In vitro production of a hybrid monoclonal antibody that preferentially binds to cells that express both HLA-A2 and HLA-B7

A hybrid mouse monoclonal IgGl having one low affinity combining site for HLA-A2 and one low affinity combining site for HLA-B7 was made by the chemical method of Nisonoff and Palmer (Science 143:376,1964). This involved selective reduction of interchain disulphides, a splitting of the IgGl into half molecules at low pH and ionic strength, and reassociation of the half molecules by neutralization. Serologically active hybrids were separated from parental IgGl by an absorbtion procedure and recovered in about 10% yield. The hybrid discriminated between cells that express either HLA-A2 of HLA-B7 from cells that express both A2 and B7. This is because it could bind bivalently to the cell with both A2 and B7 but could only bind with a single combining site to cells expressing A2 or B7. The consequence of these different modes of attachment was to give up to sevenfold greater binding to the cell expressing A2 and B7 in comparison to the cell expressing only A2 or B7.     

A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14)

Abstract: TrJ14 is a cytotoxic human IgG1Λ hybridoma mAb that recognized a novel HLA-A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the αl-domain helix. These amino acids were shared exclusively by all the identified TrJ14⁺ alleles.     

Dissection of the D-region of the human major histocompatibility complex by means of induced mutations in a lymphoblastoid cell line

This paper describes part of a mutagenic dissection of the human D-region. Twenty-six human lymphoblastoid cell mutants that had lost expressions of HLA-DR were created with a two-step procedure: (i) A mutant from which one entire haplotype had been physically deleted by gamma-rays was isolated by means of immunoselection against cells expressing a specific HLA-B antigen. (ii) This heterozygous deletion mutant was irradiated with gamma-rays or treated with ICR 191, a frameshift mutagen, and mutants that no longer expressed the remaining DR1 antigen were selected with a monoclonal antibody directed against a monomorphic DR determinant. Monoclonal antibody GENOX 3.53 was used to show that four of the gamma-ray induced DR-null mutants did not express the cis-linked MB1/MT1 locus. Since MB1/MT1 was still expressed in the other 16 gamm-ray induced and 6 ICR 191-induced DR-null mutants, the separate loss of expression of MB1/MT1 and DR1 is strong evidence that the DR1 and MB1/MT1 alloantigens are under separate genetic control in the cells we used. Since DR-null mutants bound SB2-specific monoclonal antibody ILR1, whether or not they expressed MB1/MT1, the results mean that gamma-rays resolved the genetic determinants for DR1, MB1/MT1, and SB2. Additional complexity of determinants encoded by D-region genes is indicated by the following results. The amount of MB1/MT1 antigen that was detected with ELISA tests for binding of GENOX 3.53 antibody to cells varied inversely with the number of expressed copies of DR or of a locus near DR. This could result from an increased amount of MB1/MT1 antigen or from increased binding accessibility of GENOZ 3.53-reactive antigen in DR-null mutants. Monoclonal antibodies CC 11.23 and CC 6.4 displayed patterns of binding to parental and diverse mutant cells that differed from that of GENOX 3.53, suggesting the existence of at least one additional D-region antigen that is neither SB, DR, nor MB/MT.     

Association of class II HLA alleles with susceptibility to develop immune-mediated diseases in Paraguayan patients

Genetic and nongenetic factors are involved in the pathogenesis of immune-mediated inflammatory diseases (IMIDs). The best-known genetic factor for susceptibility to IMIDs is the human leukocyte antigen (HLA). The aim of the present study was to evaluate the association of HLA class II genes with the risk of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc) in the Paraguayan population. We included 254 patients with IMIDs (101 SLE, 103 RA, and 50 SSc) and 50 healthy controls. The haplotypes of five genes corresponding to HLA class II genes and their relationship to the IMIDs studied were determined. Note that 84.6% were women, with a mean age of 43.4 ± 14 years. Among the associated HLA alleles, we found the previously identified risk factors in other populations like HLA-DRB1*03:01 and HLA-DRB1*14:02 for RA, as well as new ones not previously identified, such as DPA1*02:01 for SLE and, DB1*02:01 for RA and SSc. In the genetic association analysis, already known associations have been replicated, and unpublished associations have been identified in Paraguayan patients with IMIDs. This is the first genetic association study in Paraguayan patients with IMIDs.     

Rescue of human monoclonal antibody production from an EBV-transformed B cell line by fusion to a human-mouse hybridoma

A human-mouse cell line that is hypoxanthine-aminopterin-thymidine sensitive and ouabain resistant was derived from a fusion between human B lymphocytes and a mouse myeloma line. This new mutant, when fused to a relatively unstable EBV-transformed B cell secreting a human monoclonal anti-A (red blood cell antigen) antibody, resulted in stable hybridomas capable of long term production of the specific human monoclonal antibody. Furthermore, some of the hybrid clones secreted antibody in far greater titer than the original EBV cell line. We conclude that fusion to this human-mouse line is an efficient approach to the production of human monoclonal alloantibodies and an effective method of 'rescuing' secretion of desired antibody from EBV cell lines.     

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39

A human monoclonal IgM antibody, referred to as TU223, has been produced. The reactivity of TU223 was tested in various cells and cell lines by complement-dependent microcytotoxicity test and fluorescence-activated cell sorter analysis. The antigen defined by TU223 was expressed on Epstein-Barr virus-transformed B-cell lines and on some Burkitt's lymphoma cell lines, but was not expressed on normal T cells, B cells or erythrocytes. In addition, expression of the antigen defined by TU223 was also induced on B cells activated by Epstein-Barr virus or pokeweed mitogen, and on T cells activated by phytohemagglutinin, concanavalin A, pokeweed mitogen or recombinant interleukin-2. However, no expression of the antigen detected by TU223 was induced at all on recombinant interleukin-4-treated B cells or macrophage-like cell line U937. When the ability of TU223 and various mouse monoclonal antibodies to bind to human differentiation antigens was compared, interestingly, the reactivity of TU223 was found to be very similar to that of mouse monoclonal antibody CD23 (H107), which reacts with Fc epsilon receptor II. Two-color analysis revealed that the antigen defined by TU223 is expressed on the cell surface of certain lymphoid cells expressing CD23 antigen. However, it can be concluded that the antigen defined by TU223 is clearly distinct from Fc epsilon receptor II, based on assay of cross-blocking between H107 and TU223. The surface antigen on B85 cells recognized by TU2232 had the molecular size of 80-82 kiloDaltons as determined by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

1例心脏移植受者HLA-G5表达动态观察

目的 研究心脏移植受者外周血HLA-G5表达变化.方法 采用酶联免疫吸附法、蛋白印迹法检测1例心脏移植受者手术前、后外周血HLA-G5表达.结果 HLA-G5表达在术前和术后1周均无表达,4周后出现表达,并逐渐增高,16周后趋于稳定.结论 HLA-G5阳性表达可能与心脏移植受者的术后状况有一定的相关性,HLA-G5阳性表达可能保护移植物不受损伤,并延缓或抑制排斥反应的发生.     

A murine monoclonal antibody recognising a single glycoprotein within a human cytomegalovirus virion envelope glycoprotein complex

Nonionic detergent solubilised polypeptides from highly purified human cytomegalovirus virions were used as immunogens to produce murine monoclonal antibody secreting hybridomas. One monoclonal antibody was shown, by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), to precipitate three glycoproteins with molecular weights 52, 95, and 130 (all X 10(3)) and one minor component with a molecular weight of 50 X 10(3). When virion envelope components were first separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes, this monoclonal antibody recognised two related components with molecular weights 50 and 52 (both X 10(3)). Immunofluorescence studies suggested that these viral antigens were associated with membrane systems of virus-infected cells and were particularly abundant late in infection.     

抗人c-erbB2单克隆抗体的制备及其特异性鉴定

目的:制备小鼠抗人c-erbB2mAb,并进行特异性鉴定。方法:应用计算机软件分析人源c-erbB2抗原表位,人工合成羧基端含优势表位的13肽,与钥孔戚血蓝蛋白(KLH)偶联后,免疫BALB/c小鼠。取免疫小鼠脾细胞与Sp2/0骨髓瘤细胞常规融合,依次经HAT选择培养、间接ELISA法、克隆化和免疫组化染色法筛选出稳定分泌抗天然人源c-erbB2mAb的杂交瘤细胞株。用交叉反应试验和阻断试验检测mAb的特异性。结果:获得1株可稳定分泌抗天然人源c-erbB2抗体的杂交瘤细胞株。该mAb与已知的c erbB2抗原阳性的乳腺癌标本起反应;与其他不表达c-erbB2分子的细胞不起反应。用合成的13肽阻断后,失去与c-erbB2抗原的反应性。结论:用合成的13肽作为免疫原成功地制备出1株抗c-erbB2的mAb。     

TNF block haplotypes associated with conserved MHC haplotypes in European, Asian and Australian Aboriginal donors

Associations between major histocompatibility complex (MHC) ancestral haplotypes (AHs) and immunopathological diseases are traditionally ascribed to human leukocyte antigen (HLA) class I or class II alleles. However, polymorphisms in TNF and nearby genes in the central MHC can influence risk. We have defined TNF block haplotypes in Asian, European and Australian Aboriginal donors and shown conservation of TNF block haplotypes in geographically distinct populations, consistent with a common evolutionary origin. Here we show that most TNF block haplotypes do not align with a single MHC AH and associations often vary with ethnicity. This suggests more recent recombination events between the TNF block and the HLA alleles.     

UN1, a murine monoclonal antibody recognizing a novel human thymic antigen

Abstract: A murine monoclonal antibody (mAb) UN1 was produced on the basis of selective reactivity with human thymocytes. Characterization of UN1 by immunofluorescence gave a high intensity of labeling with the majority of human thymocytes. Expression was preferentially associated with immature thymocytes (CD3^dim) compared to mature cells, whereas only a subpopulation of peripheral blood lymphocytes was weakly stained. No specific binding to monocytes or granulocytes was detected. The T-cell lines HPB-ALL, H9 and MOLT-4 were all positively bound by UN1. Immunohistological staining of thymic tissues showed that mAb UN1 detected cells in both the cortex and medulla of fetal thymus, whereas the reaction in thymus samples from young children was mainly with medullar cells. By western blotting analysis, the antigen recognized by mAb UN1 corresponds to a membrane polypeptide with a molecular weight of approximately 120 kDa present on thymocytes and HPB-ALL cells. The mAb UN1 was submitted to the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens, Boston, 1993. UN1 did not cluster in any of the old or new clusters of differentiation discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggests that the UN1 antibody defines a previously undescribed molecule present on the cell surface of thymocytes and a minority of peripheral blood lymphocytes.     

An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58–77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38–57 and codons 78–88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.     

基因工程乳腺癌特异性人单链抗体的表达与特性

研究解决分泌人单抗的杂交瘤细胞系难于稳定持久分泌抗体的难题，制备单链抗体，使单抗分子小型化，为进一步研究其在肿瘤诊断和治疗中的应用作准备。从分泌抗乳腺癌人单抗的杂交瘤细胞CM－1总RNA中，利用RT－PCR技术分别扩增出人单抗重链可变区VH基因和轻链可变区VL基因，将扩增产物纯化后克隆于pGEM-T载体中，进行DNA测序和序列比较分析后，将两者共克隆于表达载体中诱导表达，利用斑点免疫印迹及竞争抑制法检测表达产物的抗原性。所克隆的CM－1人单抗重链可变区和轻链可变区基因片段，分别属于人免疫球蛋白IgM Ⅲ亚群，和鼠κ轻链V亚群，ⅩⅦ家族，用斑点免疫印迹法检测可见表达产物能与人乳腺癌细胞特异结合；人CM－1单克隆抗体对此单链抗体与人乳腺癌细胞的结合有竞争性抑制作用，抑制率为75．7％。结论：成功地制备了可特异结合乳腺癌细胞的CM－1单链抗体。     

The human leukocyte antigen TAP2 gene defines the centromeric limit of melanoma susceptibility on chromosome 6p

Abstract: A single human leukocyte antigen (HLA) class II allele, DQB1 * 0301 , is strongly associated with melanoma, and the HLA-DR locus provides the telomeric boundary for melanoma susceptibility in the HLA class II region of chromosome 6. However, the centromeric boundary is unknown. This study was designed to determine whether the adjacent upstream transporter associated with antigen processing (TAP) locus, TAP2 , constitutes the centromeric boundary of disease susceptibility in melanoma. Molecular oligotyping of TAP2 genes was performed for 36 Caucasian patients with melanoma and for 32 Caucasian control individuals by both amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) and PCR-sequence-specific oligonucleotide (SSO) typing. TAP2 allele frequencies in the melanoma patients were compared to those in non-melanoma Caucasian control populations, and to HLA-DQ allele frequencies determined by molecular oligotyping. While HLA-DQB1 * 0301 was more common in this group of 36 melanoma patients compared to a group of 200 controls (56 percent vs. 27 percent, Bonferoni-corrected chi-square p=0.01), no significant differences were observed in TAP2 allele frequencies between melanoma patients and controls. The TAP2 locus represents the centromeric boundary of disease susceptibility for melanoma in the class II region of chromosome 6p. These results support an etiologic role for HLA-DQB1 * 0301 in melanoma susceptibility.     

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12

Abstract: We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.     

Characterization of two human IgM monoclonal antibodies reactive with HLA-B27

Abstract: We describe here the generation and characterization of two human monoclonal IgM antibodies (UL-4F11 and UL-F6) reactive with HLA-B27. The monoclonal antibody (mAb) UL-4F11 is cytotoxic for peripheral mononuclear cells and, therefore, useful as typing reagent for HLA-B27 and HLA-B38. Protein chemistry showed that the mAb UL-4F11 precipitates HLA-B27 molecules. Epitope mapping analysis suggests that the amino acids 45, 67, 82 and 83 (alpha-1 domain) of the HLA-B27 sequence are necessary for mAb UL-4F11 reactivity. The mAb UL-F6 is suitable for complement dependent lysis of lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells with HLA-B27 (B*2701, B*2702, B*2703, B*2705, B*2707), B13, B40 (60, 61), B47 and B48 specificities. Its reactivity indicates that the amino acid valine in position 152 and glutamic acid in position 163 of the alpha-2 domain are crucial for the binding epitope.     

MICA polymorphisms and haplotypes with HLA-B and HLA-DRB1 in Koreans

Abstract
Major histocompatibility complex (MHC) class I chain-related gene A ( MICA ) is located within the human MHC, centromeric to HLA-B and telomeric to HLA-DRB1 . The location of MICA in the MHC indicates the presence of linkage disequilibrium with human leukocyte antigen (HLA). Like HLA, MICA is highly polymorphic; however, the information available for MICA polymorphisms is not as comprehensive as that for HLA polymorphisms. We estimated the allelic frequencies of MICA and haplotypes with HLA-B and HLA-DRB1 at high-resolution in a population of 139 unrelated Korean individuals by applying the newly developed method of sequence-based typing (SBT). A total of 17 MICA alleles were identified. The most frequent allele was MICA*010 (19.4%), followed by alleles *00201 (17.6%), *00801 (14.7%), *01201 (9.4%), *004 (8.3%) and *049 (7.9%). The most common two- and three-locus haplotypes were HLA-B*1501-MICA*010 (10.4%), MICA*010-HLA-DRB1*0406 (5.8%) and HLA-B*1501-MICA*010-HLA-DRB1*0406 (5.8%). This is the first study to provide such high-resolution information on the distribution of haplotypes comprising MICA , HLA-B and HLA-DRB1 in Korean individuals, a level of resolution made possible by use of the SBT method. The results of this study should help determine the mechanisms underlying diseases associated with MICA polymorphisms in Korean individuals.     

A double determinant sandwich immunoassay for quantitation of serum monoclonal anti-I-A antibody

A double-determinant sandwich radioimmunoassay (RIA) is described for the specific detection of anti-I-Ak monoclonal antibody (mAb) in the sera of non-Ighb murine hosts undergoing anti-Ia immunotherapy. This RIA utilizes 2 previously undescribed mAb reagents generated against an Ia.17-specific mAb secreted by the 10-3.6 hybridoma. The first reagent, 7.34, is specific for Ighb-linked allotypic determinants on the Fc portion of IgG2a immunoglobulins as defined by the pattern of reactivity with normal sera from a panel of inbred and Igh recombinant inbred strains. The second reagent, 58.3, is an anti-idiotypic mAb recognizing unique determinants in the combining site of 10-3.6 immunoglobulins, as determined by the specificity of the 58.3 mAb in solid-phase RIA and the capacity of this reagent to inhibit the binding of labeled 10-3.6 mAb to I-Ak-expressing spleen cells. In an RIA procedure using purified 58.3 mAb as substrate and 125I-labeled 7.34 as the detection reagent, serum concentrations of 10-3.6 as low as 1-5 ng/ml can be measured reproducibly after mathematical linearization of the sigmoid standard curve. In the present studies, the serum half-life of 10-3.6 mAb was calculated from assay data to be 3-5 h in I-Ak homo- or heterozygotes and 72 h in non-I-Ak mice. The serum level of 10-3.6 as a function of the mAb treatment protocol was also examined and results are considered with respect to the efficacy of different therapeutic regimens in prolonging transplant survival. Sandwich immunoassays of this type (RIA or ELISA) should provide a highly sensitive and specific means for monitoring serum mAb levels in individuals subjected to antibody immunotherapy for treatment of autoimmune disease, transplant rejection or tumor progression.