分泌抗人角蛋白单克隆抗体杂交瘤细胞株的建立及其抗体的初步鉴定

本研究以人角蛋白免疫的BALB/C小鼠脾细胞与NS_1骨髓瘤细胞经聚乙二醇融合、HAT选择性培养、ELISA筛选及反复克隆化,成功地获得了两株稳定分泌抗人角蛋白单克隆抗体(MCAb)的杂交瘤细胞株——AF_2和AF_6,其染色体众数分别为97和96,所产生抗体经免疫双扩散试验属小鼠IgG_1亚类。通过亲和素一生物素一过氧化物酶(ABC)免疫组     

抗人白细胞介素15单克隆抗体的制备及特性鉴定

目的 :制备鼠抗人白细胞介素 15(hIL 15)单克隆抗体 (mAb) ,并鉴定其特性。方法 :自重组人白细胞介素 15(rhIL 15)基因工程菌中 ,提取融合蛋白GST IL 15,以 12 0g/LSDS PAGE分离鉴定 ,切取含有目的条带的凝胶 ,免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2 / 0骨髓瘤细胞常规融合 ,依次进行HAT选择培养 ,间接ELISA法筛选抗体阳性的杂交瘤细胞及克隆化。对杂交瘤细胞株的稳定性及其分泌的mAb的特性进行鉴定。另外 ,以rhIL 15包涵体蛋白 (rhIL 15IBP)免疫新西兰白兔 ,制备抗hIL 15的多克隆抗体 (多抗 )。用抗hIL 15的mAb与多抗建立双抗体夹心间接ELISA。结果 :获得 1株可稳定分泌特异性抗hIL 15mAb的杂交瘤细胞。建立了双抗体夹心间接ELISA ,检测rhIL 15蛋白的敏感性达 10 μg/L。结论 :成功地制备抗hIL 15mAb ,并建立了一种可用于hIL 15检测的双抗体夹心间接ELISA。     

分泌抗人C_(1q)单克隆抗体杂交瘤细胞系的建立

以人C_(19)免疫的BALB/c小鼠脾细胞与Sp2/0骨髓瘤细胞融合,用固相C_(19)-ELISA筛选和有限稀释法克隆化以后,获得三株(A_4、B_6及D_5)分泌抗人C_(19)单克隆抗体的杂交瘤细胞系。培养上清和小鼠腹水的特异性抗体效价分别为10~(-3)、10~(-5)及10~(-7)。单克隆抗体与人IgG无交叉反应,与灭活C_(19)呈阴性反应,经人C_(19)吸收后,OD值明显下降,能阻断补体经典途径的活化,从而抑制溶血活性。根据上述结果表明,三株细胞所分泌的单克隆抗体是针对人C_(19)特异性的。经鉴定单克隆抗体A_4属小鼠IgG_(2b)、B_6及D_5属IgG_1亚类。杂交瘤细胞系核型分析表明,染色体数为87～91条。 三株杂交瘤细胞系贮存液氦6个月,体外培养传20代,仍保存分泌特异性单克隆抗体的能力。     

抗鸡IgG单克隆抗体的研究

本研究利用小鼠骨髓瘤Sp2/0细胞系与鸡IgG免疫的BALB/c小鼠脾细胞融合,建立了5个能持续分泌抗鸡IgG单克隆抗体(鸡IgG-McAbs)的杂交瘤细胞株(C_(41)、C_(42)、C_(43)、C_(44)C_(45));染色体数分别平均为99、99、101、102和99。用琼脂双扩散试验(ID)、间接ELISA和ELISA双抗体夹心法等试验证明:杂交瘤细胞所分泌的单克隆抗体只与鸡IgG发生特异性反     

抗口腔支原体单克隆抗体的制备研究

本研究通过BALB/c小鼠免疫脾细胞和小鼠骨髓瘤细胞(Sp 2/0)融合,融合率为100％(72/72),抗体阳性率为77.79％,并经筛选、克隆、建立37株分泌抗口腔支原体单克隆抗体的 杂交瘤细胞株(2A_(10)、2A_(11)、3C_8、2C_(10)、3G_3、2H_7和2H_(12)),抗体滴度介于4000～64000之间。同时,建立了检测抗体的BA法。该抗体与相应的口腔支原体以及人肺炎支原体、牛     

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。     

人白细胞介素15单克隆抗体的制备及鉴定

目的 :用rhIL 15与沙门氏菌裸菌相偶联制备IL 15单克隆抗体 (monoclonalantibody ,McAb) ,以便为疾病的诊断、治疗和发病机制的研究提供可靠的生物制剂。方法 :将纯化的rhIL 15蛋白与沙门氏菌裸菌相偶联制成免疫原 ,用脾内、腹腔、静脉三种途径相结合免疫BALB c小鼠 ,以PEG为促融剂 ,将脾细胞与SP2 0细胞进行融合 ,HAT选择培养 ,间接ELISA筛选阳性克隆 ,通过多次克隆化 ,获得稳定分泌特异性McAb的杂交瘤细胞株 ,并对所获得的细胞株及分泌的McAb特性进行了分析。结果 :获得 4株能稳定分泌特异性McAb的杂交瘤细胞系 (hybridomacell,Hc) ,ELISA法检测其效价分别为 1:10 6 、1:10 7、1:10 7、1:10 7。Dotblot检测IL 15的灵敏度为 1 5ng ,其中一株 (1 7E4 )尚可用于Westernblot检测。结论 :成功制备了 4株IL 15McAb杂交瘤细胞系。     

杂交瘤细胞(10株)染色体形态、数目与抗体分泌的关系

目的:探讨杂交瘤细胞染色体形态、数目与抗体分泌能力及稳定性的关系。方法:对我室融合成功的10株杂交瘤细胞,采用秋水仙素法制备染色体,分别计数100个完整的中期分裂相细胞,并观察其形态。制备腹水和收集细胞培养上清液,间接ELISA法检测抗体效价。结果:经染色体计数与分析,10株杂交瘤细胞中,7株瘤株细胞间染色体形态一致,分散好,既有端着丝点染色体,也见中间着丝点染色体,染色体数目的标准差小于5.0,腹水和细胞培养上清抗体效价高,分泌稳定性好;3株染色体数差异大、标准差大的瘤株中,2株抗体效价低,分泌不稳定。结论:杂交瘤细胞染色体形态、数目与其分泌单克隆抗体的能力和稳定性有关,进行染色体分析有利于了解瘤株特性,可作为筛选高效、稳定瘤株的参考依据。     

人绒毛膜促性腺激素单克隆抗体的研制

目的获得针对人绒毛膜促性腺激素单克隆抗体。方法hCG蛋白免疫Balb／c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞NS-1按8：1比例融合,间接ELISA法筛选阳性克隆,有限稀释法进行克隆化培养;制备腹水抗体;采用间接ELISA法鉴定抗体亚型和测定抗体效价。结果得到6株能稳定分泌单克隆抗体的杂交瘤细胞株;抗体经鉴定均为IgG1、κ型,效价均达10^-5以上。结论所获得的6株杂交瘤细胞株均有较强稳定分泌抗-hCG单克隆抗体的能力。这为有关hCG的检测、hCG本身相关研究以及避孕疫苗的研制打下了基础。     

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。     

Increased proportion of B cell hybridomas secreting monoclonal antibodies of desired specificity in cultures containing macrophage-derived hybridoma growth factor (IL-6)

The addition of macrophage feeder cells or conditioned medium has been shown to increase the yield of murine hybridomas obtained after the fusion of myeloma cells and activated B lymphocytes. It has been shown recently that the conditioned medium contains a growth factor (HGF) active on newly formed hybridomas and that the human HGF is similar to B cell stimulatory factor 2 which can induce the synthesis of antibodies in transformed B cells. We have compared in several fusion experiments the stimulatory effects of HGF both on the yield of hybridomas and on the number of antibody-secreting hybridomas. The results obtained clearly showed that while the stimulatory effect of HGF on the yield of growth-positive wells was variable and sometimes barely detectable, the proportion of growth-positive wells containing monoclonal antibodies was consistently much higher in the HGF-containing cultures. These results suggest that the majority of the antibody-secreting newly formed hybridomas are sensitive to HGF and indicate that HGF is a very useful culture supplement for the generation of a high number of antibody-producing hybridomas even if it may not increase significantly the yield of viable hybridomas.     

Enhancement of hybridoma production by medium supplemented with murine ascitic fluid

We have greatly improved the yield after cell fusion of antigen-specific monoclonal antibody-secreting murine hybridomas by substitution of Sp2/0 ascites for fetal bovine serum (FBS) in the culture medium. As a medium supplement for established cultures, Sp2/0 ascites at 2.5% in Dulbecco's modified Eagle's medium (DMEM) provided growth characteristics similar to media containing 10% or 20% FBS. All culture parameters associated with hybridoma fusion experiments, however, were advantageously affected in ascites-supplemented cultures. Clonal growth of hybridomas from the single cell stage was enhanced at least two-fold over 20% FBS-supplemented medium. Following fusion, both the number of colonies and hybridoma growth rates were substantially increased for ascites-containing cultures. Most importantly, the number of antigen-specific antibody-secreting hybridomas was increased in Sp2/0 ascites supplemented cultures, five-fold in the eight fusion experiments presented here. This improved performance compared to FBS-supplemented medium is reproducible from lot to lot of ascites.     

A simple,single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies

A simple method is described by which hybridomas can be selected and cloned in a single step immediately after fusion. This is done by plating the cells in semi-solid medium containing methylcellulose and the components of the HAT selection system. A number of variables have been examined in order to optimise the technique. The system is particularly suitable for isolating large numbers of hybridomas secreting different monoclonal antibodies. Evidence is presented to show that the colonies which grow in the system are in all probability clones. Thus, the need for routine recloning of the hybridomas is eliminated. In this way, the technique cuts down on the amount of tissue culture work associated with the production of monoclonal antibodies. Using this technique, it is easier to plate out large numbers of cells and to recover many independent hybridoma clones, than is the case when using cloning by limiting dilution.     

Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells

Several modifications at various stages of the standard hybridoma technique were found to increase the yield of monoclonal antibody-producing cells. Lymphocytes obtained from draining lymph nodes of mice immunized over a 10 day period with antigen injected into the foot pads were used for cell fusion. Preincubation of myeloma cells with lymphocytes in the presence of 0.25% polyethylene glycol at 37 degrees C for 90 min increased the yield of antibody-secreting hybrid colonies ten times. The use of conditioned medium from cultivated rat thymocytes ('lymphokines') as a supplement to cultivation medium made it unnecessary to use feeder cells, and increased the growth rate of the hybridomas. No change of the culture fluid was needed during the time which was necessary to grow up the cells to be tested for monoclonal antibody production. By a combination of the described procedures, the time required from the start of immunization to the screening for positive hybridomas was shortened to 23 days.     

Enhancement of hybridoma formation by addition of insulin to HAT medium (HIAT)

The effects of insulin on the formation of hybridoma clones following fusion experiments with SRBC immunized BALB/c mouse spleen cells and P3U1 mouse plasmacytoma cells were evaluated. The addition of insulin to HAT medium (HIAT) resulted in significant increases in the number and size of hybridoma colonies generated. The total number of anti-SRBC antibody-secreting clones also increased as much as sevenfold using insulin-supplemented medium compared to HAT alone. In view of the increasing interest in hybridoma technology for monoclonal antibody production, the use of insulin-supplemented medium (HIAT) may significantly expedite ongoing work by providing a more efficient method for the establishment of stable clones.     

Characterization of an established mouse-human heterohybridoma and its application for production of (mouse-human)-human triple hybridoma secreting human immunoglobulin

We report the construction of a mouse-human (M-H) heterohybridoma by fusion of the murine myeloma cell line NS-1 and human spleen cells from a 17 week old fetus. The nonsecreting, cloned hybridoma cell line II was resistant to 8-azaguanine (8-AG) and sensitive to hypoxanthine, aminopterin and thymidine (HAT) medium. It grew rapidly in 8-AG containing medium (doubling time 20 hrs.), but did not grow in HAT medium or in non-serum medium. It had a high fusion frequency with human lymphocytes from regional lymph nodes. Five human chromosomes were retained stably for over 6 months by this cell line II. Nine (mouse-human)-human ((M-H)-H) triple hybridomas secreting human IgG 1 or IgM were established by the fusion of this parental cell line II and human lymphocytes from regional lymph nodes. Immunoglobulin secretion was stable and has been maintained for over 8-10 months without recloning in these hybridomas. Secretion of immunoglobulin varies from 2.1-3.0 micrograms/10(6) cells/day, and these hybridomas contain from 3 to 16 human chromosomes, including No. 14. So, this M-H heterohybridoma II is an excellent useful parental cell line for the production of hybridomas secreting human immunoglobulin.     

Effect of the elapsed time after the final antigen boost on the specificity of monoclonal antibodies produced by B cell hybridomas

We have studied the effect of the number of days following the last antigen boost on the specificity of monoclonal antibodies produced by B cell hybridomas using spleen cells of mice immunized with human red cells of the A blood group. We showed, as previously observed by others, that the highest numbers of monoclonal anti-human red blood cells were obtained in fusions done 3 and 4 days after the final boost. However differential screening of the hybridoma cultures showed that the majority of the monoclonal antibodies reacting with the A blood group antigen were obtained in fusions done only 2 days after the last antigen injection. These results show that the delay between the final boost and the fusion experiment can influence not only the total number of antibody-secreting hybridomas but also the specificity of the antibodies produced.     

Production of monoclonal mouse IgE antibodies with DNP specificity by hybrid cell lines

IgE anti-DNP antibody-secreting hybridomas were obtained by polyethylene glycol fusion of murine myeloma cells with spleen cells from mice, primed with DNP-KLH and challenged with DNP-Nippostrongylus brasiliensis extract. The in vivo and in vitro continuously growing hybridomas are producing high amounts of monoclonal IgE anti-DNP antibodies, which are heat-labile, hapten-specific and have rat mast cell sensitizing capacity.     

Preparation and characterization of hybridomas producing monoclonal antibodies against human alpha interferon

Hybridomas producing monoclonal antibodies against human alpha interferon (hu-IFN alpha) were constructed by fusion of NSO myeloma cells with spleen cells of BALB/c mice immunized with purified hu-IFN alpha. Altogether, 527 hybridomas were prepared in two separate experiments. From this cohort of hybridomas, 51 produced monoclonal antibodies against hu-IFN alpha. Seventeen out of fifty one hybridomas produced antibodies with neutralizing capacity for IFN while 34 hybridomas produced monoclonal antibodies with binding ability not accompanied with the neutralization of biological activity of IFN. The specificity of antibodies was determined with 3 types of tests: ELISA, ELISAN (modified ELISA) and neutralization test. Using isotype analysis, it has been found that 23 monoclonal antibodies were of IgM class, 20 were of IgG1 subclass, 4 were of IgG2b and 4 of IgG3 subclass. The average number of chromosomes in hybridomas was between 61.35 and 78.55. Their average doubling time was between 13.95 and 25.76 hrs.     

Testing of monoclonal antibodies to human interferon

A comparative study of testing methods for polyclonal and monoclonal antibodies to human interferon using direct and reverse neutralization of the antiviral activity of interferon as well as ELISA was carried out. The activity of antibodies in ELISA was dozens of times higher than in neutralization tests. Polyclonal antibodies from the sera of mice immunized with alpha 2 interferon had a higher neutralizing capacity. M-5 monoclonal antibodies in specimens of ascitic fluid induced by inoculation of mice with hybrid cells exhibited an increase in both binding and neutralizing activity as compared with specimens of the culture fluid. Immunoglobulins from the ascitic and culture fluid of nonproductive myeloma cells as well as hybridomas producing monoclonal antibodies of other specificities showed practically no reaction with interferon in any of the tests under study. The screening of monoclonal antibodies intended for research and biotechnological purposes requires a composite analysis in both neutralization and binding tests in order to recover purposefully the hybrid clones producing antibodies with both or one of these properties.