EB病毒转化的人B淋巴母细胞低密度培养条件的研究

肾综合征出血热(HFRS)患者外周血单个核细胞(PBMC)经 EB 病毒转化为人 B 淋巴母细胞,其培养上清中含 IgM 和 IgG,部分为人抗 HFRS 病毒 IgM 和 IgG。采用含有高浓度白细胞介素6(IL-6)的人纤维母细胞系 CRL1506条件培养液(CM)及~(60)Co 照射 CRL1506饲养细胞对低密度培养的人 B 淋巴母细胞生长、抗体分泌有明显促进作用,并获得单个 B 淋巴母细胞克隆化成功,克隆化后人 B 淋巴母细胞系仍持续分泌抗体。本研究对于克服人单克隆抗体技术中人 B 淋巴母细胞克隆化难、抗体产量低、难于获得特异性单克隆抗体等困难有一定应用价值。     

rHuIL-6和rHuIL-2对人杂交瘤细胞增殖、克隆化以及抗体产生的影响

rHuIL6对人一鼠杂交瘤86—1、87—3和人—(人×鼠)杂交瘤C_的增殖、克隆化和抗体分泌均有明显的促进作用。rHuIL-2能提高C_8杂交瘤的增殖水平。rHuIL-6和rHuIL-6和rHuIL-2同时应用对人—鼠杂交瘤86—1和87—3细胞的克隆化效率和抗体分泌水平有明显的协同作用。     

不同培养基对人杂交瘤细胞培养效应的影响

培养液是细胞培养的基本要素,选择适当的培养液是人杂交瘤技术成功的重要因素之一。我们分析比较了三种商品化培养液RPMI1640、DMEM、IMDM对分泌人单抗杂交瘤细胞的培养效应,包括对维持细胞的活力、增殖、克隆效率及抗体分泌等参数的观察比较,发现RPMI1640对人-(人×鼠)杂交瘤细胞“87-3”与人-鼠杂交瘤“87-3”培养效应最佳,为首选培养液。     

丁酸钠对杂交瘤细胞增殖及抗体分泌的影响

用含不同浓度丁酸钠完全培养液和无血清培养液培养分泌抗组织型纤溶酶原激活剂(t-PA)单克隆抗体的杂交瘤细胞。当丁酸钠浓度为0.25～0.5 mmol/L时,对杂交瘤细胞分泌抗体能力具有刺激作用,提高抗体效价2～4倍,对细胞生长无抑制作用。表明丁酸钠可作为一种添加剂用以提高杂交瘤细胞分泌抗体的能力。     

杂交瘤腹水抗体效价的不稳定性及其对策

自1980年以来，本实验室陆陆续续共制备了针对200余种抗原的约2000余株单克隆抗体（mAb）杂交瘤细胞系。在后续工作中发现大部分抗体分泌能力和效价长期稳定，但遇到有3株出现了不同程度的抗体分泌能力下降，腹水mAb效价由原有的1：10^7降至1：10^4或更低。对此，我们采取了再次克隆化的方法，发现这些细胞系中出现高比例的抗体分泌阴性细胞。不仅证明了腹水mAb效价下降是由于mAb分泌阴性细胞竞争性扩增引起的，而且成功地挽救了这些得来不易的宝贵的杂交瘤细胞系。     

抗肾综合征出血热病毒人单克隆抗体杂交瘤细胞株的建…

以亲和层析技术和纯化的肾综合征出血热病毒结构蛋白为特异性抗原，体外免疫正常人外周血淋巴细胞，然后将体外免疫的淋巴细胞与人－鼠种间杂交瘤细胞融合，经ＥＬＩＳＡ间接法选出２株分泌抗－ＨＦＲＳＶ人单克隆抗体杂交瘤细胞株，４次克隆化后，１００％阳性。     

角蛋白和过氧化物酶双特异性杂交杂交瘤的建立

作者采用杂交杂交瘤技术制备用于免疫学测定的双特异性抗体。以8-氮鸟嘌呤处理抗过氧化物酶杂交瘤细胞株E-47,获得了HAT敏感的突变株,且保持抗过氧化物酶分泌活性,将其中一个细胞克隆O45克隆化后,与角蛋白免疫的小鼠脾细胞融合,得到了一株稳定分泌抗角蛋白和抗过氧化物酶双特异性抗体的杂交杂交瘤BKH,染色体众数为129条,分泌的抗体含有IgG(1-2a)型杂交抗体。免疫组化显示,BKH抗体可识别角化的皮肤鳞状上皮组织,而不与其它上皮组织反应。     

EB病毒转化的人外周血淋巴细胞与SHM—D33细胞系融合制备人单克隆抗体

用EB病毒转化人外周血淋巴细胞,因克隆化不成功,分泌不稳定而失败。将转化的细胞与KR—12细胞融合,因融合率低而未成功。将转化细胞与SHM—D33细胞融合,成功地建立了3株稳定分泌人单抗的杂交瘤细胞,均产生IgMλ型抗体,30～50μg/ml上清。其中1株(86—2)已连续传代18个月,未经克隆化仍稳定分泌抗体,已适应低血清培养,抗体分泌量不变;可用裸鼠制腹水。     

体外免疫建立分泌卵巢癌反应性单克隆IgG人—鼠杂交瘤

用正丁醇提取的人卵巢癌细胞ａｏ１０／１７抗原，置于含免疫反应剂的无血清培基中免疫人脾细胞。经融合、筛选和克隆化，建立了９株分泌人源性单克隆ＩｇＧ抗体（ＨｍＡｂ）的人－鼠杂交瘤细胞系ＡＦ１～ＡＦ９。酶标抗体竞争抑制试验表明，其中ＨｍＡｂＡＦ５和ＡＦ８能识别卵巢癌抗原分子上的不同表位；杂交瘤细胞系所分泌的ＨｍＡｂ的效价高，所识别的抗原表达于卵巢癌细胞的表面     

人-鼠杂交瘤制备人单克隆抗体技术的研究

本文采用小鼠骨髓瘤细胞系(Sp2/0)与破伤风类毒素(TT)免疫的人外周血淋巴细胞(PBL)进行融合,对人—鼠杂交瘤细胞系制备人单克隆抗体的技术条件进行了探讨。按免疫时间(d)及体外刺激与否分为4组。结果表明:(1)PWM+TT体外刺激可导致B细胞总数增加,且融合率高,容易建成稳定分泌人单抗的细胞株。(2)经TT免疫的人PBL,6d就有抗TT抗体(IgG)的前体细胞出现。15d及21d时,抗TT抗体前体细胞的频率更高。(3)体内免疫7d及14d并分别经体外刺激的两组共建立5株阳性株。经双扩碓定IgG2株,IgM3株。冻存10个月后复苏、传代及适当的克隆化后仍为阳性。上清液中人Ig的分泌量为0.42～1.15μg/ml。经染色体核型鉴定,碓认为人-鼠杂种细胞。     

Feline hybridoma growth factor/interleukin-6 activity

An assay system was developed to measure feline hybridoma growth factor (HGF)/interleukin-6 (IL-6) activity in biological samples containing many kinds of cytokines by using the proliferation of the newly established mouse-rat hybridoma clone, B3B1. The proliferative response of this B3B1 clone was IL-6-specific, and could not be promoted by other cytokines including IL-1, IL-2, IL-3, and granulocyte-colony-stimulating factor (G-CSF). The anti-human B-cell stimulatory factor 2 (BSF-2)/IL-6 antiserum did not neutralize feline HGF/IL-6 activity in conditioned media prepared from feline con A-stimulated splenocytes and unstimulated alveolar macrophages, indicating antigenic differences between species. Feline HGF/IL-6 was eluted into the fractions corresponding to a molecular weight of 30,000-40,000 in gel filtration, and into the fractions at a salt concentration of 0.2-0.3 M NaCl in anion exchange chromatography. The physicochemical properties of feline HGF/IL-6 were slightly different from those of murine and human IL-6.     

Production of hybridoma growth factor by human monocytes

Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-beta 2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human gamma-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.     

Production of interleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.     

A two-way interaction between hepatocyte growth factor and interleukin-6 in tissue invasion of lung cancer cell line

Although both hepatocyte growth factor (HGF) and interleukin (IL)-6 play important roles in invasion of cancer cells, interaction between these two critical factors has not been well elucidated. In the present study we demonstrated a two-way interaction between HGF and IL-6 in in vitro invasion of a lung cancer cell line. A549 lung adenocarcinoma cells were stimulated with IL-6, and this treatment induced an upregulation of c-Met/HGF receptor mRNA expression in the cells. In addition, IL-6 enhanced the HGF-induced in vitro cell invasion. This effect was abolished by pretreatment of the cells with either anti-IL-6 neutralizing antibody or with anti-c-Met/HGF receptor blocking antibody. We also found that HGF upregulated the expression of IL-6 receptor mRNA in the same cell line, and that this upregulation enhanced the IL-6-induced cell invasion. Finally, costimulation with HGF and IL-6 showed an additive effect on invasion, and this effect was mediated by production of matrix metalloproteinase (MMP)-2 and MMP-9. These results suggest that HGF and IL-6 upregulate each other's receptors, and thus would cooperatively enhance tissue invasion. They also suggest an "autocrine circuit" among cytokines and growth factors in certain cancer cells which functions to accelerate their biologic activities such as metastatic property.     

Human plasma-derived media supplement supports antibody production by hybridoma cells

Summary A new, virus-inactivated, media supplement from human plasma (GEMS) has been developed which maintains hybridoma cells at a low growth rate. The rate of antibody production per cell by hybridoma cells growing in the presence of GEMS is at least equivalent to the rate of antibody production by the same cell lines growing in fetal bovine serum.     

The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas

Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.     

Commercial serum-free media: hybridoma growth and monoclonal antibody production.

Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media. However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used. In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines. Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA). Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions. Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines. We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.     

Generation of a human hybridoma producing a pure anti-HLA-A2 monoclonal antibody

We report the production and characterization of a human monoclonal IgM hybridoma antibody recognizing antigen HLA-A2. B lymphocytes obtained from the peripheral blood of a multiparous volunteer 1 week postpartum were transformed in vitro by Epstein-Barr virus, screened by a microlymphocytotoxicity assay, and electrofused with the heterohybridoma fusion partner, K6H6/B5. A specifically anti-A2 secreting hybridoma cell line. MBW1, was then identified and cloned. The cytotoxic IgM antibody produced showed complete correlation (r = 1.00) with the A2 antigen on a large panel of unrelated donors' lymphocytes, and no cross-reactivity with A28, Aw68, or Aw69 antigens was observed.