抗A型和抗B型血型单克隆抗体的制备及其在血型鉴定中的初步应用

本试验用A型和B型人红细胞混合悬液免疫BALB/c小鼠,取免疫脾细胞与同系鼠Sp2/0骨髓瘤细胞融合,建立了5株分泌盐水凝集抗体的杂交瘤细胞株。3次克隆化后,细胞株稳定分泌McAb已6个月。经A、B、O各型红细胞鉴定及阻断试验、吸收释放试验结果证明:H_2、H_5、B_7株是特异性抗A型抗原的,D_5株是特异性抗B型抗原的。本试验制备的McAb亲合力高于人抗血清,用保护剂稀释后稳定性良好。用McAb和人抗血清对献血员血液标本692份,脐带血58份做血型鉴定所得结果完全一致。     

人类AB0血型系统-B抗原单克隆抗体的研制

血库、临床医院、法医以及在血液免疫学，人类遗传学的基础理论研究方面，都迫切需要高特异性、高亲和性(avidity)．高亲和力(affinity)的血型McAb，从80年代初始，国内外研制ABO血型McAb的工作。目前，有些国家已经商品生产，取代了ABO系统标准血清。我们应用淋巴细胞杂交瘤技术获得了3株较理想的细胞株：3Ca、5Ca、13B1，有希望取代抗B标准血清.     

抗血型M、N及抗血型糖蛋白A/B单克隆抗体的制备及其特性鉴定

目的:制备抗血型M、N及抗血型糖蛋白A/B(GPA/GPB)的单克隆抗体(mAb),并进行特性鉴定。方法:用人“O”型血红细胞作为免疫源,免疫BALB/c小鼠。采用淋巴细胞杂交瘤技术制备mAb,用谱红细胞筛选阳性克隆;采用直接、间接血凝试验检测杂交瘤细胞培养上清及腹水中mAb的效价。分别用快速定性试纸和酶处理红细胞检测mAb的Ig亚类及抗原表位。用Western blot鉴定抗GPA/GPB mAb的特异性。结果:获得4株分泌抗M、1株抗N及3株抗GPA/GPB mAb的杂交瘤细胞株。杂交瘤细胞培养上清mAb的效价介于1×2-4~1×2-8之间,腹水mAb的效价在1×2-7~1×2-12之间。除1株mAb 1C1C9C4为IgM外,其他7株mAb均为IgG。通过杂交瘤细胞培养上清与谱红细胞的反应格局,结合mAb抗原表位的检测,确定4株和1株mAb可分别特异性结合于GPA的M、N抗原表位;另3株mAb 6D7C9、7C9H4和7C9G11与“O”型血红细胞膜的Western blot结果显示,均可结合GPA、GPB蛋白。结论:成功地建立了4株分泌抗M、1株分泌抗N及3株分泌抗GPA/GPB mAb的杂交瘤细胞株,可用于MNSs血型系统的研究及鉴定,并为制备双功能抗体用于病毒、肿瘤疾病的诊断和治疗打下了坚实的基础。     

抗人IgM重链McAb的研制和应用 Ⅱ.在临床检测中的应用

抗人IgM抗血清是早期诊断临床感染的重要试剂。近年来,国内外先后制备成抗人IgM McAb。我们为配制全套抗人Ig试剂,也建立了16株分泌抗人IgM McAb杂交瘤细胞株,并对其特性进行了详细的分析,本文着重报道该类McAb的实用价值。     

妊娠相关血浆蛋白-A单克隆抗体的制备和初步鉴定

目的制备娠相关血浆蛋白-A（PAPP-A）单克隆抗体（McAb）。方法利用PAPP-A抗原免疫Balb／c小鼠，应用杂交瘤技术将免疫小鼠的脾细胞与小鼠骨髓瘤细胞（SP2／0）融合，间接ELISA方法对细胞培养上清检测、筛选，建立分泌PAPP—A单克隆抗体的杂交瘤细胞株，扩人培养后，Balb／c小鼠腹腔注射杂交瘤细胞株，产生腹水后，对腹水进行收集、纯化、签定．结果筛选出稳定分泌PAPP—AMcAb的杂交瘤细胞株，Western—blot分析证实McAb与PAPP—A有较高的特异性及敏感性。结论本实验成功制备了抗PAPP—A特异性的McAb，可用于PAPP—A免疫检测方法的建立。     

人类ABO血型系统-B抗原单克隆抗体的研制

血库、临床医院、法医以及在血液免疫学、人类遗传学的基础理论研究方面,都迫切需要高特异性、高亲和性(avidity)、高亲和力(affinity)的血型McAb,从80年代初始,国内外研制ABO血型McAb的工作。目前,有些国家已经商品生产,取代了ABO系统     

抗结核杆菌TB-15C3 McAb特异性和敏感性的研究

前文报道建立了抗结核杆菌McAb 5个杂交瘤细胞株,制备出McAb,这5种McAb采用ELISA法检测了7种抗原,除大肠杆菌外、均出现不同反应。因而有必要进一步筛选特异性强和稳定性高的抗结核杆菌McAb,以提供结核菌的菌种鉴定、分型、抗原提纯以及为结核病人血清学诊断等应用。现将筛选出来的1株TB-15C_3McAb,与6株分枝杆菌进行了交义试验,结果报告如下。     

Rh血型系统抗-E配血不合及检测

Rh血型系统是人类红细胞血型系统中最复杂的一个系统,在临床有其重要意义.该血型系统主要血型抗体有5种,抗-E抗体即是其中之一.临床因抗-E抗体引起的输血反应时有报道,本文因反复输血出现IgG抗-E1例,报告如下：     

抗人胃腺癌细胞株SGC-7901单克隆抗体的建立及初步临床应用

本文报道了抗人胃腺癌细胞株SGC-790单克隆抗体(McAb)的研究结果,探讨了McAb的特性及相应抗原的性质,并报道了初步临床应用结果。 一、抗胃癌细胞株SGC-7901杂交瘤细胞株的建立及抗体特性分析:三次融合共获4株     

人类血型-H抗原单克隆抗体20D7的研制

用鼠细胞融合杂交瘤技术,获得一株分泌抗H血型抗原的单克隆抗体杂交瘤细胞株-20D7H,为IgM类。已连续培养一年,生物学性状稳定。凡具有H抗原的红细胞与该抗体作用均发生凝集,而与H抗原缺失型:孟买、类孟买血球不发生凝集。20D7细胞分泌单克隆抗体能被O型分泌型唾液抑制;能被O型细胞吸收并释放。该单抗经O型分泌型唾液吸收后,再与红细胞谱细胞反应,这些谱细胞虽经木瓜酶处理和抗球蛋白试验等,均不再出现血球凝集反应。该单抗用血型抗原Syn-sorb试剂检测,所识别的抗原为H_2型。20D7细胞培养上清的抗H血凝效价是1:256,亲和力为5秒,都比市售多克隆标准品质量高。在不同温度下保存,或经反复冻融其效价稳定,可用于临床检测。     

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination.     

High efficiency creation of human monoclonal antibody-producing hybridomas

The native human antibody repertoire holds unexplored potential for the development of novel monoclonal antibody therapeutics. Current techniques that fuse immortal cells and primary B-lymphocytes are sub-optimal for the routine production of hybridomas that secrete human monoclonal antibodies. We have found that a murine cell line that ectopically expresses murine interleukin-6 (mIL-6) and human telomerase (hTERT) efficiently forms stable human antibody-secreting heterohybridomas through cell fusion with primary human B-lymphocytes. The hybrid cells maintain secretion of human antibodies derived from the primary B-lymphocytes through multiple rounds of cloning. Using splenic B-lymphocytes from a patient immunized with a Streptococcus pneumoniae capsular polysaccharide vaccine, we have succeeded in creating hybridomas that secrete human monoclonal antibodies specific for S. pneumoniae antigens. Using peripheral blood lymphocytes, we have similarly cloned a human antibody that binds a viral antigen. These experiments establish that SP2/0-derived cell lines ectopically expressing mIL-6 and hTERT will enable the rapid cloning of native human monoclonal antibodies.     

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Peripheral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody/antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.     

In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection.     

Production and the characterization of monoclonal antibody against CD43, K06

A monoclonal antibody against human CD43 has been developed and designated as K06. Its reactivity in the lymphoid organs was different from that of known anti-CD43 monoclonal antibodies suggesting that this may recognize a novel epitope of human CD43 molecule. The CD43 epitope detected by anti-K06 monoclonal antibody was highly expressed in cortical thymocytes, platelets, and myeloid cells of normal peripheral blood, and its reactivity was comparable to that of known anti-CD43 monoclonal antibodies. However, the density of this epitope was lower in the medullary thymocytes. Biochemical studies indicated that anti-K06 monoclonal antibody could recognize glycosylated moiety of CD43 antigen. The expression profile of anti-K06 monoclonal antibody in several cell lines was somewhat different from that of known anti-CD43 antibodies. In addition, CD43 ligation through the K06 epitope appeared to induce apoptosis in human leukemic cell line, Molt-4. We therefore assume that K06 epitope of human CD43 might have some role in T-cell development.     

Accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic ribosome display

What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using ribosome display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro ribosome display for enrichment of human monoclonal antibodies against recombinant gp120^{K530}, derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 μg of total RNA, the equivalent of 3-5 mL of human blood.     

Sheep erythrocyte rosette formation by B chronic lymphocytic leukemia cells as a consequence of monoclonal surface immunoglobulin with "Forssman-like" antibody activity

Peripheral blood and splenic lymphocytes from an elderly man with chronic lymphocytic leukemia (Rai stage 4) were shown to have monoclonal surface immunoglobulin (IgM+, IgD+, kappa+), Ia-like antigen and receptors for unsensitized sheep red blood cells. The sheep red blood cell receptor was not blocked by monoclonal antibodies that bind to the classic T lymphocyte rosette receptor (OKT-11 and Lyt-3) or by anti-human IgG or antilambda antibodies. However, the sheep red blood cell receptor was blocked by antihuman IgM and kappa antisera and by soluble guinea pig kidney antigen (Forssman antigen). It is concluded from these and other observations that our patient has a B-cell lymphoproliferative disorder expressing monoclonal surface immunoglobulin with anti-"Forssman-like" antibody activity.     

Human Monoclonal Antibodies against Blood Group Antigens Preferentially Express a VH4-21 Variable Region Gene-Associated Epitope

An anti-idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti-I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4-21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies. Using this antibody, epitope expression was investigated in a panel of 72 human monoclonal allo-antibodies specific for human blood group antigens, as compared with a control panel of 39 randomly selected human monoclonal IgM antibodies of unknown specificities. The anti-blood group panel included 44 IgM and 28 IgG monoclonal antibodies against a variety of blood group antigens including the A antigen, Rh C, c, D, E, e, G antigens, and the Kidd antigens Jka and Jkb. The epitope was expressed by 64% (28/44) of the IgM anti-blood group antibodies and by 21% (6/28) of the IgG antibodies, but by only 7.7% (3/39) of the control IgM antibodies. These data indicate that the human alloimmune response to blood group antigens is biased in the use of VH gene families, with a preference for the VH4-21 gene segment of the VHIV family, or closely related gene segments. The fact that this mirrors the findings for the autoimmune cold agglutinins suggests a link in immunoglobulin gene usage between antibodies against structurally diverse antigens on the red cell surface.     

人源抗乙肝表面抗原基因工程抗体的研究

目的 研制人源抗乙肝表面抗原(HBsAg)基因工程抗体.方法 采集多个HBsAg加强免疫后表面抗体阳性(HBsAb+)志愿者的外周血,分离淋巴细胞,构建人源抗HBsAg Fab噬菌体抗体基因文库.用纯化的HBsAg对抗体库进行富集筛选,经过序列测定确定抗体轻重链型别,分别克隆入真核细胞表达载体pAc-FcR和HL51-14,转染昆虫Sf9细胞和293T细胞,利用杆状病毒/昆虫细胞系统和哺乳动物细胞系统实现全抗体的分泌型表达.结果 成功筛选出20株抗HBsAg的人源Fab抗体并制备出其中6株的IgG全抗体,竞争性ELISA结果显示6株全抗体针对的是HBsAg 3个不同表位.结论 成功筛选并获得了6株针对3个不同表位的抗HBsAg的IgG全抗体,为治疗性抗体和新疫苗的研制奠定了基础.     

Detection of monoclonal antibodies against cell surface antigens: the use of antiglobulins coupled to red blood cells

An antiglobulin-coupled red cell assay is described for screening monoclonal antibodies against cell surface antigens. A monoclonal antibody specific for rat immunoglobulin kappa chains was coupled to red blood cells and used to detect binding of rat monoclonal antibodies to cells attached to the wells of microtitre plates. The method was found to be simpler and more rapid than the alternative enzyme-linked binding assay and useful for rapid screening and selection of antibodies for use as differentiation markers of human and mouse haemopoietic cells.