Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas (original) (raw)
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Enhanced Monoclonal Antibody Production in Hybridoma Cells by LPS and Anti-mIgG
Biotechnology Progress, 2007
This paper reports on a methodology for increasing proliferation and monoclonal antibody (mAb) production in hybridoma cultures. The 55-6 murine B cell hybridoma line (CD40 and CD19deficient expression) was treated with increasing concentrations of lipopolysaccharide (LPS). Expression of CD69, CD40, and CD19 surface antigens on 55-6 cells did not show significant changes from untreated cells. The specific growth rate decreased at higher concentrations of LPS, but the monoclonal antibody production rate was highest at the highest LPS concentration assayed. These data are in agreement with the lowest growth rate found at this concentration of LPS. Furthermore, cells were cultured with anti-mouse surface immunoglobulin G antibody (anti-mIgG) plus LPS to find out whether LPS-derived signals and anti-mIgG stimuli are synergistic. CD69, CD40, and CD19 expression was greater than for either untreated cells (control culture) or cells stimulated with LPS alone. Moreover, LPS stimulation in combination with anti-mIgG enhanced both the growth rate and IgG2a production over the control culture and cells stimulated with LPS alone. Maximum antibody concentration increased almost 500% compared to the control and about 100% with respect to culture stimulated with LPS alone. The maximum specific IgG2a production rate was about 300% higher than in the control culture and about 30% higher than in culture stimulated only with LPS.
Hydroxyurea and cell-cycle kinetics of cultured antibody-forming cells
Cellular Immunology, 1971
Cell-cycle kinetics of precursors to antibody-forming cells was studied in mouse spleen cell cultures immunized with sheep red cells. Hydroxyurea (0.5 m.iV) added on the day of culture did not interfere with the response if removed within 24 hr. When it was added on Day 4 of culture, during log growth, 76% of the antibody-forming cells were killed with treatment lasting 8 hr or less. Treatment of 12 hr or more resulted in death for 98% of the cells. The shape of the survival curve showed the proportion of cells killed in S (.74), the duration of S (6-7.5 hr), and generation time 8.5-10 hr. This technique is particularly useful for the study of a very small population of differentiating cells at a time prior to the expression of the unique character by which they are detected.
Biotechnology Progress, 2007
The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (µ max) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demostrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.
Journal of Immunological Methods, 1988
Anti-/~ preparations differ greatly in their ability to stimulate mouse B cells to incorporate tritiated thymidine (TdR). We have found that these differences may be due in part to different levels of lipopolysaccharide (LPS) content. In this report we show that LPS concentrations as low as 0.025 ng/ml stimulate the proliferation of T-depleted (C57BL/6 × DBA/2)F 1 (B6DEF1) spleen cells, provided that 5 × 10-5 M 2-mercaptoethanol is also present. Each of six commercial anti-/~ preparations tested for LPS content contained more than this amount. We describe a technique that uses polymyxin B-agarose to remove nanogram quantities of LPS from anti-/x preparations. In B6DEF 1 B cells, LPS-depleted anti-# preparations induced much more uniform tritiated thymidine incorporation than did non-depleted preparations; but there was little difference between the two preparations when tested on B cells from C3H/HeJ (LPS hyporesponsive) mice.
Cytotechnology, 2013
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 cocultures 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 largescale 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.
The Journal of Immunology
Proliferation of single B cells was observed in response to the combination of LPS and DXS when spleen cells were placed into Terasaki plates at dilutions where the majority of wells containing cells contained one cell. Clones derived from a single cell exhibited varying morphologies and growth characteristics. Neither filler cells nor conditioned medium was required for these responses. Since isolated cells were stimulated, we conclude that B cells can be activated to clonal growth in the absence of accessory cells. Limiting dilution analysis showed that the frequency of cells responding to the combination of LPS + DXS was about 80% of the frequency of input B cells. When only LPS or DXS was present, the frequency of response was less than 25% and less than 5% of the input B cell frequency, respectively. This synergy in mitogenesis is consistent with previous kinetic and cytofluorometric analyses of mitogen-stimulated growth which have indicated that the combination of LPS + DXS re...
Cell growth and monoclonal antibody production in the presence of antigen and serum
Biotechnology Progress, 1995
The impact that the continuous presence in the fermentation broth of the cognate antigen has on the serum-supplemented hybridoma cell cultures was investigated. Both soluble and immobilized antigen a t various concentrations was applied. The cell line (ATCC TIB191) was cultured in a serum-supplemented PFHM-I1 medium in T-flasks. Sepharose gel beads provided the immobilization matrix, and bovine y-globulin was the carrier protein upon which the antigen, picric acid, was conjugated. Produced antibody, after elution from the beads by displacement with free picric acid, was measured with a n ELISA. Soluble antigen-carrier protein conjugates showed no effect on the cultures, but the immobilized antigen had a strong influence on them.
Hydroxyurea interferes with antigen-dependent T-cell activation in vitro
European Journal of Clinical Investigation, 2000
Background Hydroxyurea is believed to inhibit human immunodeficiency virus type 1 (HIV-1) in HIV disease by decreasing the amount of intracellular deoxynucleotides needed for viral replication. A plasma concentration of 400 mmol L ¹1 is tolerated in oncological diseases. The present study focused on the possible interference of hydroxyurea with antigen-dependent T-cell activation as an alternative explanation for inhibiting HIV replication in vivo.
Enhanced antibody production of human-human hybridomas by retinoic acid
Cytotechnology, 2000
The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10(-7) M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.