Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG(1), and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r(2) (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.19647347

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G(1) monoclonal antibody

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me2+-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn2+ complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose. (C) 2004 Elsevier B.V. All rights reserved.8164167125926

Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG(1) Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand-CM-Asp-was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.160721482165Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions

In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II (R) stop and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the calculation of kinetic parameters. The results show that S2 cells metabolism was able to adjust to different environmental situations, as determined by medium formulation, as well as by the particular situation resulting from the culture conditions. Cells attained a 163% higher final cell concentration (1.4 x 10(7) cells mL(-1)) in SF900 II (R) medium, when compared to serum-supplemented TC-100 medium. Also, a maximum specific cell growth rate 52% higher in SF900 II (R) medium, when compared to serum-supplemented TC-100 one, was observed. Glutamine was the growth limiting factor in SF900 II (R) medium, while glucose, sometimes associated with glutamine, controlled growth in serum-supplemented TC-100 medium based formulation. The different pattern of lactate production is an example of the versatility of the metabolism of these cells. This by-product was produced only in glutamine limitation, but the amount synthesized depended not only on the excess glucose, but on other medium components. Therefore, in serum-supplemented TC-100 medium a much smaller lactate amount was generated. Besides, glucose was identified not only as a growth limiting factor, but also as a viability limiting factor, since its depletion accelerated cell death.571233