抗人重组SCF单克隆抗体杂交瘤细胞系的建立及初步鉴定

本研究利用PEX31B作为载体,在大肠杆菌表达出重组人SCF融合蛋白。用该蛋白作为抗原,免疫BALB/c小鼠。通过鼠-鼠杂交瘤技术,成功地获得4株分泌抗人重组SCF单克隆抗体(下称单抗)的杂交瘤细胞系。经检测,它们所分泌的抗体亚类均为IgG1,免疫印迹试验和抗体特异性鉴定证实,4株单抗均能特异性地识别可溶性SCF。抗SCF单抗杂交瘤细胞系的建立,为深入研究SCF的生物学特性、功能以及SCF产品的纯化,提供了有力的工具。     

抗人BPI23单克隆抗体的制备和鉴定

目的 采用杂交瘤技术制备抗人BPI23单克隆抗体,并对其应用进行初步分析。方法 免疫小鼠脾细胞与小鼠骨髓瘤细胞按常规方法融合;用间接ELISA法和Western - blot筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法亚克隆3次获得稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注入小鼠腹腔后制备腹水;纯化腹水中的单抗并对抗体类型进行鉴定;用Western-blot分析抗体的特异性;用间接ELISA法测抗体效价;将分离纯化的正常人外周血中性粒细胞和单个核细胞制成涂片,用抗人BPI23单克隆抗体进行免疫染色。结果 获得3个（1B4、9C12和2H11）稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株,所分泌的单抗类型分别为κ型IgM、κ型IgG1和κ型IgG1;抗体效价分别为1.28×105、1.28×105和4.1×106,纯化后抗体含量分别为0.208g/L、2.03g/L和3.88g/L;3种纯化抗体均能与本实验制备的人BPI23和市售人BPI55标准品特异性结合,而不能与小鼠BPI25和人LBP结合;在免疫组化实验中,1B4、9C12和2H11单抗均能与人中性粒细胞中的BPI特异性结合。结论 成功制备了人BPI23特异性单克隆抗体,为BPI检测试剂盒的研制奠定了基础。     

PD-L1 mAb的制备、鉴定及其特性的研究

目的制备抗程序化死亡配体-1(Programmed Death-Ligand 1,PD-L1)的mAb杂交瘤细胞系并对其分泌的抗体特性进行研究。方法用我室纯化的PD-L1／IgG4 Fc融合蛋白免疫Balb／c小鼠,取小鼠脾细胞与骨髓瘤细胞SP2／0进行融合,经筛选和克隆化后,建立杂交瘤细胞系。以Western-blot等方法研究抗体特性。结果得到3株能够稳定分泌抗体,效价高的杂交瘤细胞系2D10、1C3和1A6,Western-blot显示2D10、1C3和1A6能与PD-L1(Mr为5800)结合。结论得到杂交瘤细胞系稳定分泌的mAb,在抗病毒免疫、抗肿瘤反应和自身免疫性疾病的免疫诊断和／或治疗方面可能有广阔的应用前景。     

抗恶性疟单抗独特型决定簇的第二单克隆抗体的制备和鉴定

本文以分泌抗恶性疟疾红内期抗体的小鼠杂交瘤细胞系94D1诱生的腹水,通过Protein A-Sepharose cL-4B亲和层析法,纯化IgG,并以此作为免疫原皮下多点免疫Wistar大鼠。加强免疫后3天取脾与小鼠Sp2/0瘤细胞进行异种间的细胞融合,经间接ELISA法检测筛选和5次克隆化,最终得到一株生长稳定,连续分泌特异抗体的异种杂交瘤细胞系41RF5。经过多次冷冻和复苏以及体外连续传代培养6个月,其稳定性与小鼠×小鼠杂交瘤细胞相近。选择8株单抗和Sp2/0 IgG作为包被抗原,即6株抗恶性疟单抗:21E3、92D4(IgG2b)、93A3(IgG_1)、94B5(IgG_1)、94C_3(IgG_1)和94D1(IgG2b),1株抗猪尾猴疟原虫(Plas mc-dium inui)红内期单抗32A12(IgG_1)和1株抗登革热Ⅲ型病毒单抗215D3(Ig-G2b),用间接ELISA法对41RF5特异性进行鉴定,结果显示,41RF5单抗只与94D1呈阳性反应,与其余各种包被抗原均呈阴性反应,表明41RF5具有良好的抗94D1单抗独特型决定簇的特异性,41RF5培养上清中特异的抗体效价为1:1,28O。     

抗人重组SCF单克隆抗体杂交瘤细胞系的建立及初步鉴定

本研究利用ＰＥＸ３１Ｂ作为载体，在大肠杆菌表达出重组人ＳＣＦ融合蛋白。用该蛋白作为抗原，免疫ＢＡＬＢ／ｃ小鼠，通过鼠－鼠杂交瘤技术，成功地获得４株分泌抗人重组ＷＳＣＦ单克隆抗体的杂交瘤细胞系。经检测，它们所分泌的抗体亚类均为ＩｇＧ１，免疫印迹试验和抗体特异性鉴定证实，４株单抗均能特异性地识别可溶性ＳＣＦ。抗ＳＣＦ单抗杂交瘤细胞系的建立，为深入研究ＳＣＦ的生物学特性、功能以及ＳＣＦ产品的纯化，提供了     

用CD38单抗纯化膜抗原制备分泌不同同种型CD38单抗的杂交瘤

根据单抗的应用目的不同,人们常常需要免疫球蛋白不同类或亚类的单抗。因此,应用已知某一类型免疫球蛋白的单抗纯化相应抗原,建立分泌不同免疫球蛋白类或亚类的杂交瘤细胞系,具有实际应用价值。本文介绍用已知ISG_1亚类CD 38单抗建立分泌IgG2a亚类单抗杂交瘤细胞系的方法。     

抗人肝癌单克隆抗体HL2的制备及鉴定

本文以贯序免疫法依次用人肝癌细胞株QGY-7703、BEL-7402、SMMC-7721免疫BACB/c小鼠,取其脾细胞与小鼠骨髓瘤Sp2/0细胞融合,经检测细胞抗原的EL-ISA方法筛选,连续克隆4次后,获得了1株能稳定分泌肝癌单抗的杂交瘤株HL_2。其     

日本脑炎病毒内影像组抗独特型单克隆抗体的制备及初步鉴定

目的 制备具有内影像作用的分泌日本脑炎病毒（JEV）抗独特型单克隆抗体（AId mAb)杂交瘤细胞系。方法 以传统免疫法和硝酸纤维素膜（NC膜）皮下包埋免疫法,用具有高中和活性的抗JEV mAb 2H4和2F2分别免疫Balb/c小鼠,按常规方法融合,经多种ELISA、表面等离子共振和动物免疫实验筛选并鉴定具有内影像作用的AId mAb。结果 获得了3株可分泌AId mAb的杂交瘤细胞系。结论 所获AId mAb均能模拟JEV抗原,为JEV受体的分离、纯化提供了条件。     

抗T4单克隆抗体的制备和特性鉴定

采用T4 BSA联结物免疫BALB/c小鼠 ,通过杂交瘤技术 ,获得 4株稳定分泌高特异性抗T4单克隆抗体的杂交瘤细胞系 ,并制取到含高效价单克隆抗体的腹水。放免分析结果显示 ,所得的 4株单抗对T4均表现高亲和性和高特异性 :Ka>10 8L/Mol,与T2交叉反应未测出 ,与T3交叉反应 <0 4 0 % ,与rT3交叉反应为 <0 2 2 %。采用T4 10D11株单抗替代多抗应用于放免药盒 (RIA )与商品药盒进行比较 ,同时测定 96例临床血样 ,结果表明 ,两者之间有极显著的正相关 (Y =1 12X 3 15 ,r=0 985 0 ) ,为这些单抗的应用提供了基础     

抗人胃癌单克隆抗体的研究——Ⅰ.分泌抗人胃癌细胞单克隆抗体杂交瘤细胞系的建立

用国内胃癌细胞株GGC-81-40免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞Sp2/0-Ag融合,经ELISA法和IFS法筛选,获得了5个产生抗人胃癌细胞单克隆抗体的杂交瘤细胞系。这些杂交瘤细胞系具有双亲代生物学性质,现已传代培养一年多,染色体数为103～105条,免疫球蛋白分型:二株分泌的单抗为IgG_1,其它三株为IgM。它们均分泌高效价的抗体,ELISA测定效价为10~(-5) ,IFS效价为1:500;细胞结合试验与GGC-81-40呈现强阳性反应,但不与正常骨髓细胞,扁桃体T、B细胞以及外周血细胞反应。     

Estimation of heterokaryon formation and hybridoma growth in murine and human cell fusions

Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions.     

Lymphocytes from enlarged iliac lymph nodes as fusion partners for the production of monoclonal antibodies after a single tail base immunization attempt

A novel method of preparing hybridomas producing mouse monoclonal antibodies was -established, called "the mouse iliac lymph node method". Lymphocytes from enlarged iliac lymph nodes from mice injected intramuscularly at the tail base with an emulsion of antigen and Freund's adjuvant were used for cell fusion. For the most part, lymph node lymphocytes from two mice were used for a single cell fusion attempt. Ovalbumin was used as the antigen and the results of fusion were compared with the results of a previous report (Cell Struct. Funct. 20; 151-156, 1995). Approximately 100 positive wells producing antibody of interest were identified using this method. By comparison, approximately 10 positive wells were identified using the more conventional mouse spleen method after three immunization injections. The relative proportions of hybridomas producing IgM, IgG1, IgG2a, IgG2b, and IgG3, following fusion using iliac lymph node lymphocytes obtained 14 days after injection were 14.0%, 78.7%, 3.2%, 3.5% and 0.5%, respectively. This method demonstrated the following advantages: (1) a single injection of the antigen emulsion was sufficient, (2) the lymph nodes were ready for use 14 days after injection, and (3) a high yield of positive hybridomas was obtained.     

Efforts to produce human cytotoxic T-cell hybridomas by electrofusion and PEG fusion

Cytotoxic human T cells from different sources were fused with different types of human T-lymphoma cells and mouse B-myeloma cells using variations of the polyethylene glycol (PEG) method and electrofusion. Both techniques yielded proliferating hybridomas. The frequency of wells with proliferating hybridomas depended on the tumor fusion partner used; the best results were obtained with HSB-1, whereas fusions with JURKAT-1 and HPB-1 did not yield any hybridomas. For one tumor cell line (HSB-1), considerably more hybridomas were obtained with electrofusion than with the PEG fusion (with or without heat shock). There was no consistent relationship between the presence or absence of cytotoxic activity of the T lymphocytes against the tumor fusion partner and the yield of hybridomas. In human-human as well as in human-mouse hybridomas most of the lymphocyte derived chromosomes were lost. Four of the more than 600 hybridomas tested showed transient cytotoxic activity, but in none of them this function could be immortalized. Two of the hybridomas obtained with CEM-1 as tumor fusion partner expressed low levels of lymphocyte-derived CD3 antigens. Two hybridomas obtained with HSB-1 were highly invasive in vitro in rat hepatocyte cultures, whereas HSB-1 tumor cells were not.     

Preparation of Hybridomas producing monoclonal antibodies against human interferon

To prepare hybridomas secreting monoclonal antibodies (MoAb) against human alpha-interferon (alpha-IFN), BALB/c mice were immunized with IFN produced in Namalwa cells. Native alpha-IFN, as well as partially purified or on cellulose adsorbed alpha-IFN preparations were used for immunization. Seven hybridomas continuously secreting IgG against human alpha-IFN were prepared by fusion of splenocytes from immunized donors with the mouse myeloma cells. MoAb reacted in ELISA as well as in neutralization test with human lymphoblastoid, leukocytic and recombinant alpha-IFN.     

Efficient generation of stable antibody forming hybridoma cells by electrofusion

A variety of modified electrofusion protocols designed to improve the efficiency of hybridoma production have recently appeared in the literature. We undertook to maximize the number of antibody secreting murine hybridomas by optimizing the temperature and fusion strength parameters of the conventional electrofusion technique. Anti-DNP secreting hybridomas were generated by fusing SP2/0 to immunized mouse splenic lymphocytes using an unmodified electrofusion protocol consisting of washing in a weakly conducting sorbitol fusion medium supplemented with bovine serum albumin, calcium and magnesium ions. This was followed by dielectrophoretic alignment and application of 3 short duration, high intensity field pulses in helical chambers. Optimal efficiencies of hybridomas were generated by the application of 2000 V/cm pulses at 25 degrees C (2.45 hybridomas x 10(-4) splenocytes) and as many as 63% of resulting hybridomas secreted anti-DNP monoclonal antibodies, the majority of which were IgG's. These data show that modification of the electrofusion protocol by pretreatment of fusion partners with proteolytic enzymes or the use of antigen bridging is not required for the successful and efficient production of specific monoclonal antibodies by electrofusion.     

Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6.

The addition of auxiliary feeder cells or conditioned medium has been shown to augment the yield of mouse hybridomas obtained following the cell-cell fusion of myeloma and B lymphocytes. The addition of one of these factors, interleukin-6 (IL-6) has been found to increase the proportion of hybridomas secreting monoclonal antibodies of desired specificity. As an alternative genetic approach, we have examined the efficacy of a retroviral infectant of Sp2/0 cells that constitutively expresses recombinant murine IL-6 (Sp2/mIL-6) as fusion partner. The results demonstrated that the yields of both viable Ig-secreting hybridomas, and antigen-specific monoclonal antibodies were increased 3-15-fold and 5-9-fold, respectively, with the Sp2/mIL-6 relative to Sp2/0 or Sp2/neo cells as fusion partner. Sp2/mIL-6 cells generated hybridomas with comparable growth rates, stability, and Ig production. The results of staining nascent hybridoma colonies immunohistochemically for Ig production suggest that Sp2/mIL-6 cells as a fusion partner increased the viability and/or stability of nascent hybrid cells that are producing Ig. Thus the Sp2/mIL-6 cells are an improved myeloma parent for the generation of large numbers of antibody-producing hybridomas against specific antigens.     

Bispecific IgA/IgM antibodies and their use in enzyme immunoassay.

Two hybrid hybridomas secreting polymeric bispecific antibodies to human chorionic gonadotropin and calf intestinal alkaline phosphatase were produced by fusion of IgA- and IgM-secreting mouse hybridomas. Both hybrid antibodies were purified from ascitic fluid by size exclusion chromatography. An IgM-like fraction was shown to exhibit bispecific activity. Bispecificity was completely lost following mild reduction and alkylation. Both bispecific antibodies were used to develop a sensitive enzyme immunoassay for hCG.     

Flow sorting of hybrid hybridomas using the DNA stain Hoechst 33342

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described for the selection of hybrid hybridomas producing bispecific monoclonal antibodies. Parental hybridoma cells were first labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC); heterofluorescent cells were recovered after fusion. After a period of growth in culture, the cells were then stained with the DNA-specific dye bis-benzimidazole Hoechst 33342 and sorted on the basis of their DNA content. The staining conditions (10 micrograms/ml of Hoechst 33342, 90 min incubation time at 37 degrees C) were found to be optimal for obtaining a well resolved DNA histogram with minimal effect on the growth properties of cells from different mouse hybridoma lines. Employing this method we have isolated hybrid hybridomas synthesizing bispecific monoclonal antibodies reacting with human low density lipoprotein and alkaline phosphatase from calf intestine.     

Specific monoclonal antibodies to the Mycoplasma-type pathogenic agent of grapevine flavescence dorée

Three hybridomas producing monoclonal antibodies specific for the mycoplasma-like organism (MLO), pathogenic agent of grapevine flavescence dorée, were obtained after fusion between spleen cells from a mouse immunized against flavescence dorée MLO and Sp2/O-Ag-14 mouse myeloma cells. These hybridomas were selected in an indirect sandwich ELISA in which antibodies from two different animal species were used (rabbit and mouse). This assay is convenient for anti-MLO monoclonal antibody screening, because of its sensitivity, specificity and good preservation of antigens. The three monoclonal antibodies were examined for reactivity towards the MLO causal agents of 15 plant yellows. None of the three were implicated in a serological relationship between the MLO of these plant yellows and flavescence dorée-MLO. Reactivity of the three monoclonal antibodies was checked towards two other isolates of flavescence dorée. One of the three antibodies detected the wild isolates.     

Mink-mouse hybridomas that secrete mink immunoglobulin G

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.