Continuous antibody production by phytohemagglutinin-L-aggregated hybridoma cells

Phytohemagglutinin-L (PHA-L) induced aggregation of SP2 myeloma and antibody-producing hybridoma cells. The aggregates were of diverse shapes, and major-axis length of the hybridoma aggregates was mostly 50-80 microm at 1 microg/ml PHA-L and 100-150 microm at 5 microg/ml. PHA-L did not suppress growth rate at these concentrations. The aggregated cells had almost the same antibody productivity as that of non-aggregated cells in a static culture. Essentially, identical results were obtained with soybean agglutinin (SBA). On the other hand, pokeweed mitogen (PWM) did not induce apparent aggregation at a concentration of 10 microg/ml. These results suggest that lectin binding to particular carbohydrate moiety on the cell surface is necessary for cell agglutination. Using PHA-L, a 200-ml suspension culture of aggregated hybridoma cells was successfully performed in a spinner flask over 10 days. The aggregates were mainly globular with a diameter of 50-100 microm at 1-2 microg/ml PHA-L. The aggregation greatly enhanced settlement of hybridoma cells by gravity, and medium exchanges were thereby easily performed in a short period. During a course of the semi-continuous culture, antibody concentrations of culture medium were maintained at approximately 10 microg/ml which was comparable to that of static culture of the aggregated hybridoma cells. Thus, the lectin aggregation is applicable to the separation of culture medium from anchorage-independent cells like hybridomas, and can be employed in a large-scale commercial production of biologically active proteins.     

Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens.

Human monoclonal antibodies which bind Schistosoma mansoni worm and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nien cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni.     

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.     

Cell activation by CpG ODN leads to improved electrofusion in hybridoma production

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.     

Integration vectors for antibody chimerization by homologous recombination in hybridoma cells

Gene targeting in hybridoma cells provides a tool for generating chimeric antibodies with great ease and at high yield. We present an evaluation of integration vectors for the chimerization of the immunoglobulin heavy chain locus which are universally applicable to hybridomas of different isotypes and mouse strains. There are three problems arising with vector integration: (i) the frequent persistence of the parental isotype; (ii) an isotype-dependent aberrant replacement-like recombination giving rise to antibodies devoid of the C_H1 domain; and (iii) secondary recombinations leading to excision of the integrated sequence. To overcome these problems, we have systematically evaluated the consequences of extending the vector flank. Although the homology length clearly determines the recombination frequency, this effect is counteracted by the secondary recombination, which also correlates to the homology length. In contrast, the truncating recombination events are not dependent on the homology length and never lead to re-excision of the construct. To take advantage of the increased genetic stability obtained with short flanks, we constructed an enrichment vector which yields high recombination efficiencies despite using a short flanking sequence. In addition, irradiation of the cells enhanced homologous recombination. The problem of the co-production of two isotypes was overcome by a two-step targeting reaction.     

Production of anti-RNA antibody by hybridoma cells: Purification from mixed immunoglobulin products

Fusion between spleen cells from an autoimmune NZB/NZW mouse and the Balb/c drugresistant MPC-11 myeloma resulted in the formation of a hybridoma-secreting RNA-specific IgG-3 antibody and the parental IgG-2b myeloma. Analysis of the mixed immunoglobulin assembly products made by the hybridoma cells showed efficient pairing of IgG-2b and IgG-3 heavy chains and did not show a marked preferential assembly of the homologous heavy and light chains. Partial purification of the anti-RNA antibody from the mixed assembly products was achieved by utilizing an antigen affinity column (RNA-Sepharose). The use of a heavy chain-specific affinity column (anti-IgG-2b-Sepharose) increased the purity of the desired antibody, but parental light chains were still present after this step. A complete purification of the RNA-binding protein could be achieved by papain cleavage of the total IgG fraction and binding of the resulting Fab fragments to RNA-Sepharose. This procedure may, therefore, be employed as a general method for purifying antibodies from hybridomas that continue to produce their parental myeloma chains.     

Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion

The fusion of a mouse-human heteromyeloma with a mouse hybridoma is used as a model to define parameters to generate human hybridomas. Electrofusion of these cells in 300 mosM and 75 mosM solutions showed that strong hypo-osmolar conditions resulted in a dramatic increase in the efficiency of hybridoma formation. In contrast to iso-osmolar electrofusion, a high hybrid yield could be obtained by injection of only a single field pulse. The field strength was adjusted in proportion to the increased size of the cells in hypo-osmolar solutions. Hypo-osmolar electrofusion allowed the generation of approximately 0.45% hybrids at a suspension density of 1.75 X 10(5) mouse-human cells/ml corresponding to an input number of 3.5 X 10(4) mouse-human cells. A further increase in the efficiency of hybridoma formation to about 0.6% was achieved by cell alignment in an alternating field of modulated field strength. Experiments in which the total cell number per fusion chamber was decreased at constant optimum suspension density showed that a further increase in the efficiency of hybridoma formation in hypo-osmolar solution was not possible because of the increasing influence of the heterogeneity of the cell lines with decreasing cell number. The results allow the conclusion that hypo-osmolar electrofusion is a potential tool to enhance successful immortalisation of human B lymphocytes.     

电融合技术与半固体培养技术结合制备抗人肠道腺病毒单克隆抗体

将１×１０７个经肠道腺病毒（Ａｄ４０／４１）免疫的小鼠脾细胞与３×１０６个骨髓瘤细胞进行电融合，再置ＩＭＤＭ甲基纤维素半固体选择培养基中进行选择培养，一次性得到１８个杂交瘤细胞克隆，并从中筛查出７株高阳性抗体分泌克隆。从而成功地运用电融合与半固体培养相结合的方法以少量的材料制得了抗肠道腺病毒单克隆抗体。     

Cloning hybridoma cells by limiting dilution

Summary A method is described for cloning hybridoma cells by limiting dilution. This technique utilizes a feeder layer of murine splenocytes to enhance the growth of clones.     

电融合技术与半固体培养技术结合制备抗人肠道病毒单…

Ten million splenocytes from the enteric adenovirus (Ad40/41) immunized mice were fused with 3 x 10(6) myeloma cells by electrofusion. Then, the cells were selectively cultured in methyl cellulose semi-solid medium. Eighteen clones of hybridoma were directely picked out from the culture plates and 7 of them could secret high level specific antibodies to enteric adenovirus. So far, the anti-Ad40/41 virus monoclonal antibodies were successfully generated by the protocal of electrofusion accompanied with selective culture in methyl cellulose semi-solid medium.     

Augmentation of the antibody forming cell response to neuraminidase-treated cells by myxoviruses.

Following the inoculation of Newcastle Disease virus, Sendai virus and influenza virus CBA mice made a specific plaque-forming cell response to virus-treated sheep red blood cells but not to virus-treated L1210 cells. This non-specific response to virus-treated L1210 could be exactly reproduced by treatment of L1210 with bacterial neuraminidase. Neuraminidase-treated cells from eight of nine vertebrates including chickens and syngeneic mice also registered plaques. Inoculations of the above viruses were considered to have evoked separate antibody forming cell responses to viral antigens, foreign cell components in the virus inoculum and to neuraminidase revealed cell membrance antigens.     

The generation of Ig-secreting UC 729-6 derived human hybridomas by electrofusion

UC 729-6, a 6-thioguanine resistant human lymphoblastoid B cell line, was fused with human lymphocytes by electrofusion. Resulting human-human hybridomas were tetraploid, expressed markers derived from both fusion parents, and secreted approximately 1 microgram Ig/10(6) cells/ml/day. Cells to be used for electrofusion were washed in 0.3M mannitol, and fusions were performed with glass slides, 1.0 ml, and 50.0 ml chambers. Fusion sequences consisted of alignment, compression, and the fusion event itself. The optimal cell concentration for electrofusion was 5 X 10(6)/ml. Fusion efficiencies of human lymphocytes were ranked as follows: lymphoma cells greater than lymph node lymphocytes greater than PBL. 80% of the lymphoma hybridomas, 60% of the lymph node hybridomas, and 40% of the PBL hybridomas were Ig secretors. These data demonstrate that the UC 729-6 cell line is a suitable vector for generating human-human hybridomas by electrofusion.     

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.     

Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC. Demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells.

In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility. However, there are some potential problems in the preparation of monoclonal antibodies. A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture. Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen. Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture. The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea. HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody. This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.     

Immunogenicity and therapeutic efficacy of dendritic-tumor hybrid cells generated by electrofusion

Dendritic cells (DCs) are potent antigen-presenting cells capable of inducing strong immune responses to weak tumor-associated antigens. Among various DC-based approaches, cancer immunotherapy with DC-tumor fusion hybrids offers advantages of polyclonal stimulation of a diverse array of tumor antigens. However, prevalent fusion methods using chemical fusogens such as polyethylene glycol often result in toxicity and low fusion efficiency. In this article, we describe an electrofusion technique, applicable to processing large numbers of cells with consistent and high fusion efficiency. Generation of fusion hybrids was verified by unequivocal experimental evidence. In animal models, fusion hybrids expressed the mature DC-like phenotype. They stimulated both CD4 and CD8 tumor-specific T cells to secrete interferon-gamma in vitro. In immunotherapy, a single vaccination with DC-tumor fusion cells along with interleukin-12 as an adjuvant eradicated tumors established in the skin nd lung. These results provide an impetus for treating cancer patients with similarly generated cells.     

Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice

Incomplete Freund's adjuvant was used as a priming agent prior to the injection of hybridoma cells in mice to expand monoclonal antibody production. Two hybridoma cell lines, FDO28B (IgG1) and FDO31C (IgM), which produce monoclonal antibodies reactive towards human placenta, were used. Monoclonal antibody was detected in the ascites fluids by agarose gel electrophoresis. It was found that the time interval between adjuvant priming and cell injection could be reduced to 1 day, allowing collection of ascites fluid containing monoclonal antibody within 2 weeks of priming. In addition, as few as 1 X 10(5) hybridoma cells were needed to collect approximately 5-7 ml of ascites fluid containing antibody detectable by gel electrophoresis. Thus priming with incomplete Freund's adjuvant enables production of large amounts of monoclonal antibody in a short time using a low number of hybridoma cells.     

Development of hybridoma cells producing monoclonal antibody against human epsilon (epsilon) chain

Epsilon (epsilon) chain is one of the five classes of immunoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of epsilon chain have high priority in development of diagnostic kits. In this study, an attempt was made to produce monoclonal antibodies against human epsilon chain. Balb/c mice were immunized with semipurified epsilon chain and spleen cells were fused with Sp2/0 mouse myeloma cell line in the presence of poly ethylene glycol. Supernatant of hybridoma cells were screened for detection of antibody by Enzyme-linked Immuno Sorbent Assay (ELISA) method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clones (monoclones) were selected by ELISA and confirmed by immunoblot. The subclasses of the chosen monoclonal antibodies were determined and the clones freezed and kept in liquid nitrogen. During this study three successful fusions were carried out, which resulted in over than 100 clones with high production of anti- epsilon chain. Twelve clones with the highest titers were selected for cloning. After limiting dilution more than 50 monoclonal antibodies were produced and the unsuitable one (C1F2) displayed higher absorbance in reaction with purified IgE, relatively high cross-reactivity with IgM, and the highest cross-reactivity with IgG. In Immunoblotting, presence of relatively high-density band in reaction with IgE was confirmed. The unsuitable monoclonal antibody was shown to be IgG1 subclass with kappa light chain. It seems that, this monoclonal antibody could not be successfully useful in diagnostic kits.     

Efficient generation of reciprocal signals by inhibition

Reciprocal activity between populations of neurons has been widely observed in the brain and is essential for neuronal computation. The different mechanisms by which reciprocal neuronal activity is generated remain to be established. A common motif in neuronal circuits is the presence of afferents that provide excitation to one set of principal neurons and, via interneurons, inhibition to a second set of principal neurons. This circuitry can be the substrate for generation of reciprocal signals. Here we demonstrate that this equivalent circuit in the cerebellar cortex enables the reciprocal firing rates of Purkinje cells to be efficiently generated from a common set of mossy fiber inputs. The activity of a mossy fiber is relayed to Purkinje cells positioned immediately above it by excitatory granule cells. The firing rates of these Purkinje cells increase as a linear function of mossy fiber, and thus granule cell, activity. In addition to exciting Purkinje cells positioned immediately above it, the activity of a mossy fiber is relayed to laterally positioned Purkinje cells by a disynaptic granule cell → molecular layer interneuron pathway. Here we show in acutely prepared cerebellar slices that the input-output relationship of these laterally positioned Purkinje cells is linear and reciprocal to the first set. A similar linear input-output relationship between decreases in Purkinje cell firing and strength of stimulation of laterally positioned granule cells was also observed in vivo. Use of interneurons to generate reciprocal firing rates may be a common mechanism by which the brain generates reciprocal signals.     

Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining

Methods are described for the immunocytochemical staining of cryostat sections of lymphoid tissue with enzyme conjugates of antigen, idiotype (Id) and anti-idiotype. Results established this as a useful approach, for simultaneously detecting Id and anti-Id antibody forming cells (AFC) in situ. As a model, the 5AF6 Id family associated with the BALB/c mouse antibody response against the p-azophenyl-arsonate (Ar) epitope was examined by two-color immunocytochemical staining, allowing the simultaneous detection of both Id+ and Id- anti-Ar AFC. Spleens from mice secondarily immunized with Ar antigen but not normal mice contained anti-Id AFC stained with the 5AF6 Id but not with another immunoglobulin of the same isotype. A sequential staining method was developed which allowed the detection of both Id and anti-Id AFC in the same tissue, thus providing a means of examining Id and anti-Id antibody networks in intact lymphoid tissues.