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Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1–8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57–60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits. [ABSTRACT FROM AUTHOR]

The spread of the disease caused by monkeypox virus (MPox) since 2022 has shown the urgency of developing countermeasures. The development of modern methods of clinical laboratory diagnostics of MPox contributes to this. Enzyme-linked immunosorbent assay (ELISA) is an accessible and sensitive platform for developing diagnostic tools. Detection of MPox antigens using ELISA kits based on monoclonal antibodies (MAbs) is promising due to the quick time of analysis and minimal requirements for sample preparation. We have developed and deposited two strains of Escherichia coli that produce recombinant proteins. Mice were immunized with the AgPOX protein, which contains unique antigenic sequences of MPox. The Trx + A29 protein for selecting MAb producers includes the original amino acid sequence A29L. The absence of antibody crossover to Trx protein and native preparations of variola virus and vaccinia virus tested by ELISA. As a result of hybridization of splenocytes from immunized mice, MAb producers were obtained. Fifteen MAb-producing hybridomas were selected based on ELISA results with three specific MPox antigens and three nonspecific ones. Three hybridomas were selected for deposit according to the productivity criteria. The possibility of detection by means of its MAbs of the native MPox antigen at various concentrations was tested and method sensitivity was determined. The MAbs a-A29L_MPoxV of three hybridomas detected the native antigen MPox at a concentration of 102 PFU/mL. It is likely that the method is even more sensitive when selecting analysis conditions. Based on labeled MAbs a-A29L_MPoxV, it is possible to develop a sensitive and specific indirect two-step ELISA kit for immunodiagnostics of MPox. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are an established treatment for many illnesses, including cancer, toxicity and infectious diseases. The goal of this work was to optimize the conditions for obtaining hybridomas secreting human monoclonal IgG antibodies. A new protocol has been developed for efficient electrofusion of human B lymphocytes with partner cells. Electrofusion parameters were optimized, and the preferred partner cell line and ratio of cells involved in the fusion were selected. Two myeloma cell lines, K6H6/B5 and SHM-D33, were tested in detail, and the highest fusion efficiency was observed for K6H6/B5. Three hybridomas that secreted fully human monoclonal IgG antibodies were obtained using the optimized electrofusion protocol; the secretion of antibodies was observed for 1 month, which indicates the stability of the clones and the absence of chromosome segregation. [ABSTRACT FROM AUTHOR]

The purpose of this work was to obtain genus-specific monoclonal antibodies against the Legionella spp. recombinant PAL protein, which will subsequently allow to use them as a basis for the development of new express tests for pathogenic legionella detection. A short three-week immunization protocol for Wistar rats was used to generate rat–mouse heterohybridomas producing antibodies against PAL. Mouse myeloma cell line Sp2/0-Ag14 served as the fusion partner. Hybridization was performed using two methods: PEG-mediated fusion and electrofusion. Subsequent screening was performed by indirect solid-phase ELISA against the target protein rPAL. Specificity analysis was performed by dot-blot using a panel of lysates obtained from 39 pure cultures of different strains, which included closely related and heterologous microorganisms among others. No difference in the efficiency of stable hybridoma clones production by the two indicated cell-fusion methods was detected. Twelve clones producing specific rat monoclonal antibodies were obtained based on the screening results. The obtained rat monoclonal antibodies are highly specific towards the PAL protein of L. pneumophila of different serological groups and other pathogenic legionella and are good candidates to be used as the components of diagnostic test systems for the detection of pathogenic representatives of the Legionella genus. [ABSTRACT FROM AUTHOR]

Purpose: The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. Methods: Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. Results: Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. Conclusion: Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy. [ABSTRACT FROM AUTHOR]

This paper focuses on the production of a high-affinity monoclonal antibody (mAb) that can efficiently detect and block purinergic ligand-gated ion channel 7 receptor (P2X7R). To achieve this goal, the extracellular domain of human P2X7R, P2X7R-ECD, was used as an immunogen for BALB/c mice, inducing them to produce spleen lymphocytes that were subsequently fused with myeloma cells. Screening of the resultant hybridoma clones resulted in the selection of one stable positive clone that produced a qualified mAb, named 4B3A4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purity of the purified 4B3A4 mAb was above 85%, with prominent bands corresponding to molecular weights of 55 kDa (heavy chain) and 25 kDa (light chain), and the BCA assay showed that the concentration of the purified 4B3A4 mAb was 0.3 mg/mL. Western blot analysis revealed that the 4B3A4 mAb could specifically recognize and bind both P2X7R-ECD and the full-length P2X7R protein. Laser scanning confocal microscopy (LSCM) revealed that the 4B3A4 mAb specifically bound to P2X7R on the membrane of human peripheral blood mononuclear cells (PBMCs). P2X7R expression was significantly different between healthy individuals and people with certain cancers as determined by flow cytometry (FCM). In addition, the 4B3A4 mAb significantly reduced ATP-stimulated Ca2+ entry and YO-PRO-1 uptake, which indicated that the 4B3A4 mAb effectively blocked P2X7R activity. These data indicate that the 4B3A4 mAb can be further used as not only an antibody to detect cell surface P2X7R but also as a therapeutic antibody to target P2X7R-related signaling pathways. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (McAb) has been established as one of the most successful therapeutic strategies for the treatment of cancer. M1A2 (McAb) as a new monoclonal antibody was designed to recognize heat shock protein (HSP60), but its optimum production condition has not been studied. In this study, the cell culture conditions for both Roswell Park Memorial Institute Medium (RPMI 1640) and Dulbecco's Modified Eagle Medium (DMEM) were optimized using artificial neural network (ANN) analysis to obtain maximum production of IgM McAb by hybridoma M1A2 cells. By using a central composite design, an experimental matrix with cultivation parameters of incubation time, temperature and fetal bovine serum (FBS) concentration on IgM McAb production was designed. The results was analysed by ANN network with different learning algorithms. From the analysis, batch back propagation (BBP) trained ANN composed of eight hidden nodes using a hyperbolic tangent sigmoid transfer function was capable to provide the highest McAb production for both RPMI and DMEM media. Under optimum conditions of 12.5% of FBS, at 33 °C after 3(1/2) days of incubation, maximum McAb production (1132.69 μg/ml) in DMEM was achieved. With PRMI 1640 medium, maximum McAb production (1105.12 μg/ml) was achieved at optimum conditions of 11% of FBS, at 33 °C after 4 days of incubation. The results of this study will provide information for optimum culture conditions of M1A2 McAb production in both DMEM and RPMI 1640 media and also give some clues for the other hybridoma excreting antibodies in the development of in vitro cell culture. [ABSTRACT FROM AUTHOR]

The ubiquitously expressed protein RapGEF1 (C3G) regulates multiple cellular activities and is essential for early embryonic development in mammals. It has functions dependent on its catalytic activity as well as protein interaction domain and regulates β-catenin signaling. This study describes the generation of a novel monoclonal antibody, 3F6mAb and its characterization for recognition of RapGEF1. Mice were immunized with recombinant protein having only the Crk binding region of RapGEF1 and hybridoma clones created by fusion of immunized spleen cells with Sp2/0 myeloma cells. This antibody recognizes human, primate and murine RapGEF1 protein. Based on the recognition of various deletion constructs, we have mapped its epitope to 580-648 amino acids. Isotyping showed that it belongs to IgG1 class of heavy chain and Kappa light chain. 3F6mAb is suitable for detecting cellular RapGEF1 by western-blotting, immunofluorescence and immunoprecipitation. It has an advantage over most of the commercially available antibodies as it can detect N- and C-terminal truncated forms of RapGEF1. Using this antibody to detect mobility shift, we show that RapGEF1 is phosphorylated on tyrosine as well as S/T residues in its Crk binding domain. This monoclonal antibody is a valuable tool that will aid in understanding functions of cellular RapGEF1. [ABSTRACT FROM AUTHOR]

Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell's metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells. [ABSTRACT FROM AUTHOR]

The nuclear receptor Rev-erbβ is a potent transcriptional factor whose functional study has been limited by the lack of suitable antibodies against it. To better understand Rev-erbβ's biological roles, we generated five hybridoma cell lines secreting antibodies against human Rev-erbβ in mice immunized with the purified, prokaryotically expressed recombinant Rev-erbβ-6His fusion protein. Using Western blotting and immunofluorescence analyses, all the five monoclonal antibodies (MAbs) showed strong immunoreactivity to both prokaryotically and eukaryotically expressed recombinant Rev-erbβ. An immunoprecipitation study showed that all five monoclonal antibodies against Rev-erbβ were able to pull down the recombinant Rev-erbβ-Flag protein, but only one of the MAbs against Rev-erbβ, 37H8, could pull down the endogenous Rev-erbβ protein. Furthermore, domain specificity of these MAbs was characterized. Due to the high similarities between Rev-erbα and Rev-erbβ in the C and E domains, those C and E domain-specific anti-Rev-erbβ antibodies can react with human Rev-erbα as well. The MAbs produced in the study will provide a valuable tool for investigating the function of Rev-erbβ. [ABSTRACT FROM AUTHOR]

IL-37 has been described as a natural inhibitor of immune responses. Monoclonal antibody (mAb) against human IL-37b with high affinity and specificity can serve as a molecular probe to detect IL-37 and study IL-37 functions, mechanisms and related signal pathways in inflammatory diseases. However, there are very few such mAbs against human IL-37 commercially available so far. In the current study, monoclonal antibodies against human IL-37b were developed by fusing splenocytes from immunized mouse with SP2/0 myeloma cells and polyethylene glycol. Then the antibodies were screened with prokaryotic expressed human IL-37b protein and eukaryotic expressed human IL-37b protein subsequently. Western blot and flow cytometry analysis revealed that selected mAb clons were able to recognize human IL-37 with high specificity. And more importantly, the IL-37b mAbs were fluorescently labeled and can be directly used in flow cytometry and immunohistochemistry. In conclusion, the current study developed new mAbs against human IL-37b, which are applicable in flow cytometry and immunohistochemistry. [ABSTRACT FROM AUTHOR]

Over the last decade, isolation and purification of monoclonal antibodies, for diagnostic analysis, have been carried out using the hybridoma expression system. The present study describes a novel example of a detection system using hybridoma cells containing antibody against O1 antigen directly for V. cholerae diagnosis, which is a major health problem in many parts of the world, especially in developing countries. This method has advantages such as simplicity, ease of process, and it does not require manipulation of hybridoma cell. For this approach, an efficient amount of fluorescence calcium indicator, fura 2-AM, was utilized, which emitted light when the intracellular calcium concentration increased as result of antigen binding to specific antibody. More reliable results are obtained via this method and it is considerably faster than other methods, which has the response time of less than 45 s for detection of V. Cholerae O1. Also, the limit of detection was computed to be 50 CFU/mL (<13 CFU per assay). In addition, no significant responses were observed in the presence of other bacteria with specific hybridoma or other cell lines exposed to V. cholerae O1. Furthermore, this method was successfully applied to V. cholerae O1 detection in spiked environmental samples, including water and stool samples without any pretreatment. All results reveal that hybridoma cells can provide a valuable, simple, and ready to use tool for rapid detection of other pathogenic bacteria, toxins, and analytes. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability. [ABSTRACT FROM AUTHOR]

Balb/С mice were immunized with recombinant Ebola virus glycoprotein. Following the selection, screening, and cloning of murine hybridomas, we obtained five genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from the ascitic fluid of Balb/С mice using Protein G affinity chromatography (for IgG) and euglobulin precipitation (for IgM). To select at least three candidate antibodies for testing in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of the epitope specificity of the isolated antibodies. Based on the data of immunoblotting and sandwich ELISA, it became evident that the epitope recognized by GPE 534 differs from the epitopes recognized by the monoclonal antibodies GPE 118 and GPE 325. The last two antibodies also have different epitope specificity: it follows from the immunoblotting data and from the data on the binding of these antibodies with the intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the biological activity studies and the development of recombinant counterparts, we selected three candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325. [ABSTRACT FROM AUTHOR]

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration. [ABSTRACT FROM AUTHOR]

The cell growth and monoclonal antibody production of the 55-6 hybridoma cell co-cultured with the murine thymoma cell line EL-4 at different initial 55-6:EL-4 ratios were investigated. Both populations were seeded in co-culture without previous stimulation and therefore with low constitutive CD40 and CD40 ligand (CD154) expression levels, and in the absence of exogenous co-stimuli. Viable cell density and growth rate data seem to suggest a competition for nutrients, which is detrimental for both cells in terms of biomass production and also of growth rate for 55-6. Final concentrations of antibody and specific antibody production rates were affected by the initial 55-6:EL-4 ratio. The 4:1 ratio yielded the highest IgG2a concentration, whereas the highest specific antibody production rate was obtained at the 2:1 ratio. Changes mainly in CD154 and also in CD40 expression in co-cultures could suggest cross-talk between both populations. In conclusion, different types of interactions are probably present in this co-culture system: competition for nutrients, cognate interaction and/or autocrine or paracrine interactions that influence the proliferation of both cells and the hybridoma antibody secretion. We are hereby presenting a pre-scale-up process that could speed up the optimization of large-scale monoclonal antibodies production in bioreactors by emulating the in vivo cell-cell interaction between B and T cells without previous stimulation or the addition of co-stimulatory molecules. [ABSTRACT FROM AUTHOR]

This study considers the problem of manipulating in an optimal way the perfusion and bleed flow rates of a continuous culture of hybridoma cells, so as to achieve a fast transient start-up and reject potential disturbances. The proposed solution makes use of an analysis of the properties of the steady state solutions of the nonlinear dynamic model of the cell culture, and in particular the relationship between the two main limiting substrates, glucose and glutamine. The solution is implemented using extended prediction self-adaptive control. Simulation results demonstrate the approach potentiality. [ABSTRACT FROM AUTHOR]

Recent outbreaks of orf in China have been attributed to a novel strain of Orf virus (ORFV) designated ORFV-Jilin. Currently, monoclonal antibodies (Mabs) have not yet been developed against this specific pathogen even though such entities could have potential applications regarding the diagnosis and characterization of ORFV-Jilin. Therefore, the current study was undertaken to generate Mab against the immunodominant ORFV059 protein of this virus. For this purpose, the ORFV-Jilin ORFV059 protein was expressed in Escherichia coli and subsequently used as an antigen to immunize mice and for the initial screening of hybridomas prepared from the mice for their ability to produce anti-ORFV059 protein Mabs via an indirect ELISA. Ten, positive hybridomas were identified in this manner and verified based on the ability of their released Mab to react specifically with both naturally and artificially expressed ORFV059 protein in Western blots. The two hybridomas with the greatest propensity to secrete Mab were subcloned three times before being introduced intraperitoneally into mice. Afterwards, both Mab were separately purified from the mice's ascetic fluids and found to successfully recognize the ORFV-Jilin ORFV059 protein in a variety of immunological assays. Thus, the widespread utility of these Mab as a diagnostic core reagent should prove invaluable for further investigations regarding the mechanisms of orf pathogenesis and the control of this disease. In this regard, it should be noted that Mab A3 was used to confirm the predicted late expression of the ORFV-Jilin ORFV059 protein during virus replication. [ABSTRACT FROM AUTHOR]

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions. [ABSTRACT FROM AUTHOR]

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis. [ABSTRACT FROM AUTHOR]

Human nuclear respiratory factor 2 alpha subunit (NRF-2α) is fundamentally important to cell function and the development. We aimed to establish the monoclonal antibody (MAb) against the human NRF-2α protein and to investigate its distribution in human hepatocellular carcinoma (HCC) and tumor-adjacent tissues. The 6× His-NRF-2α fusion protein was successfully induced and purified. One monoclonal antibody (MAb) against human NRF-2α, 1-D10-E1-B11-G3 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. Using immunohistochemical analysis, the expression of NRF-2α was investigated in 38 cases of HCC specimens and 14 cases of tumor-adjacent specimens. Staining was found positive in 9 cases of HCC tissues (23.7%) and 8 cases of normal hepatic tissues (57.1%). The higher-grade frequency of expression of NRF-2α in tumor-adjacent tissues was significantly higher ( P < 0.01) than that in tumor tissues, suggesting that NRF-2α may play important roles in carcinogenesis of HCC. [ABSTRACT FROM AUTHOR]

Alpha-thalassaemia 1 genetic disorder occurs when there is a deletion of two linked alpha-globin genes. The interaction between these abnormal genes leads to the most severe type of thalassaemia disease, haemoglobin (Hb) Bart's hydrops fetalis. The identification of alpha-thalassaemia 1 carriers and genetic counselling are essential for the prevention and control of severe thalassaemia diseases. In this study, we have developed a rapid screening method for identifying alpha-thalassaemia 1. A sandwich-type immunochromatographic (IC) strip test was developed, using the generated monoclonal anti-Hb Bart's antibody, to trace the Hb Bart's in haemolysates. When assayed by our IC strip test, all alpha-thalassaemia 1, HbH disease, HbH-Constant Spring (H-CS) disease, HbH-CS and heterozygous HbE (CSEA) Bart's disease, and homozygous alpha-thalassaemia 2 showed positive results. No false negative results were observed in these blood samples. In alpha-thalassaemia 2 heterozygotes, 83% of them showed positive reactivity. Among HbE (both homozygotes and heterozygotes), beta-thalassaemia (heterozygotes, homozygotes and beta-thalassaemia/HbE) and normal subjects, the IC strip test revealed negative reactivity of 100, 85 and 97%, respectively. These results indicate that this novel immunodiagnostic kit, in combination with red blood cell indices, is suitable for screening and ruling out mass populations for the presence of alpha-thalassaemia 1. [ABSTRACT FROM AUTHOR]

Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding domain from the C1303 cells. Genes encoding an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, VH) — linker — (light chain variable region, VL)] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly4-Ser)3 was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432 bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific drug compounds or the detoxification of drug abusers by immunotherapy. [ABSTRACT FROM AUTHOR]

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO2 supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities. [ABSTRACT FROM AUTHOR]

The subcommissural organ (SCO) is an ependymal differentiation located in the dorsal midline of the caudal diencephalon under the posterior commissure. SCO cells synthesize and release glycoproteins into the cerebrospinal fluid (CSF) forming a threadlike structure known as Reissner’s fiber (RF), which runs caudally along the ventricular cavities and the central canal of the spinal cord. Numerous monoclonal antibodies have been raised against bovine RF and the secretory material of the SCO. For this study, we selected the 4F7 monoclonal antibody based on its cross-reactivity with chick embryo SCO glycoproteins in vivo. E4 chick embryos were injected with 4F7 hybridoma cells or with the purified monoclonal antibody into the ventricular cavity of the optic tectum. The hybridoma cells survived, synthesized and released antibody into the CSF for at least 13 days after the injection. E5 embryos injected with 4F7 antibody displayed precipitates in the CSF comprising both the monoclonal antibody and anti-RF-positive material. Such aggregates were never observed in control embryos injected with other monoclonal antibodies used as controls. Western blot analysis of CSF from E4-E6 embryos revealed several immunoreactive bands to anti-RF (AFRU) antibody. We also found AFRU-positive material bound to the apical surface of the choroid plexus primordia in E5 embryos. These and other ultrastructural evidence suggest the existence of soluble SCO-related molecules in the CSF of early chick embryos. [ABSTRACT FROM AUTHOR]

IL-6 has been known to modulate the growth of many hybridoma cells and also promote resultant antibody productivity. However, IL-6 is so expensive that the use of IL-6-dependent hybridomas for industrial antibody production is not practical. In this study, we aimed at designing antibody/gp130 and antibody/EpoR chimeras which could tightly control cell growth in response to more affordable cognate antigen. Retroviral vectors encoding VH or VL region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2/transmembrane domains of erythropoietin receptor (EpoR) and cytoplasmic domains of either EpoR or gp130, were constructed, and a homodimeric or a heterodimeric pair of chimeric receptor combinations (VH-gp130 and VL-gp130 or VH-gp130 and VL-EpoR) were expressed in an IL-6-dependent hybridoma 7TD1. The chimeric receptor-derived growth signal was observed in both combinations, while some residual growth signal was observed in the absence of HEL. To reduce interchain interaction between the two receptor chains, we introduced mutations to the transmembrane domain of both chimera combinations. Consequently, the heterodimeric combination of VH-gp130 and VL-EpoR showed clear HEL-dependent cell growth, while the homodimeric combination of VH-gp130 and VL-gp130 showed reduced cell growth in the absence of HEL. This is the first report that an EpoR-gp130 cytoplasmic domain heterodimer could transduce a growth signal in hybridoma cells, indicating tight and economical growth control of hybridoma cells via our chimeric receptors. [ABSTRACT FROM AUTHOR]

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR. Activities of caspases 2, 3, 8 and 9 were also analyzed. Increase in the activities of the caspases was observed with up-regulation in the expression of FAS (6-8–fold) and PEG3 (2.5-fold), suggesting that the cells experienced apoptotic cell death via both the death receptor and mitochondrial pathways. [ABSTRACT FROM AUTHOR]

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca2+ and Mg2+ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl2 and MgCl2 concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca2+/Mg2+ feeding to 117 h with Ca2+/Mg2+ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 106 and 9.12 × 106 cells ml−1, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture. [ABSTRACT FROM AUTHOR]

A simple and rapid screening methodology based on the in vitro culture of murine hybridoma and human T-lymphocytes was developed to assess the potential immunomodulatory activity of water-soluble extracts (WSE) from cheese. The two immune cell lines were cultured in microplates with or without cheese WSE. The proliferation and the metabolic activity of cells were monitored at their different growth phases by the BrdU (5-bromo-2′-deoxyuridine) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide] assays, respectively. WSE from Abondance cheese enhanced DNA synthesis and the metabolic activity of the hybridoma cells (1.2-and 1.3-to 1.6-fold of the control, respectively) and also of the T lymphocytes (1.5-fold of the control) for almost all of the dilutions tested. To evaluate the validity of the results obtained following this screening methodology, hybridoma and T-lymphocyte cells were cultivated in 50 ml flasks: the maximal cell density was increased by about 10–16% in presence of cheese WSE for both cell lines and the antibody production by the hybridoma cells was increased by 50%. [ABSTRACT FROM AUTHOR]

The combination of BD Cell MAb medium with the CL-1000 flask is increasingly being used to generate a few hundred milligram of antibody for early stage research projects. Cells are inoculated at 2 million per ml, and the antibody is harvested after 15 days or when the antibody concentration reaches above 10 mg ml−1, whichever comes first. Currently, there is no means to scale up beyond this production level using this technology. In this study, we evaluated hollow fiber technology as the scale up alternative. The hollow fiber system was run in batch mode to mimic the method used for the CL-1000 with BD MAb medium. The FL-NS murine hybridoma cell line was simultaneously inoculated at 2 million cells per ml in a CL-1000 and the Maximizer hollow fiber bioreactor system, a 21-fold theoretical scale up over the CL-1000. The Maximizer produced 23-fold more antibody, very close to the expected theoretical amount. However, production was complete after 9 days in the Maximizer, while the CL-1000 required the full 15 days for production. In summary, these results demonstrate successful scale up of antibody production from the CL-1000 to a hollow fiber system. [ABSTRACT FROM AUTHOR]

Lactate dehydrogenase (LDH) derived from rabbit muscle enhanced IgM production by human-human hybridoma HB4C5 cells 12.4-fold at 320 μg/ml under serum-free conditions. LDHs from pig muscle and pig heart also accelerated IgM production 8.4- and 6.4-fold, respectively. The immunoglobulin production stimulating activity of LDH was not accompanied by activation of cell proliferation. LDH from rabbit muscle facilitated IgM and IgG production by human peripheral blood lymphocytes. This means LDH stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers. LDH from rabbit muscle enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D. The immunoglobulin production-stimulating factors (IPSFs) effect of LDH was slightly weakened by sodium fluoride (translation inhibitor) treatment of HB4C5. Moreover, the amount of intracellular IgM of monensin-treated HB4C5 cells was obviously enhanced by LDH. This result means that the IPSF effect of LDH is irrelevant to the post-translation activity of target cells. It is expected from these findings that LDH from rabbit muscle accelerates the translation step to enhance immunoglobulin productivity. The immunoglobulin production-stimulating activity of LDH was inhibited by colchicine, endocytosis inhibitor. This fact suggests that it is necessary for LDH to be taken by target cells to act as an IPSF. [ABSTRACT FROM AUTHOR]

In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect) which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. [ABSTRACT FROM AUTHOR]

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis. [ABSTRACT FROM AUTHOR]

Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways. [ABSTRACT FROM AUTHOR]

In the present study an approach has been developed in order to examine the consequence of essential and non essential amino acid supplementation on VO208 hybridoma cells behaviour. The effect of amino acid enrichment has been studied taking into account the culture process, i.e., batch or continuous culture mode and the medium composition, i.e., a home made serum-free medium or a serum containing one. A group of 4 amino acids, i.e., Ser, Pro, Gly and Arg presented atypical evolution pattern of their extracellular concentration depending on the type of the medium and on the culture mode. Some amino acids were probably involved in the limitation of the cellular proliferation. Met was one of the amino acids that appears to may have been at limiting concentration in all cases. In continuous culture mode, an enrichment of amino acids resulted in a rapid improvement of the viable cell density in both media, with or without the presence of serum. For most amino acids, supplementation during continuous culture induced an increase of the amino acid uptake rate. A comparative analysis of amino acids utilisation, depending on the culture conditions studied in the present study, has been performed in order to propose an overall picture of amino acids metabolism by VO 208 Hybridoma cell line. [ABSTRACT FROM AUTHOR]

The ectopic expression of several members of the Bcl-2 family of anti-apoptotic proteins is a promising strategy to improve the viability of hybridoma cells in culture. However, the impact of post-translational modifications on the function of these proteins in murine hybridomas is unknown. To address this issue, the anti-apoptotic properties of a mutant of Bcl-xL devoid of the so-called “loop domain„ (Bcl-xL▵ 46-83) were investigated using the Sp2/ O-Ag14 hybridoma model. Clones of Sp2/ O-Ag14 cells expressing Bcl-xL▵ 46-83 exhibited resistance against L-glutamine deprivation to similar levels than cells expressing the wild type protein. In contrast, protection against the cytotoxic effects of cycloheximide (CHX) was highly dependent on the level of expression of the Bcl-xL▵ 46-83 mutant. Analysis of the growth behaviour of the transfected cells showed that Bcl-xL▵ 46-83 was superior to the wild type protein in prolonging Sp2/ O-Agl4 cell viability in stationary batch culture. Furthermore, the prolongation of cell viability in batch culture was directly proportional to the level of expression of the mutated protein. Our results indicate that removal of the loop domain improves the anti-apoptotic activity of Bcl-xL in hybridoma cells grown in stationary batch culture. [ABSTRACT FROM AUTHOR]

An analog of aromatic cytokinins, the 2,6,9-trisubstituted purine derivative bohemine, was applied to cultures of mouse hybridoma cells in order to analyze its capacity of suppressing cell growth and maintaining or enhancing the production of monoclonal antibody. Addition of bohemine at concentrations in the range of1–10 μM resulted in a short-term arrest of growth and of monoclonal antibody production. The short-term suppression of cell functions was followed by a significant temporary increase of specific growth rate and of specific production rate. The steady-state viable cell density values, found in semicontinuous cultures, showed a certain stimulation of cell growth in the range of micromolar concentrations of bohemine, and inhibition of growth at 10 and 30 μM concentrations. The profiles of cell cycle phases indicated that hybridoma cells are retarded both at the G1/S boundary and at the G2/M boundary, depending on bohemine concentration. The existence of the sequence of events,from suppression to stimulation, suggests that bohemine probably modulates more than one regulatory pathway in the cell. [ABSTRACT FROM AUTHOR]

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. [ABSTRACT FROM AUTHOR]

Five human human-mouse hybridomas secreting human monoclonal antibodies to rice allergens were established. Antibodies secreted from the hybridomas reacted with 14–16 kDa rice major allergen proteins. Analysis with overlapping peptides synthesized on a multi-pin apparatus revealed a binding sequence of the major rice allergen protein RA17. The antigenic determinant was located in the C-terminus region of the RA17 protein. [ABSTRACT FROM AUTHOR]

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures. [ABSTRACT FROM AUTHOR]

In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system. [ABSTRACT FROM AUTHOR]

The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations >1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate the de novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism. [ABSTRACT FROM AUTHOR]

The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10-7 M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas. [ABSTRACT FROM AUTHOR]

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed. [ABSTRACT FROM AUTHOR]

Glutamic acid was found to be growth inhibitory to a murinelymphocyte hybridoma in a concentration-dependent manner from 3to 12 mM glutamate. At 12 mM glutamate there was a 70% decreasein the specific growth rate of the cells. Attempts to alleviateinhibition or adapt cells to growth in glutamate-based mediawere unsuccessful. It is proposed that elevated glutamate levelsimpair adequate uptake of cystine, a critical amino acid for thesynthesis of glutathione. Glutathione is required by cells toprevent intracellular oxidative stress. The measured rate ofuptake of U-14C L-cystine into the cells was found to havethe following parameters: Km = 0.87 mM, Vmax = 0.9nmole/mg cell protein per min. The uptake was sodiumindependent and resembled the previously described x- ctransport system, with elevated glutamate levels causingextensive inhibition. Glutamate at a concentration of 1.4 mMcaused a 50% decrease in cystine uptake from the serum-freegrowth medium. Glutamate was taken up from the external medium(Km = 20 mM and Vmax = 12.5 nmole/mg cell protein permin) by the same transport system in a stereo specific, sodiumindependent manner. Of the amino acids examined, it was foundthat cystine and homocysteic acid were the most extensiveinhibitors of glutamate uptake and that inhibition was competitive. Metabolic profiles of the cells grown in culturescontaining enhanced glutamate levels revealed an overallincrease in net production of alanine, serine, asparagine andaspartate. A substantially increased specific consumption ofglutamate was accompanied by a decreased consumption of cystine,valine and phenylalanine.The combined kinetic and metabolic results indicate thatglutamate and cystine are taken up by the anionic transportsystem x- c. The increasing levels of glutamate in themedium result in a decreased transport of cystine by this systemdue to competitive inhibition by glutamate. [ABSTRACT FROM AUTHOR]

Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. [ABSTRACT FROM AUTHOR]

The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 106 cells/ml. However, when the cell density was over 107 cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed. [ABSTRACT FROM AUTHOR]

The murine hybridoma (CC9C10) was subjected to high shear rates in a spinner flask to determine the effect of various culture additives on cell survival. At 500 rpm, the half-life of the viable cell concentration in a low protein serum-free medium was 50 min. Both bovine serum albumin and Pluronic F-68 had a significant effect in protecting cells under these conditions. The effects of the two supplements were additive, so that in the presence of both supplements there was minimal cell damage at 500 rpm. The survival rate of cells grown in media supplemented with linoleic acid improved significantly under high stirring rates. Cells grown for one passage in 50 μM linoleic acid and stirred at 500 rpm had a significantly higher survival rate than control cells. For cells grown over 5 passages in 25 μM linoleic acid, the survival rate at 470 rpm was ×3 greater than that determined for control cells. This difference gradually decreased at higher stirring rates up to 610 rpm when the half-life of the viable cell population was reduced to ∼10 min. Supplementation of cultures with linoleic acid has previously been shown to result in incorporation into all three cellular lipid fractions - polar, non-polar and free fatty acid (Butler et al., 1997). Our explanation for the increased survivability of the cells at high agitation rates in the presence of linoleic acid is that the structural lipid components of the cell including the outer membrane attained a higher unsaturated/saturated ratio which was more robust than that of control cells. [ABSTRACT FROM AUTHOR]

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. [ABSTRACT FROM AUTHOR]