mAb Titer Research Articles

Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".

Antibodies specific to infectious bronchitis in broilers in Ceará state, Brazil

The experiment was carried out to determine the antibody levels to infectious bronchitis virus (IBV) in 1120 broilers of two broiler flocks, both from the same parental flock and free from previous vaccination. Forty chicks of each line were alloted to the control group and the sera were tested by indirect ELISA. The vaccination program consisted on the administration of commercial vaccines against IBV at 10 and 25 days of age. Chicks with low levels of maternal antibodies (Mab) did not show significant titers to the first vaccinal stimulus. They presented a vaccinal response to the second vaccinal stimulus reaching the top around GMT 1100 at 45 days. Chicks with high Mab titers did not show significant titers to the primary and secondary vaccinal stimuli, reaching peak levels of GMT 500 at 45 days. No antibody response was detected after the primary vaccination at day 10. A delayed antibody response was detected after the secondary vaccination on day 25, indicating no previous priming. The maternal antibody titers can interfere on the response to the first and second vaccinal stimulus promoting the neutralization of the first vaccination and a different response to the second one, according to high or low maternal antibodies.

Large Scale Production and Characterization of Anti-Human IgG Monoclonal Antibody in Peritoneum of Balb/c MICE

Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers. For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human IgG were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 mL Pristane. After 10 days, approximately 3 mL ascitic fluid was harvested from the peritoneum of each mouse. Ascitic fluid was assayed for the titer of monoclonal antibody in reaction with human IgG and its cross reactivity in reaction with IgM and IgA. The titer of mAb was 100,000 and didn’t show cross reactivity with IgM and IgA. Immunobloting was done for confirming the ELISA method. In immunobloting, only one sharp band in the heavy chain position of IgG was developed. The subclass of antibody was IgG1 and its light chain was kappa. Ascitic fluid was purified by ion exchange chromatography and the purified monoclonal antibody was conjugated with HRP. The conjugated monoclonal antibody could had application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of all other infectious diseases.

Conditioning of Neonatal Pigs Using Low-Dose Chemotherapy and Murine Fetal Tissue before Murine Hybridoma Transplantation

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.

Neuron-binding human monoclonal antibodies support central nervous system neurite extension.

Two human IgMs (sHIgM12 and sHIgM42) were identified that supported in vitro central nervous system (CNS) neurite extension equal to the potent neurite stimulatory molecule laminin. Both IgMs bound to multiple cell types in unfixed CNS tissue and to the surface of neurons in culture. Both monoclonal antibodies (mAbs) overrode the inhibitory effect of CNS mouse myelin on granule cell neurite extension. Neither mAb bound to the surface of mature oligodendrocytes or strictly colocalized with myelin proteins. Sialidase treatment eliminated the neuronal surface binding of both mAbs, whereas blocking sphingolipid synthesis with Fumonisin B1 or removing GPI-linked proteins with PIPLC did not. When used as substrates for mixed neuron/glia aggregates, sHIgM12 and sHIgM42 supported robust neurite extension while astrocytes remained in the aggregates. In contrast, laminin supported astrocyte migration and spreading. Human mAbs that support neurite extension are novel factors that may be of use in encouraging axon repair following injury while minimizing glial cell infiltration. Both human mAbs were isolated from individuals with monoclonal gammopathy. Each individual has carried high mAb titers in circulation for years without detriment. sHIgM12 and sHIgM42 are therefore unlikely to be systemically pathogenic.

Neutralization of dermonecrotic and lethal activities and differences among 32–35 kDa toxins of medically important Loxosceles spider venoms in Brazil revealed by monoclonal antibodies

Neutralization of dermonecrotic and lethal activities and differences among the principal toxic proteins (32–35 kDa) of medically important Loxosceles spider venoms in Brazil ( Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia) were studied using monoclonal antibodies (MAbs) produced against the dermonecrotic component (35 kDa) of L. gaucho venom. MAb titers were 512,000 to homologous venom, between 2000 and 64,000 for L. intermedia venom and between 1000 and 64,000 for L. laeta venom. By Western blotting, MAbs could recognize mainly the 35 kDa protein of L. gaucho venom and with less intensity the 35 kDa protein of L. intermedia venom. These MAbs also recognized weakly or did not recognize the 32 kDa component of L. laeta venom. Only MoALg 1 showed high affinity for L. gaucho venom and neutralized in vivo 90–97% of the dermonecrotic activity, besides delaying the lethality induced by homologous venom. MoALg 1 maintained its capacity to neutralize the dermonecrotic activity, even when administered (i.v.) 6 h after envenoming (i.d.). All MAbs obtained failed to neutralize the toxic activities of the heterologous venoms. These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms, and confirm the participation of other venom components during the local reaction process. This study also confirms the importance of antibodies for neutralization of dermonecrotic activity, even when administered some hours after envenoming, and emphasizes the differences of composition and toxicity of medically important Loxosceles venoms. These findings must be considered in order to improve loxoscelism immunotherapy.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

The effect of fatty acids on hybridoma cell growth and antibody productivity in serum-free cultures

A murine B-lymphocyte hybridoma (CC9C10) was adapted for growth in a serum-free medium. Supplementation of the medium with cis-unsaturated fatty acids (10–50 μM) improved the cell yield in the order oleic/linoleic > linoleic > oleic. Initial supplementation with the fatty acids also caused a significant increase (58%) in the volumetric Mab titre. Continued growth of the cells in the fatty acid supplemented media over five culture passages resulted in a gradual deterioration of the Mab yield concomitant with the appearance of lipid inclusions in the cytosol. The higher Mab yield could be restored by a limited period of growth of the lipid-loaded cells in fatty acid-free medium. These effects were independent of growth rate. This suggests that the optimal intracellular lipid content is finely balanced between a reduced and an overloaded state. Specific glucose and glutamine utilisation rates were unaffected by the presence of fatty acids. Also, the optimal glucose and glutamine concentrations for growth were independent of the fatty acids.

In pursuit of the optimal fed-batch process for monoclonal antibody production.

Fed-batch culture currently represents the most attractive choice for large scale production for monoclonal antibodies (MAbs), due to its operational simplicity, reliability, and flexibility for implementation in multipurpose facilities. Development of highly productive cell lines, maximization of cell culture longevity, and maintenance of high specific antibody secretion rates through genetic engineering techniques, nutrition supplementation, waste product minimization, and control of environmental conditions are important for the design of high-yield fed-batch processes. Initially simple supplementation protocols have evolved into sophisticated serum-free multi-nutrient feeds that result in MAb titers on the order of 1-2 g/L. Limited research has been published to date on the effect of various culture parameters on potentially important quality issues, such as MAb glycosylation and stability. Although most fed-batch protocols to date have relied on relatively simple control schemes, increasingly sophisticated algorithms must be applied in order to take full advantage of the potentially additive effects of manipulating nutrient and environmental parameters to maximize fed-batch process productivity.

Alteration in the level of interferon-γ results in acceleration of Theiler's virus-induced demyelinating disease

Intracerebral (i.c.) inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. We examined the role of interferon (IFN)-γ in this virally induced pathogenesis. Intraperitoneal (i.p.) injection of susceptible mice with an IFN-γ neutralizing monoclonal antibody (mAb), DB-1, resulted in a significantly accelerated onset of disease. The anti-IFN-γ mAb-treated animals showed a strong delayed-type hypersensitivity (DTH) response to the virus similar to that of control mAb-treated animals. Treatment with anti-IFN-γ mAb appeared to decrease TMEV-specific mAb titers in one of the protocols used. Intracerebral injection of the anti-IFN-γ mAb had no significant effect on the clinical course of disease. However, intracerebral administration of recombinant IFN-γ significantly accelerated the onset of TMEV-induced disease, as well as enhanced TMEV-specific T cell proliferation and DTH responses. The enhancing effect of IFN-γ was completely abrogated by simultaneous treatment with anti-IFN-γ mAb. Collectively, our data suggest that the level of IF-γ plays a key role in the TMEV-induced inflammatory respopnse and a perturbation of this balance may result in an alteration in the course of the demyelinating disease.

Enhanced IgG production in eRDF media with and without serum: A comparative study

The performance of three basal media RPMI, DMEM/F12 (DF) and eRDF (enhanced RDF, RPMI:DMEM:F12 in 2:1:1) were evaluated in cultures with and without serum with respect to cell proliferation, metabolism and monoclonal antibody (Mab) productivity. Based on the ease of adaptation, growth rate, maximum cell density and Mab production, the media were ranked as follows: eRDF > DF > RPMI. This was true for serum-free (SF) and serum supplemented (SS) media in static and shaker cultures. Growth performances in static and shaker cultures were consistently 20–50% lower in all three SF media compared to the corresponding SS conditions. Antibody titres in DF/SF and RPMI/SF cultures, irrespective of the culture condition, were generally similar or slightly lower than their SS counterparts. However, eRDF/SF medium yielded a much higher Mab titre (193 mg 1 −1) compared to eRDF/SS medium (145 mg 1 −1). This was also six times higher than the lowest titre of 30 mg 1 −1 in RPMI/SF medium. Hybridomas in eRDF/SF were further adapter to media without bovine serum albumin (eRDF/SF-BSA). Maximum cell densities in these cultures improved wiith scale up, from 1.1 × 10 6 ml − in static, to 1.9 × 10 6 ml −1 in shaker flasks, to 2.5 × 10 6 ml −1 in bioreactors. However, Ig levels remained between 100–130 mg 1 −1 which were much lower than in eRDF/SF medium. Thus BSA appears to be necessary for Ig production. The manufacturing cost (excluding purification) of Ig using eRDF was calculated to be between 17–50% of the price of the other two media and therefore this is regarded as the best medium for Ig production.

Age-related resistance to experimental autoimmune myasthenia gravis in rats.

Abstract The influence of age on the induction of experimental autoimmune myasthenia gravis (EAMG) was investigated. Immunization with acetylcholine receptor (AChR) or injection of varying amounts of anti-AChR mAb 35 into young adult (10-12 wk) BN rats induced severe signs of EAMG including weight loss and decrement of muscle action potential, whereas aged BN rats (120-130 wk) did not show any clinical signs of EAMG. Serum anti-AChr mAb titers were not significantly different in young and aged rats up to 24 h after administration of mAb. No significant AChR loss was demonstrated in aged rats, whereas similarly treated young rats showed extensive AChR loss. In contrast to young rats, no degradation of the postsynaptic membrane could be demonstrated by electron microscopy in aged rats. C component C3 and C5b-9 membrane attack complex could be demonstrated at the neuromuscular junction in both young and aged mAb-treated rats. However, infiltrating macrophages and necrotic muscle fibers were seen only in young rats. These results suggest that the postsynaptic membrane in aged rats is resistant to autoantibody attack. AChR degradation by antigenic modulation may be less efficient in aged rats as a result of altered AChR density and distribution or rigidity of the postsynaptic membrane. Age-related resistance in the EAMG model can provide more information about the factors that determine the severity of myasthenia gravis. Manipulation of AChR density or lipid composition of the postsynaptic membrane may be of therapeutic interest in myasthenia gravis.

Cross-reactivity between a monoclonal antibody that recognizes a tumor-associated antigen on bovine lymphosarcoma cells and blood lymphocytes from various mammalian species

Summary Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (mab). By use of flow cytometry and radioimmunoprecipitation, 1 of the mab (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (bl) from various mammalian species. By use of flow cytometry, the c143 mab reacted with 10 to 49% of bl derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 mab with bl from horses, pigs, human beings, and llamas ranged between 1:6.0 × 104 and 1:5.3 × 105; titer associated with bl of goats and sheep was 1:1.6 × 106; and that associated with bl of cattle was 1:4.3 × 107. The c143 mab specifically immunoprecipitated 3 homologous proteins from cell extracts of caprine, ovine, and bovine bl (32-, 34-, and 36- to 37-kDa bovine proteins; 31-, 32-, and 36- to 37-kDa caprine proteins; and 31.5-, 33-, and 36- to 37-kDa ovine proteins), but none was immunoprecipitated from human, murine, canine, porcine, and llama bl. These results indicate that the avidity of the c143 mab in binding to bl from ruminants (eg, goats, sheep, and cattle) is higher than that to bl from human beings, mice, dogs, horses, pigs, and llamas. In sheep, the c143 mab could immunoprecipitate the aforementioned proteins from bl of the Suffolk breed, but not bl from the Corriedale breed, whereas the c143 mab immunoprecipitated apparently identical proteins from bl of 4 breeds of cattle.

Passive protection of suckling infant mice against F41-positive enterotoxigenic Escherichia coli strains by intravenous inoculation of the dams with monoclonal antibodies against F41.

Ten monoclonal antibodies (MAbs) against five different epitope clusters of adhesion factor F41 (two MAbs per cluster) were tested for protection of infant mice against an oral challenge with F41-positive enterotoxigenic Escherichia coli (ETEC) B2C and B41M. Infant mice suckling dams intravenously inoculated with MAbs were orally challenged, and the survival rates were measured for 12 days after inoculation and challenge. Irrespective of their epitope specificity, all F41 MAbs given in a single dose of 4 mg per dam had a protective effect against both ETEC strains. In contrast, one K99 MAb of the same isotype and given in the same dose as the F41 MAbs did not protect infant mice at all. A reduction in the dose of F41 MAbs to 0.032 mg per dam resulted in a decrease in protection. Two different MAbs against the same epitope cluster were not necessarily equally protective. Combining MAbs two by two, whether the MAbs recognized the same epitope cluster or not, resulted in protective activity essentially similar to that obtained with each MAb separately, without any improvement. Therefore, one MAb against any epitope may be sufficient for protection. Enzyme-linked immunosorbent assay (ELISA) titers of MAbs in the serum of dams were similar, irrespective of the epitope specificity of the MAbs, and gradually decreased from day 1 to day 12 after inoculation. We found a good correlation between colostrum and milk ELISA titers of MAbs and serum ELISA titers of MAbs. Colostrum and milk MAb titers were 10-fold lower than corresponding serum MAb titers and stayed high until day 5 after inoculation. The most protective MAb had the highest ELISA titers in colostrum and milk for the first 5 days after inoculation. ETEC strain B2C colonized the intestines of infant mice suckling MAb-inoculated mothers until day 12 after challenge. Intestinal levels of the challenge strain were high on day 2 but never reached the very high numbers (10(9) to 10(10)) described previously in a diarrheic infant mouse model. MAbs did not eliminate the challenge ETEC strain from the intestines of infant mice.

Avoiding rapid growth at high cell densities: a potentially important optimisation criterion for hybridoma cultures.

Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002-0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4 x 10(9) cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.

Age related resistance to experimental autoimmune myasthenia gravis in rats

The influence of age on the induction of experimental autoimmune myasthenia gravis (EAMG) was investigated. Immunization with acetylcholine receptor (AChR) or injection of varying amounts of anti-AChR mAb 35 into young adult (10-12 wk) BN rats induced severe signs of EAMG including weight loss and decrement of muscle action potential, whereas aged BN rats (120-130 wk) did not show any clinical signs of EAMG. Serum anti-AChr mAb titers were not significantly different in young and aged rats up to 24 h after administration of mAb. No significant AChR loss was demonstrated in aged rats, whereas similarly treated young rats showed extensive AChR loss. In contrast to young rats, no degradation of the postsynaptic membrane could be demonstrated by electron microscopy in aged rats. C component C3 and C5b-9 membrane attack complex could be demonstrated at the neuromuscular junction in both young and aged mAb-treated rats. However, infiltrating macrophages and necrotic muscle fibers were seen only in young rats. These results suggest that the postsynaptic membrane in aged rats is resistant to autoantibody attack. AChR degradation by antigenic modulation may be less efficient in aged rats as a result of altered AChR density and distribution or rigidity of the postsynaptic membrane. Age-related resistance in the EAMG model can provide more information about the factors that determine the severity of myasthenia gravis. Manipulation of AChR density or lipid composition of the postsynaptic membrane may be of therapeutic interest in myasthenia gravis.

Puerperal thyroid gland dysfunction in healthy patients

Elevated thyroid antibodies (AB) have been described in clinically healthy women indicating a postpartum thyroid dysfunction. We evaluated the incidence of the postpartum thyroid dysfunction in Hannover, FRG. 121 women were examined 1-5 days pp and 2-4 months later; 76 were restudied 5-7 months pp. Every time T3, T4, TSH, TBG, microsomal AB and thyroglobulin AB were determined. Six patients showed increased TAB and 10 increased MAB titers. Severe clinical symptoms were not complained. In some patients these elevated titers turned to normal subsequently. Further studies must evaluate the prognosis of this disease.