Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG1 and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells (original) (raw)
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Journal of Immunological Methods, 2000
. Ž . Fetal calf serum FCS was depleted of its immunoglobulin G IgG in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap Ž . protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS G-FCS were Ž used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G anti-HBs IgG1 mAb producing hybridoma . Ž . cell and CAy-M anti-HBs IgM mAb producing hybridoma cell , which secreted hepatitis B virus surface antigen Ž . Ž . HBsAg -reactive IgG1 and IgM monoclonal antibodies mAbs , respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMIrG-FCS with that of RPMIrFCS and serumrprotein-free Hybri Max Ž . Sigma, MO, USA hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMIrG-FCS were similar to culture in RPMIrFCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in Ž . RPMIr10% G-FCS RPMI-1640 medium supplemented with 10% G-FCS . SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMIr10% G-FCS was more than 99%. q
Journal of Biotechnology, 1991
During batch cultivation of the 1.13.17. hybridoma cell line, there is a 15 to 20-fold decrease in the levels of cytoplasmic and membrane-bound mAb, and a 7 to 10-fold decrease in the cellular levels of kappa and gamma chain mRNAs, as the cells pass from the exponential into the decline phase of growth. The profile of the specific mAb production rate does not correlate with the kinetics of either the cytoplasmic mAb or the specific mRNAs throughout the culture. Flow cytometry analyses have revealed that dead cells, which account for 40 to 70% of total cells during the decline phase, might significantly interfere with the determination of cytoplasmic mAb levels of cell-lysates ELISA and with the calculation of the specific mAb production rate. Possible influences of these parameters on mAb synthesis and secretion during hybridoma batch culture are discussed.
Journal of Immunological Methods, 2004
Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 Al of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05-5 ng/spot. The assay is simple, reproducible and can process simultaneously a large number of samples.
In this study, the effects of different sample preparation techniques on the separation of monoclonal antibody IgG1 were investigated experimentally. Monoclonal IgG1 was obtained from hybridoma cell line TB/C3 transfected with bcl-2 carrier plasmid, which was grown in serum-free medium. Three different pre-treatment techniques prior to Protein G affinity chromatography have been used in order to concentrate and partial purify the monoclonal antibody. The pre-treatments researched in this paper are precipitation of the antibody by ammonium sulfate, dilution of the antibody in the binding buffer of affinity chromatography and ultrafiltration through an Amicon Ultra-15 filter with molecular weight cut-off at 100 kDa. Purification through direct application of the antibody onto the Protein G affinity column without pre-treatments was used as a control method. The results indicate that the ultrafiltration through an Amicon filter was an effective method for both concentration and partial...
Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.
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.
Two new murine monoclonal antibodies rised against human IgG
Journal of the Serbian Chemical Society
Many pathological conditions are accompanied with changes in the concentration of the total IgG or some of its fraction. For this reason there is great interest in the production of reagents specific for IgG. In this paper, the binding characteristics of two new murine monoclonal antibodies (MoAb), assigned MoAb 15 and MoAb 22, are reported. These MoAbs were produced by hybridoma technology. By performing ELISAs and Western blots analyzes, it was demonstrated that both MoAbs interact specifically with human IgG. Cross reactivity with other sera proteins was not observed. In order to precisely localize the epitopes recognized by MoAb 15 and MoAb 22, the Western blots interactions of these MoAbs with electrophoreticaly separated IgG-fragments, obtained by the action of proteolytic enzymes (papain, pepsin, trypsin), were analyzed. According to the results of these experiments, both MoAbs interacted with epitopes in the Cg3 domain. The affinity constants, calculated from Scatchard plots...
A microELISA for the quantitation of mouse monoclonal IgM in hybridoma culture supernatants
Biotecnología Aplicada, 2000
A microELISA has been set up which is able to sensitively quantitate mouse monoclonal IgM in hybridoma culture supernatants in a suitable range of concentrations. This assay uses ion exchange chromatography-purified rabbit anti-mouse IgM polyclonal antibodies, both in coating capture step and conjugated to horseradish peroxidase in revealing step. It was possible to detect concanavalin A-Sepharose affinity-purified and gel filtration-highly purified mouse monoclonal IgM in the range 190 ng/mL to 12360 ng/mL (sensitivity 125.5 ng/mL). This assay showed a variation coefficient of 4.50% for intraplate repeatability and 7.50% for interplate reproducibility. When compared with an alternative ELISA test that uses a commercial goat anti-IgM-alkaline phosphatase conjugate a correlation coefficient r = 0.92 was found. With this assay, the quality control program of the authors’ hybridoma unit is completed, which was developed with their own resources.
DOAJ (DOAJ: Directory of Open Access Journals), 2015
Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Methods: Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. Results: The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. Conclusion: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.