸(W)、脯氨酸(P)和谷氨酰胺(Q)结构(-WP-Q-),且均具有40%以上的抑制率;其余4个克隆没有共性,抑制率较低,可能为非特异性结合.Western blot分析表明,被选定的噬菌体能被抗Rh(D)血清特异识别,具有类似于Rh(D)蛋白的抗原性.结论 利用抗Rh(D)单克隆抗体筛选噬菌体随机肽库,成功获得含有(-WP-Q-)结构的Rh(D)抗原模拟表位,为进一步探讨Rh(D)新生儿溶血病的发病机制和疫苗的研究奠定基础.     

Molecular basis of two novel high-prevalence antigens in the Kell blood group system, KALT and KTIM

BACKGROUND: The Kell blood group system consists of 25 antigens that result from single-nucleotide polymorphisms. Most polymorphic Kell antigens reside on the N-terminal domain of Kell before the zinc-binding catalytic motif, which is the major site for endothelin-3-converting enzyme activity. Kell antigens are important in transfusion medicine owing to their strong immunogenicity, and the corresponding antibodies are clinically significant. Two probands were studied whose serum samples contained antibodies to different high-prevalence Kell antigens. STUDY DESIGN AND METHODS: Standard hemagglutination methods were used for serologic testing of Proband 1 and Proband 2. DNA was prepared from both probands and family members. The 19 exons and the intron-exon regions of KEL from both probands were amplified by polymerase chain reaction, and the sequences were compared with that of common KEL. The identified substitutions were located on a three-dimensional model of Kell generated based on the crystal structure of neutral endopeptidase, a homolog of Kell. RESULTS: In Proband 1, a homozygous 1988G>A mutation (Arg623Lys) in Exon 17 was present. One sibling of Proband 1 was homozygous for 1988G>A. In Proband 2, a homozygous 1033G>A mutation (Asp305Asn) in Exon 8 was present. Three siblings of Proband 2 were heterozygous for 1033G>A. CONCLUSION: The identified KEL mutations of the two probands are novel and inherited. The antigen absent from the red blood cells (RBCs) of Probands 1 and 2 are named KALT and KTIM, respectively. KALT is unique in that it is the only Kell antigen sensitive to treatment of RBCs by trypsin.     

Comparison of human and chimpanzee Kell blood group systems

Kell antigens on chimpanzee (Pan troglodytes) red cells were determined using specific human alloimmune and murine monoclonal antibodies. After avoidance of interspecies reactions, chimpanzee red cells were found to react with most Kell system antibodies. The chimpanzees had phenotypes similar to those of humans. The main difference was that all of 27 chimpanzee red cell samples tested were of the K:6, -7, phenotype, while in humans most are K:-6, 7. The most common chimpanzee Kell blood group phenotype was K:-1,2,-3,4,5,6,-7,11,12,13,14, 15,18,19,22. Murine monoclonal anti-K2 and -K14 immunoprecipitated a 97-kD protein from chimpanzee red cells and a 93-kD protein from human red cells. Enzymatic deglycosylation yielded proteins of about 79 kD for humans and 77 kD for chimpanzees. Both human and chimpanzee Kell proteins reacted equally well on Western blots with polyclonal rabbit antibody to human Kell protein, which indicated close homology.     

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.     

Human monoclonal antibodies to human blood group antigens Kidd Jka and Jkb

Summary. Three IgM human monoclonal antibodies to Jk^b, and one IgM human monoclonal antibody to Jk^a were produced from the lymphocytes of two immunized donors. Two of the anti-Jk^b monoclonal antibodies (MS-7 and MS-9) are of the IgM(κ) isotype and one (MS-8) is an IgM(λ). The anti-Jk^a monoclonal antibody (MS-15) is of the IgM(κ) isotype. They are all specific for their respective antigens, and give positive agglutinations in saline by the immediate spin technique, even against Jk(a + b +) cells. The heterohybridomas have been shown to be suitable for bulk culture and produce levels of antibody that reach 18 μg/ml in the spent culture supernatant. They offer considerable advantages over currently available reagents in terms of stability, simplicity and speed of use, and will provide a reliable and unlimited supply of what are at the moment rare and unsatisfactory antibodies.