mAb Secretion Research Articles

Serum low replacement medium versus serum rich replacement medium for production of Anti-Schistosoma Monoclonal antibody: A comparative study

Objectiveto assess the production of Anti-Schistosoma Monoclonal Antibodies by growing IgM producing hybridoma cell line in serum low medium and to evaluate their diagnostic potential to replace the old conventional method in animal ascetic fluid. MethodsHighly reactive and specific IgM MAb secreting hybridoma cell line to S. mansoni SEA was cultured in three different media; serum rich replacement medium (conventional), serum low replacement medium (SLRM) and serum free replacement medium (SFRM). ResultsCell count and viability were better in conventional medium and SLRM than SFRM. The lower detection limit of the assay Mab of SLRM culture was 1.1 ng/ml of S. mansoni SEA. SFRM was excluded because of the low concentration and reactivity. MAb was tested against 34 human samples. Sensitivity and specificity of the assay for each were 92% and specificity 90% for SLRM respectively. The diagnostic efficacy of the assay was 94%. Conclusionsthe production and diagnostic efficacy of antischistosomal MAbs was comparable on usage of either in vitro cell culture supernate in SLRM or conventional media.

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics.

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation "hotspot". The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.

Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture.

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.

Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells.

Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro.

AIMTo investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb).METHODSDCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs’ viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a 51Cr releasing test. T cell responses induced by RNA-loaded DCs were analyzed by measuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α and IL-12.RESULTSThe anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DC-tumor-anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-γ and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01).CONCLUSIONDCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

Long-term in vivo expression of trastuzumab following intramuscular electrotransfer of the encoding DNA in mice

In vivo antibody expression is at crossroads between monoclonal antibody (mAb) and gene therapy. Following a single intramuscular injection of the DNA that encodes a therapeutic mAb, the muscle is turned into a ‘bioreactor’, resulting in prolonged mAb secretion in circulation [1-3]. This innovative approach addresses several challenges associated with conventional mAb proteins. In R&D, in vivo mAb expression allows production and evaluation of leads directly into animal models. In the clinic, it can improve treatment efficacy and patient comfort by avoiding repeated high-dose injections, and provide a cost-effective answer to the increasing need for mAb combination therapies - in the field of cancer immunotherapy and beyond. This study outlines the development and delivery of a trastuzumab-encoding plasmid for in vivo mAb expression. To improve intramuscular DNA delivery in mice, we first established an optimal electrotransfer protocol using novel reporter plasmids. Following the optimized intramuscular electrotransfer of the trastuzumab-encoding plasmid, trastuzumab was detected with a commercial ELISA at therapeutically relevant concentrations (1-15 µg/ml) in the sera of athymic nude mice (n=16) for the full duration of the ongoing follow-up (>3 months). A cell viability assay demonstrated similar activity of the expressed versus commercial trastuzumab in the BT-4747 breast cancer cell line. In conclusion, we achieved proof of concept for the long-term in vivo expression of biologically active trastuzumab in mice. Ongoing work focuses on optimizing in vivo mAb expression for clinical application and evaluation for combination therapy.

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies.

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.

A monoclonal antibody against neem leaf glycoprotein recognizes carcinoembryonic antigen (CEA) and restricts CEA expressing tumor growth.

Carcinoembryonic antigen (CEA) is one of the promising tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immunotherapy. Neem leaf glycoprotein (NLGP), an effective immunomodulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a monoclonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti-NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, Western blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA(+) human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA(-) human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better survivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA(+) cancer cells. 1C8 has no direct influence on proliferation and migration of CEA(+) cells, however, NK cell-dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA(+) cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA(+) tumors.

A Dual-Mode Surface Display System for the Maturation and Production of Monoclonal Antibodies in Glyco-Engineered Pichia pastoris

State-of-the-art monoclonal antibody (mAb) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. This results in a separation between antibody affinity maturation and full-length mAb production platforms. Here, we report for the first time, a method in Glyco-engineered Pichia pastoris that enables simultaneous surface display and secretion of full-length mAb molecules with human-like N-glycans using the same yeast cell. This paradigm takes advantage of homo-dimerization of the Fc portion of an IgG molecule to a surface-anchored "bait" Fc, which results in targeting functional “half” IgGs to the cell wall of Pichia pastoris without interfering with the secretion of full length mAb. We show the utility of this method in isolating high affinity, well-expressed anti-PCSK9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain IgG libraries. Coupled with Glyco-engineered Pichia pastoris , this method provides a powerful tool for the discovery and production of therapeutic human mAbs in the same host thus improving drug developability and potentially shortening the discovery time cycle.

An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer

In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) monoclonal antibody production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell-specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell--where control of productivity is inherently protein product and cell line specific.

The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

Integrin adhesion in regulation of lacrimal gland acinar cell secretion

The extracellular microenvironment regulates lacrimal gland acinar cell secretion. Culturing isolated rabbit lacrimal gland acinar cells on different extracellular matrix proteins revealed that laminin enhances carbachol-stimulated secretion to a greater extent than other extracellular matrix proteins investigated. Furthermore, immunofluorescence indicated that integrin subunits, potentially functioning as laminin receptors are present in acinar cells. Among these, the integrin α6 and β1 subunit mRNA expression was also confirmed by RT–PCR and sequence analysis. Secretion assays, which measured β-hexosaminidase activity released in the culture media, demonstrated that function-blocking integrin α6 and β1 monoclonal antibodies (mAbs) induce a rapid, transient and dose-dependent secretory response in cultured cells. To determine the intracellular pathways by which integrin α6 and β1 mAbs could induce secretion, selected second messenger molecules were inhibited. Although inhibitors of protein kinase C and IP 3-induced Ca 2+ mobilization attenuated carbachol-stimulated secretion, no effect on integrin mAb-induced release was observed. In addition, protein tyrosine kinases do not appear to have a role in transducing signals arising from mAb interactions. Our data clearly demonstrate, though, that cell adhesion through integrins regulates secretion from lacrimal gland acinar cells. The fact that the integrin mAbs affect the cholinergic response differently and that the integrin β1 mAb secretion, but not the α6, was attenuated by the phosphatase inhibitor, sodium orthovanadate, suggests that each subunit utilizes separate intracellular signaling pathways to induce exocytosis. The results also indicate that the secretory response triggered by the β1 integrin mAb is generated through dephosphorylation events.

Inducible expression of Bcl-XL inhibits sodium butyrate-induced apoptosis in hybridoma, resulting in enhanced antibody production.

Sodium butyrate (NaBu) can increase the specific Mab production rate of hybridomas by enhancing histone hyperacetylation and influencing the cell cycle, but it can also inhibit cell growth and induce apoptosis. Thus, the beneficial effect of NaBu on Mab secretion is compromised by its cytotoxic effect. In the present study, expression of the anti-apoptotic protein human Bcl-XL was made inducible in hybridoma H18 to overcome the cytotoxic effect of NaBu, circumventing the detrimental effects of constitutive high-level expression. We constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drove the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in H18 hybridoma cells. Our data showed that stably transfected H18.D4 cells expressed high levels of Bcl-X(L), which was induced within 24 h of addition of ZnSO4. NaBu (0.4 mM) increased antibody production by more than 3-fold in H18.D4. This effect resulted from the suppression of NaBu-induced apoptosis, allowing the H18.D4 cells to grow at higher viability and extending culture longevity by >3 days.

MHC-Class I antigens regulate both the function and the survival of human peripheral blood NK cells: role of endogenously secreted TNF-alpha.

The cytotoxic activity and survival of NK cells are negatively regulated in the presence of antibodies directed against the FCgammaRIII (CD16) surface receptor. The addition of MHC-class I monoclonal antibody (mAb) in combination with CD16 mAb resulted in a significant potentiation of the inhibition of NK cell cytotoxic function and both accelerated kinetics and synergistic induction of cell death in both resting and IL-2-treated human peripheral blood purified NK cells. The potentiating effect of class I MHC monoclonal antibody was specific as monoclonal antibodies directed against other cell surface antigens on NK (e.g., LFA-3, LFA-1, and CD56) had no effect. A correlation was observed between the levels of secreted TNF-alpha and the frequency of NK cell death and the addition of anti-TNF-alpha antibody inhibited NK cell death. The levels of death promoting gene products such as the Fas receptor and the Fas ligand were upregulated in NK cells in the presence of anti-class I and anti-CD16 antibodies. However, anti-Fas antibodies did not block cell death. These findings demonstrate that MHC-class I antigens regulate both TNF-alpha secretion and cell death of anti-CD16 mAb treated NK cells. These results also suggest that MHC-class I antigens regulate the function and survival of activated NK cells.

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions.

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAbtrade mark. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAbtrade mark accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAbtrade mark resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAbtrade mark. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAbtrade mark with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells.

Use of the weighted jackknife method to calculate the variance in cellular-specific protein secretion rate: Application to monoclonal antibody secretion rate kinetics in response to osmotic stress

The specific secretion rate (q, microg protein secreted/viable cell-h) and its variance are very useful to compare the capability of cell lines for protein secretion. An assessment of specific secretion rate variability is also beneficial and important when the specific secretion rate is to be used as an on-line process parameter to monitor culture production behavior or for in-process decisionmaking. Experimental errors in mammalian cell culture (e.g., protein concentration measurement and cell counting) and estimation error in the method of calculating q contribute to the total variance of the specific secretion rate. Although the variance of q is essential for comparing the differences between cell lines and the response of the same cell line to different nutrient or environmental conditions, few methods for calculating the variance of the specific secretion rate have been reported. As a model system, we have used the weighted jackknife method and the delta method to calculate the variance in the specific secretion rate of a murine monoclonal antibody (q(mAb)) determined by a differential method. These methods were applied to calculate q(mAb) and its standard deviation to determine the change in q(mAb) kinetics during batch culture of the 9.2.27 hybridoma in response to growth in hyperosmotic media or osmotic stress. Without osmotic stress, during exponential growth in DMEM + 5% FBS spinner culture, the estimate of q(mAb) decreases at least threefold. Results indicate that the 9.2.27 hybridoma responds to hyperosmotic media (400 mOsm, 470 mOsm) by significantly reducing the degree of q(mAb) decrease in the exponential phase, thus maintaining a higher q(mAb) through the stationary phase. The trend of q(mAb) during the batch cultures studied is further confirmed by t-test. Osmotic stress is statistically shown to be able to alter significantly the hybridoma-specific mAb secretion kinetics during batch culture. Determination of the variance of specific secretion rate using the weighted jackknife method offers a powerful approach for establishing the confidence limits of specific protein secretion rate between cell cultures in different nutritional or osmotic environments.

Alteration of hybridoma viability and antibody secretion in transfectomas with inducible overexpression of protein disulfide isomerase.

Monoclonal antibody (mAb)-secreting transfectomas with dexamethasone inducible expression of the mammalian endoplasmic reticulum foldase and chaperone protein disulfide isomerase (PDI, ERp59) were generated from the murine 9.2.27 hybridoma in order to obtain in vivo evidence of whether alteration of the level of PDI, believed to be involved in immunoglobulin (Ig) assembly, results in alteration of mAb secretion kinetics. Using an RNase refolding assay, the specific activity of endogenous PDI in the 9.2.27 hybridoma was found to be constant during batch growth. An expression vector for glucocorticoid-inducible overexpression of PDI, pMMTVPDI, was constructed from pMAMneo using a rat PDI cDNA. Cell lysates of stable transfectomas contained 2-4-fold higher levels of PDI mRNA and increased levels of PDI protein, detected by immunoblotting, following induction with 0.1 microM dexamethasone. Monoclonal antibody secretion kinetics were evaluated in 12.5 mL shake flasks, a 100 mL spinner, and a 1 L aerated batch reactor. A transfectoma was found with altered mAb secretion kinetics during cell growth following dexamethasone induction of PDI overexpression. Specific mAb secretion rate was not significantly increased following dexamethasone induction; however, hybridoma viability was sustained longer during the stationary phase of cell growth and hence total antibody yield was increased in comparison to the parent 9.2.27 hybridoma.

Effect of l-carnitine and acylcarnitine derivatives on the proliferation and monoclonal antibody production of mouse hybridoma cells in culture

The effect of l-carnitine (Cn) on cell growth metabolism and antibody production rates was investigated using the murine hybridoma cell line, Mark3, in batch and fed harvest cultures. Two acylcarnitine derivatives were also tested: palmitoyl l-Cn and acetyl- dl-Cn. The addition of 20 μM l-Cn to cultures of Mark3 hybridoma cells that had been adapted to l-Cn significantly stimulated monoclonal antibody (mAb) production without affecting cell growth. In contrast, mAb secretion slightly decreased when l-Cn was added to culture of cells that had not been adapted to l-Cn. Palmitoyl l-Cn also stimulated mAb production by adapted cells, whereas the acetyl- dl-Cn, reduced mAb secretion. The presence of l-Cn in the medium did not affect glucose consumption or lactate production, but the metabolism of some amino acids was altered. The medium concentrations of valine, leucine, isoleucine and lysine were enhanced from 22% to 41% according to amino acids, whereas those of alanine, glycine and proline decreased. The mechanism by which l-carnitine affects mAb production and the metabolism of some amino acids is unknown. This effect is likely to be indirect, since there was no net entry of l-[ 3H]carnitine into the cells.

A solid-phase assay for the detection of anti-sperm antibodies.

ELISA is an ideal assay method for a large-scale screening of anti-sperm antibodies among a large number of infertile males. However, conventional ELISA with whole spermatozoa needs time-consuming steps of centrifugation. A solid-phase assay used for detecting anti-sperm antibodies was established. This assay is suitable not only for detecting circulating anti-sperm antibodies of IgG, IgM, and IgA subclass simultaneously but also for screening hybridomas secreting anti-sperm monoclonal antibodies (mAbs). The microtiter plates, on which solubilized sperm antigens are fixed, can be stored at -80 degrees C for up to six months without losing reactivity with anti-sperm antibodies. Using this assay, 53 sera (13 were proven positive and 40 were proven negative for sperm agglutination antibody) were tested. Although the false-negative rate was 0%, the false-positive rate was 32%. One thousand one hundred sixty-five supernatants from hybridomas constructed with splenocytes of mice who were hyperimmunized with human sperm and nonsecreting myeloma cells were tested by this solid-phase assay and two anti-sperm mAb secreting clones were selected and established. It is recommended that for research work this assay could be used for the first screening of the hybridoma secreting anti-sperm mAb, and for clinical use this assay might be suitable for the first screening of sera of infertile patients. However, conventional bioassays should follow to confirm the biological meaning of the positivity.

The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas

Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the booster injection of Newmarket/79 virus, the inclusion of Freund's incomplete adjuvant and the use of an aminopterin-sensitive primary heterohybridoma as the fusion partner, improved the production of HIg-secreting heterohybridomas. After two clonings eight cell lines were established which maintained anti-Newmarket/79 antibody secretion for over a year. FACS analysis of the cell lines provided a useful means of predicting breakdown of MAb secretion by the cell lines, thus enabling re-cloning to be carried out in time.