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Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions.

Дипломний проєкт: 135 с., 26 рис., 12 табл., 13 формул, 90 посилань. Робота присвячена уніфікації та вдосконаленню технології масового напрацювання виробництва моноклональних антитіл у біореакторі, шляхом іммобілізації клітин продуцента на поверхні напівпроникних мембран та перфузійного типу подачі поживного середовища. На основі порівняльних характеристик, основним продуцентом було обрано найбільш стабільний та високо імуногенний штам гібридомних клітин, отриманих шляхом злиття клітин мієломи та спленоцитів мишачого походження. Штам 95E1 характеризується титром 1:1000 у культуральній рідини та ізотопом антитіл Ig G2α. У роботі наведений опис основних етапів технології з наведенням біохімічних характеристик та фізико-хімічних параметрів. Серед конструкцій біореакторів, що використовуються в технології, було обрано мембранний ферментер половолоконного типу, культивування в якому забезпечує високий вихід цільового продукту. На основі наявної моделі біореактору, було проведено технологічні, конструкційні та гідравлічні розрахунки, з метою модернізації технологічних параметрів та виробничих показників., Diploma work: 135 p., 26 figures, 12 tables, 13 formulas, 90 references. The work is devoted to the unification and improvement of the technology of mass production of monoclonal antibodies in the bioreactor, by immobilization of producer cells on the surface of semipermeable membranes and perfusion type of nutrient medium. Based on comparative characteristics, the main producer was selected by the most stable and highly immunogenic strain of hybridoma cells obtained by fusion of myeloma cells and splenocytes of mouse origin. Strain 95E1 is characterized by a titer of 1: 1000 in the culture fluid and an isotope of Ig G2α antibodies.Presented data describes the main stages of technology with biochemical characteristics and physicochemical parameters. From between of the designs of bioreactors used in the manufacturing technology, based on the index of a high yield of the target product, was chosen membrane bioreactors with hollow fiber cartridges type. In result of using the real model of the bioreactor as prototype, the technological, structural and hydraulic calculations were performed in order to modernize the technological parameters and production indicators.

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.Copyright © 2019, The Author(s).

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.Copyright © 2019, The Author(s).

Commercial antibodies against varying proteins are often not optimal for identification of proteins in their amyloid fibril forms. Reasons can be the different conformation but also a variety of modifications like N- or C-terminal truncation. Therefore, development of own monoclonal antibodies against amyloid fibril proteins may be advantageous. This chapter gives suggestions of how to be successful in such approaches.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

For about four decades, hybridoma technologies have been the “work horse” of monoclonal antibody production. These techniques proved to be robust and reliable, albeit laborious. Over the years, several major improvements have been introduced into the field, but yet, antibody production still requires many hours of labor and considerable resources. In this work, we present a leap forward in the advancement of hybridoma-based monoclonal antibody production, which saves labor and time and increases yield, by combining hybridoma technology, fluorescent particles and fluorescence-activated cell sorting (FACS). By taking advantage of the hybridomas’ cell-surface associated antibodies, we can differentiate between antigen-specific and non-specific cells, based on their ability to bind the particles. The speed and efficiency of antibody discovery, and subsequent cell cloning, are of high importance in the field of infectious diseases. Therefore, as a model system, we chose the protein LcrV, a major virulence factor of the plague pathogen Yersinia pestis, an important re-emerging pathogen and a possible bioterror agent.

For about four decades, hybridoma technologies have been the “work horse” of monoclonal antibody production. These techniques proved to be robust and reliable, albeit laborious. Over the years, several major improvements have been introduced into the field, but yet, antibody production still requires many hours of labor and considerable resources. In this work, we present a leap forward in the advancement of hybridoma-based monoclonal antibody production, which saves labor and time and increases yield, by combining hybridoma technology, fluorescent particles and fluorescence-activated cell sorting (FACS). By taking advantage of the hybridomas’ cell-surface associated antibodies, we can differentiate between antigen-specific and non-specific cells, based on their ability to bind the particles. The speed and efficiency of antibody discovery, and subsequent cell cloning, are of high importance in the field of infectious diseases. Therefore, as a model system, we chose the protein LcrV, a major virulence factor of the plague pathogen Yersinia pestis, an important re-emerging pathogen and a possible bioterror agent.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

The request and the process of procurement and preparation of biological speciments was considered for the following events with reference to the IML (International Microgravity Laboratory)-2 experiment definition document. The result was compiled on the document of request and process for/of procurement and preparation of biological speciments: (1) PS (Payload Specialist) training in Japan; (2) hanger L simulation (rehearsal) at KSC (Kennedy Space Center); and (3) flight experiment and ground experiment. With reference to the above document, biological speciments were procured and prepared for PS training in Japan which were executed during March to May in 1993. The speciments were prepared until the time of deliver and they were delivered to the Kobe Shipyard and Machinery Works of Mitsubishi Heavy Industries, Ltd. (MHI)., IML(国際微小重力実験室)-2実験計画書をもとに、下記の宇宙実験などに使用する生物試料の調達・調製要求および手順について検討するとともに、代表研究者との調製を支援し、その結果を反映して生物試料調達・調製要求書および手順書としてまとめた。(1)日本におけるPS(搭乗科学技術者)訓練、(2)KSC(ケネディ宇宙センター)ハンガーLリハーサル、および(3)軌道上実験および地上対照実験。前項で作成した調達・調製要求書および手順書にしたがって、日本におけるPS訓練(1993年3〜5月実施)用生物試料を調達・調製し、入手から納入までの期間維持し、さらにPS訓練実施時に三菱重工業(株)神戸造船所に納入した。

Increasing demand on more effective cell culture reactors has driven optimization works to increase output of products. This has led to development of soft sensors that uses mathematical formulas to increase the available information for the parameters during runs. In the project two parameters was evaluated for use in such a soft sensor, viability by measuring on-line capacitance with Aber probe and L-lactate production using BioSenz apparatus. To determine how well these could be used both were used on batch reactors measuring on a mouse-mouse B cell hybridoma culture which produced IgG1. On-line measurements were performed by probes which measured directly on the cell suspension or withdrew sterile sample from the reactor. Measuring viability gave results with low error, which can be concluded to the variation in reference cell count, but it could not be determined if measuring L-lactate production with BioSenz works in reactors of this size. More work needs to be done on other types of reactors, like fed-batch or perfusion, or lower working volumes.

Increasing demand on more effective cell culture reactors has driven optimization works to increase output of products. This has led to development of soft sensors that uses mathematical formulas to increase the available information for the parameters during runs. In the project two parameters was evaluated for use in such a soft sensor, viability by measuring on-line capacitance with Aber probe and L-lactate production using BioSenz apparatus. To determine how well these could be used both were used on batch reactors measuring on a mouse-mouse B cell hybridoma culture which produced IgG1. On-line measurements were performed by probes which measured directly on the cell suspension or withdrew sterile sample from the reactor. Measuring viability gave results with low error, which can be concluded to the variation in reference cell count, but it could not be determined if measuring L-lactate production with BioSenz works in reactors of this size. More work needs to be done on other types of reactors, like fed-batch or perfusion, or lower working volumes.

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed alpha, beta, and gamma subunits of high-affinity immunoglobulin E (IgE) receptor (FcERI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of 1L-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-alpha, and cyclooxygenase 2, and production of prostaglandin D2 and leukotriene C4 in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-alpha expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcERI- and TLR-mediated effector functions of mast cells.

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. © 2013 Springer Science+Business Media Dordrecht

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I50 value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food. © Taylor & Francis Group, LLC.

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I50 value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food. © Taylor & Francis Group, LLC.

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is commonly associated with the A3243G mitochondrial DNA (mtDNA) mutation encoding the transfer RNA of leucine (UUR) (tRNA (Leu(UUR))). The pathogenetic mechanisms of this mutation are not completely understood. Neuronal functions are particularly vulnerable to alterations in oxidative phosphorylation, which may affect the function of the neurotransmitter glutamate, leading to excitotoxicity. In order to investigate the possible effects of A3243G upon glutamate homeostasis, we assessed glutamate uptake in osteosarcoma-derived cytoplasmic hybrids (cybrids) expressing high levels of this mutation. High-affinity Na(+)-dependent glutamate uptake was assessed as radioactive [(3)H]-glutamate influx mediated by specific excitatory amino acid transporters (EAATs). The maximal rate (V(max)) of Na(+)-dependent glutamate uptake was significantly reduced in all the mutant clones. Although the defect did not relate to either the mutant load or magnitude of oxidative phosphorylation defect, we found an inverse relationship between A3243G mutation load and mitochondrial ATP synthesis, without any evidence of increased cellular or mitochondrial free radical production in these A3243G clones. These data suggest that a defect of glutamate transport in MELAS neurons may be due to decreased energy production and might be involved in mediating the pathogenic effects of the A3243G mtDNA mutation.

As part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new method that enables researchers to quickly evaluate a huge range of potentially useful antibodies, thereby bypassing the more costly and time-consuming hybridoma technique. The Tomlinson I and J phage libraries were used to select phage antibodies exhibiting affinity for sulfur mustard adducts on keratins, isolated from human callus. Two kinds of phage antibodies were obtained: antibodies recognizing keratin and antibodies recognizing keratin which was exposed to sulfur mustard. These phage antibodies retained activity after repeated culturing and culturing in larger volumes. For the first time antibody phage display was successfully applied for immunodiagnostics of a chemical warfare agent. © 2007 The Authors.

The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of M(r) 33-226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals

H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutamine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. H-1/N-15 NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorporated into alanine. (NH4+)-N-15 was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that the [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance., QC 20100525

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359-368; E. Suzuki, D.F Ollis, Biotechnol. Bioeng. 34 (1989) 1398-1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61-69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture. (C) 2000 Elsevier Science S.A. All rights reserved.

Batch and fed-batch cultures of a murine hybridoma cell line (AFP-27) were performed in a stirred tank reactor to estimate the effect of feed rate on growth rate, macromolecular metabolism and antibody production. Macromolecular composition was found to change dynamically during batch culture of hybridoma cells possibly due to active production of DNA, RNA and protein during the exponential phase. Antibody synthesis is expected to compete with the production of cellular proteins from the amino acid pool. Therefore, it is necessary to examine the relationship between cell growth in terms of cellular macromolecules and antibody production. In this study, we searched for an optimum feeding strategy by changing the target specific growth rate in fed-batch culture to give higher antibody productivity while examining the macromolecular composition. Concentrated glucose (60 mM) and glutamine (20 mM) in DR medium (1:1 mixture of DMEM and RPMI) with additional amino acids were fed continuously to the culture and the feed rate was updated after every sampling to ensure exponential feeding (or approximately constant specific growth rate). Specific antibody production rate was found to be significantly increased in the fed-batch cultures at the near-zero specific growth rate in which the productions of cellular DNA, RNA, protein and polysaccharide were strictly limited by slow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359-368; E. Suzuki, D.F Ollis, Biotechnol. Bioeng. 34 (1989) 1398-1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61-69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture. (C) 2000 Elsevier Science S.A. All rights reserved.

Batch and fed-batch cultures of a murine hybridoma cell line (AFP-27) were performed in a stirred tank reactor to estimate the effect of feed rate on growth rate, macromolecular metabolism and antibody production. Macromolecular composition was found to change dynamically during batch culture of hybridoma cells possibly due to active production of DNA, RNA and protein during the exponential phase. Antibody synthesis is expected to compete with the production of cellular proteins from the amino acid pool. Therefore, it is necessary to examine the relationship between cell growth in terms of cellular macromolecules and antibody production. In this study, we searched for an optimum feeding strategy by changing the target specific growth rate in fed-batch culture to give higher antibody productivity while examining the macromolecular composition. Concentrated glucose (60 mM) and glutamine (20 mM) in DR medium (1:1 mixture of DMEM and RPMI) with additional amino acids were fed continuously to the culture and the feed rate was updated after every sampling to ensure exponential feeding (or approximately constant specific growth rate). Specific antibody production rate was found to be significantly increased in the fed-batch cultures at the near-zero specific growth rate in which the productions of cellular DNA, RNA, protein and polysaccharide were strictly limited by slow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359-368; E. Suzuki, D.F Ollis, Biotechnol. Bioeng. 34 (1989) 1398-1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61-69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture. (C) 2000 Elsevier Science S.A. All rights reserved.

Batch and fed-batch cultures of a murine hybridoma cell line (AFP-27) were performed in a stirred tank reactor to estimate the effect of feed rate on growth rate, macromolecular metabolism and antibody production. Macromolecular composition was found to change dynamically during batch culture of hybridoma cells possibly due to active production of DNA, RNA and protein during the exponential phase. Antibody synthesis is expected to compete with the production of cellular proteins from the amino acid pool. Therefore, it is necessary to examine the relationship between cell growth in terms of cellular macromolecules and antibody production. In this study, we searched for an optimum feeding strategy by changing the target specific growth rate in fed-batch culture to give higher antibody productivity while examining the macromolecular composition. Concentrated glucose (60 mM) and glutamine (20 mM) in DR medium (1:1 mixture of DMEM and RPMI) with additional amino acids were fed continuously to the culture and the feed rate was updated after every sampling to ensure exponential feeding (or approximately constant specific growth rate). Specific antibody production rate was found to be significantly increased in the fed-batch cultures at the near-zero specific growth rate in which the productions of cellular DNA, RNA, protein and polysaccharide were strictly limited by slow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.

The Mycobacterium leprae and M. tuberculosis 10000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species-specific determinants localized within amino acid residues 21-40 and 49-72. Analysis of the molecular determinants of species-specificity for the M. leprae GroES sequence 25-40, using T-cell hybridomas and major histocompatibility complex (MHC)-binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H-2A(d) (24-34) or H- 2E(d) (28-34). Furthermore, the site recognized by the M. leprae-specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25-31 and 25-29. In conclusion, we demonstrated that immunodominant species-specific T- and B-cell epitopes can be found in a mycobacterial heat- shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae- specific determinants for a composite T-cell immunodiagnostic reagent for tuberculoid leprosy. Chemicals/CAS: Antigens, Bacterial; Chaperonin 10; Epitopes; H-2 Antigens; Peptide Fragments

Contemporary biochemical methods for protein purification were applied for the isolation of anti-IgE monoclonal antibody E1 from mouse ascitic fluid. The yield of monoclonal antibody E1 in each of the isolated fractions was estimated by determining the total protein concentration and the specific concentration of IgG1. The homogeneity and purity of the isolated protein was determined by SDS PAG electrophoresis. Precipitation methods (ammonium sulphate, polyethylene glycol, caprylic acid, rivanol) were used, alternatively or successively, as the first step in the isolation of MoAb E1. Higher levels of purity of monoclonal antibody E1 were obtained using chromatographic methods (ion exchange chromatography, gel filtration and affinity chromatography). The best result (97% purity and 77% yield) was obtained by affinity chromatography on Staphylococcal protein A.

Contemporary biochemical methods for protein purification were applied for the isolation of anti-IgE monoclonal antibody E1 from mouse ascitic fluid. The yield of monoclonal antibody E1 in each of the isolated fractions was estimated by determining the total protein concentration and the specific concentration of IgG1. The homogeneity and purity of the isolated protein was determined by SDS PAG electrophoresis. Precipitation methods (ammonium sulphate, polyethylene glycol, caprylic acid, rivanol) were used, alternatively or successively, as the first step in the isolation of MoAb E1. Higher levels of purity of monoclonal antibody E1 were obtained using chromatographic methods (ion exchange chromatography, gel filtration and affinity chromatography). The best result (97% purity and 77% yield) was obtained by affinity chromatography on Staphylococcal protein A.

Efficient and accurate diagnostic assays are essential for the design and evaluation of control measures of the potato cyst nematodesGlobodera rostochiensis andG. pallida by means of resistance. The hybridoma technology and the polymerase chain reaction (PCR) offer in potential various possibilities to design such diagnostic tests for routine purposes. We set out to devise a refined advisory system based on biochemical assays by using the following stepwise approach.

Immune response to proteins necessarily involve the recognition by T lymphocytes of a peptide or peptides derived from a a protein complexed with a major histocompatibility antigen. Th T-cell response of BALB/c mice to the bacteriophage lambda cI repressor protein (residues 1-102) is directed predominantly towards the epitope contained within a single peptide encompassing residues 12-26 (refs 1, 2). Similar phenomenon of immunodominance of a particular peptide have also been observed in other protein systems. The mechanism that have been suggested to account for the focusing of the T-cell response are partial deletion in the T cell repertoire, biased antigen processing, and competition for binding to the presenting molecule, the major histocompatibility complex encoded class II transplantation antigen. In a model system with a polypeptide containing two synthetically linked immunologically active epitopes, we now demonstrate the existence of a hierarchy between these epitopes, so that the immune response elicited is directed mainly towards the more immunogenic epitope whereas the less immunogenic epitope elicits little or no T cell reactivity. in addition the same hierarchy of dominance is also apparent when the polypeptide id used to induce tolerance in the periphery in adult mice.

Hybridoma-derived monoclonal antibodies (MoAb) specifically reactive with lymphocyte cell surface molecules have been of great value in the analysis of lymphocyte differentiation and lymphoid neoplasia. MoAb reactive with human neutrophils have been developed and are potentially important tools for the study of granulocyte function, leukemic cell origins, and granulopoiesis. Antibodies against the My-1 human granulocyte antigen react with morphologically identifiable neutrophil precursors, but not with colony-forming cells of the granulocyte-monocyte lineage (CFC-GM). The binding of five antineutrophil monoclonal autobodies, AHN-1, -2, -3, -3, and -8, to normal and leukemic bone marrow cells was studied. AHN-7 bound to many granulocytic precursors, particularly myelocytes, and both lymphoid and blast cells in normal marrow, and to most but not all granulocyte-macrophage progenitors (CFC-GM). AHN-8 bound only to late (band and segmented) neutrophilic cells and not to CFC-GM. AHN-1, - 2, -3 bound to morphologically identifiable neutrophil precursors, but not to (day-14) CFC-GM. Approximately half of nonlymphoid leukemia specimens were positive with AHN-1 or AHN-7; by contrast, lymphoid leukemia specimens were rarely positive. EHN-8 was rarely found on leukemic cells. These antineutrophil antibodies appear to detect distinct granulopoietic subsets and may be useful in the analysis of hematologic differentiation and in the subclassification of leukemias. Reprints.

Rhesus monkey is a good experimental system for man in bone marrow transplantation. Attempts were made to adopt the human progenitor culture system to the rhesus bone marrow. Conditioned medium from phytoagglutinin stimulated human lymphocytes was suitable for the growth of rhesus progenitors in the standard methylcellulose culture assay. The major modification was the need to read BFU-E and CFU-GEMM at day 12 due to the degeneration of cells in the colonies. Monoclonal antibodies against human nonlymphoid leukemia cell lines which have reactivities against human progenitors did not show significant cross-reactivities against the rhesus marrow. Monoclonal antibodies to rhesus bone marrow cells were made. Although no antibodies specific for the stem cells were obtained, an antibody reactive with lymphoid as well as more mature myeloid cells was useful to enrich for progenitor cells. The bone marrow cells depleted of reactive cells to the monoclonal antibody was functional in vivo in that they reconstituted the bone marrow of irradiated autologous and allogeneic recipients. Monoclonal antibodies against rhesus T cells and B cells were also established. These anti-bone marrow and anti-lymphocyte antibodies may be useful for further studies of the rhesus model system. Keywords: Hybridoma, Hemopoietic cells, Hemopoiesis.

An ELIZA method using immune chicken IgG coated plates does not appear to be specific for microbial adhesion enhancing macromolecules (MAEM). Hyperimmune mouse IgG made against MAEM( but not normal mouse IgG) reacts significantly with coated plates in the presence or absence of MAEM antigens. Assays yield titers of approximately 100,000 for the hyperimmune mouse sera over that of normal control. Hyperimmune mouse sera consistently showed this property despite modifications in the assay method. The very high titer of hyperimmune mouse serum suggests that immunization has produced an antibody(ies) against MAEM antigens, however, the assay could not discriminate specific from nonspecific reactions. Efforts will continue to focus on the problem, by examining the effects of D-galactose and other simple sugars on this binding. Elevated salt concentrations will be examined as a means of minimizing non-antigen mediated reactions. Keywords: Hybridoma.

The developmental stages of plasmodia receiving the most attention in the search for a malaria vaccine are sporozoites, blood stages, and gametes. The liver stages have been the least studied. Nonetheless, recent evidence has shown that liver stages(LS) of Plasmodium falciparum have their own unique stage-specific antigens and conceivably the liver stages may eventually provide antigens with demonstrated capability of inducing protective immunity at the level of the liver. In addition, the presence of circumsporozoite (CS) antigen in trophozoites that develop from irradiated and nonirradiated sporozoites implicates the liver stages in the development of malarial immunity. The indirect fluorescent antibody test (IFAT) has been applied in assessing immune responses to malaria infections and in developing hybridomas secreting antibodies directed against stage-specific antigens. The IFA technique applied to LS relies on the use of either thin frozen liver sections or sections cut from blocks of liver fixed in Carnoy's solution as the antigen. The present report describes a rapid, simple method of preparing high-quality antigen slides of LS of Plasmodium yoelii. The method can also be applied to any rodent malaria parasite. Keywords: Reprints; Bioassay; Fluorescent antibody techniques., Pub. in Jnl. of Parasitology, v73 n6 p1268-1270 Dec 1987.

Methods and compositions are provided for the detection of a particular low density lipoprotein which has been found to be a marker for patients suffering from type IV hypertriglyceridemia. A monoclonal antibody capable of specifically binding to a characteristic epitopic site on this LDL subspecies can be utilized in a wide variety of immunoassays., Supersedes PAT-APPL-608 750-84.

Clostridium difficile, the etiological agent of pseudomembranous colitis in humans and animals, produces two toxins, designated toxin A and toxin B, that are cytotoxic for tissue-cultured mammalian cells. Toxin B is approximately 1,000-fold more cytotoxic than toxin A per mg of protein. Toxin A also possesses enterotoxin activity and causes a fluid response when injected into ligated rabbit intestinal loops. These antibodies produced by a family of murine hybrid cell lines are cross-reactive with both toxin A and toxin B of Clostridium difficile. The monoclonal antibodies are useful in an immunoassay for toxin A and toxin B of C. difficile.

Myelomas and hybridomas were observed to proliferate normally when trapped in a fibrin clot. The fibrin clot was obtained by including fibrinogen in the cell culture medium and thrombin in the plastic dish. A clot formed within seconds and cloning efficiency was around 100%. This technique has all the advantages of semi-solid medium cloning but avoids toxicity to the cells and the exposure to high temperature associated with the soft-agar cloning technique. © 1988.