SHORT COMMUNICATION: Isolation of Polyclonal Monospecific Anti HuIFN-γ Antibodies from an Antiserum Directed Against Human IFN-γ and Lymphokines (original) (raw)
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6th International Cytokine Conference Proceedings
The natural HuIFN-α represents a heterologous mixture of pleiotropic macromolecules, with more or less predominant: antiviral, antiproliferative, antitumor, radioprotective and other biological activities.The most widley used method for interferon (IFN) quantitation is based on virus cytophatic effect (CPE) inhibition. The purpose of this study was to introduce the Calf intestinal epithelilal (CIEB) cell line for measurment of the biological activity of HuIFN-α and HuIFN-γ. Cells were cultivated in Eagle2 s Minimal Essential Medium (EMEM) supplemented with 8% of SR-2.0552P (Serum replacement based on Porcine ocular fluid). Different concentrations (I.U.D ml) of HuIFN-α or γ were added. Cells were incubated overnight at 37oC, IFN samples were removed, and pre-titered VSV (Vesicular Stomatitis Virus) was added. After 18h incubation at 37oC, the 50% CPE was scored microscopically and in addition cells were stained with 0,1% of crystal violet and bound color ex-tracted with the mixture PBS:70% ethanol (1:1)
Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988
The human interferon (hulFN) 8-4 a2s_62/a164.s~ is a genetically engineered hybrid that consists of residues 5-62 of huIFN a2 and residues 64-166 of hulFN al. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring hulFN a subtypes. This novel recombinant hybrid was purified from Eschedchia coil to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAF_~Sepharose chromatography. The purified protein that was treated with 2-mercaptuethanol exh|bited two closely migrating bands on sodium dedecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17800 and 17 100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7.107 uni|s/mg when assayed on human fibroblast ceils and, in distinction to the parental hulFN a2, it also demonstrated antiviral activity on routine [,929 cells. The level of antiproliferative activity of hulFN 8-4 a2s.6z/a164_ls s on various cell lines of different histological origin appeared to be more comparable to that of huWN or1 than hu|FN a2. The data suggest that huIFN 8-4 a2s_62/o~164_t66 hybrid may be a useful tool for understanding huIFN structure-function relations. 0167-4838/88/$03.50
Biotechnology and Applied Biochemistry, 2007
Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both Hu IFN α2b (human interferon α2b) and Hu IFN α8 were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a costeffective purification procedure. After purification, both recovered proteins were biologically active. The Hu IFN α8 subtype induced 1.46-fold more antiviral activity than Hu IFN α2b using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.
6th International Cytokine Conference, Vienna, Austria, August 27-31, 2006
The natural HuIFN-α represents a heterologous mixture of pleiotropic macromolecules, with more or less predominant: antiviral, antiproliferative, antitumor, radioprotective and other biological activities. The most widely used method for interferon (IFN) quantitation is based on virus cytopathic effect (CPE) inhibition. The purpose of this study was to introduce the Calf intestinal epithelial (CIEB) cell line for measurement of the biological activity of HuIFN-α and HuIFN-γ. Cells were cultivated in Eagle2 s Minimal Essential Medium (EMEM) supplemented with 8% of SR-2.0552P (Serum replacement based on Porcine ocular fluid). Different concentrations (I.U.D ml) of HuIFN-α or γ were added. Cells were incubated overnight at 37oC, IFN samples were removed, and pre-titered VSV (Vesicular Stomatitis Virus) was added. After 18h incubation at 37oC, the 50% CPE was scored microscopically and in addition, cells were stained with 0,1% of crystal violet and bound colour extracted with the mixture PBS:70% ethanol (1:1). Photometric reading was performed at 600nm. The following results were obtained: (1) CIEB cells are sensitive for HuIFN-α and HuIFN-γ. (2) In the case of HuIFN- α the lowest amount that can be measured was 3-5 I.U.D ml. (3) The response to HuIFN-is dose-dependent between 5 and 1000 I.U.D ml. (4) In the case of HuIFN-γ, the lowest amount that can be measured was 5-10 I.U.D ml. (5) The response to HuIFN-γ is dose-dependent between 10 and 800 I.U.D ml.
Systemic Review on Pharmacy, 2022
As a type I Interferon’s, Human leukocyte Interferon-alfa (HuIFN-αN3) is a complex pleiotropic molecule composed from several natural subtypes, showing:Antiviral, pro- or anti-inflammatory and antiproliferative activity in vitro. The present work was aimed to detect, analyze and compare the natural subtype’s composition of HuIFN-αN3 from EGIS (Budapest, Hungary)or IMZ (Zagreb, Croatia) and to measure theirs pro- or anti-inflammatory, antiproliferative and cytocidal activity in vitro. HuIFN-αN3 from EGIS, (Budapest, Hungary) or IMZ, (Zagreb, Croatia) contains seven “minor” (a-g) and eight “major” (1-8) subtypes with the isoelectric points: 8.12, 7.40, 7.02, 6.90, 6.45, 6.06, 4.45, and 5.80, 5.65, 5.50, 5.30, 5.05, 4.88, 4.72, 4.53. Subtypes show different pro and anti-inflammatory effects. Those separated in the range of pI 8.0 to 6.0 show pro-inflammatory activity. Those separated in the range 5.80 to 3.50 show anti-inflammatory activity. The subtypes isolated from HuIFN-αN3 (IMZ, Zagreb, Croatia) show very weak pro and strong anti-inflammatory activity. The strongest anti-inflammatory activity has subtype 3. The antiproliferative assays on Human Embryonic Fibroblasts (HEF) or Human amnion cells line (FL) cells shows, that the total antiproliferative activity of HuIFN-αN3 is bigger than this from different subtypes, with the exception of subtype a. The cytocidal activity measured as IU/ml needed to get the 50% cytocidal effect on the Adult pig kidney cell line (PLA) cells shows: In the subtype a with 78.1, 1 with 312.5, 2 with 625, 3 with 0, 4 with 1.250, 5 with 0, 6 with 2.500, 7 with 625 and 8 with 0. When different HuIFN-αN3’s were tested, this from IMZ (Zagreb, Croatia) and SAV (Bratislava, Slovakia) has 156.2. HuIFN-αN3 from EGIS, (Budapest, Hungary) has 312.5, HuIFN-γ has 156.2, rHuIFN-α1 (recombinant Interferon's) has 2500 and rHuIFN-α2 has 5000. The results show, that all of the subtypes (a-g, 1-8) can be neutralized with the polyclonal anti-IFN-αN3. The values of NI were in the range from: -1.15 till -2.21. It can be concluded that this is the picture of different natural subtypes’ content in both preparations from EGIS or IMZ because of different technology. EGIS use concentrated purified preparation, while IMZ use concentrated non purified one. Keywords: Human Interferon alfa, Natural subtypes, Antiproliferative activity, Cytocidal activity, Chromatofocusing, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis
As a type I Interferon's, Human leukocyte interferon-Alfa (HuIFN-αN3) is a complex pleiotropic molecule composed from several natural subtypes, showing: antiviral, pro-or anti-inflammatory and antiproliferative activity in vitro. The present work was aimed to detect, analyze and compare the natural subtype's composition of (Budapest, Hungary) has 312.5, HuIFN-γ has 156.2, rHuIFN-α1 has 2500 and rHuIFN-α2 has 5000. The results show, that all of the subtypes (a-g, 1-8) can be neutralized with the polyclonal anti-IFN-αN3. The values of NI were in the range from:-1.15 till-2.21. It can be concluded that this is the picture of different natural subtypes' content in both preparations from EGIS or IMZ because of different technology. EGIS use concentrated purified preparation, while IMZ use concentrated non purified one.
An anti-cytokine bioactivity assay for interferons-α, -β and -ω
Journal of Immunological Methods, 1996
Interferons-a and -p @'N-a and $1 are cytokines that are widely known to induce potent anti-viral activity. However, it has become increasingly apparent that IFN-CY and -/3 exert a variety of other biological effects, including anti-tumour and immunomodulatory activities, and are increasingly used clinically to treat a range of malignancies, myelodysplasias and autoimmune diseases, e.g. IFN-l3 for multiple sclerosis. The most widely used bioassays for the IFNs are based on their anti-viral activity, but these do not predict the biological activity of the IFNs in anti-tumour and immunomodulatory therapies. Thus, we have developed anti-cytokine-based bioassays that may be more reflective of such activity and which have several advantages over existing anti-viral bioassays. The anti-cytokine bioassay is based on the ability of IFN-a, -p and -o to inhibit granulocyte-macrophage-colony-stimulating factor (GM-CSF) induced proliferation of the erythroleukaemic cell line TF-1. This assay can take only 24 h, is sensitive to 200 fg (0.04 IU) IFN-a or -p and 100 fg (0.02 IU> IFN-w and is able to detect down to these levels in serum or plasma samples. The usefulness of anti-cytokine bioassays for IFN-a, -p and -o is not restricted to the GM-CSF/TF-1 cell format and other alternatives are available, such as erythropoietin (EPO)/TF-1 cells and EPO/UT-7-EPO cells. These assays can be made specific for each of the IFNs by including neutralising antibodies in the bioassay.
Monoclonal antibodies against the human interferon-γ receptor(s)
Molecular Immunology, 1990
Enriched human interferon-y (HuIFN-y) receptor preparations were obtained by affinity chromatography of non-ionic detergent solubilized COLO 205 cell membranes on immobilized recombinant HuIFN-y. The active fractions, identified by a competition ELISA, were used as the immunogen in a BALB/c mouse. Fusion of its splenocytes with myeloma cells yielded several hybrids secreting antibodies that inhibit the antiviral activity of HulFN-y; the two most active ones were selected for further characterization.
The Journal of biological chemistry, 1978
Mouse L cell interferon induced by Newcastle disease virus was purified by a combination of salt precipitation, ion exchange chromatography, gel filtration, and affinity chromatography using anti-interferon anti-body. The preparation was labeled with 125I at a later step of purification, which served as an index of protein concentration. Two main species of interferon molecules differing in molecular weight were separated from each other, and the final preparations obtained were shown to be essentially pure by polyacrylamide gel electrophoresis in the absence as well as in the presence of sodium dodecyl sulfate. The highest specific activities obtained were 2.6 X 10(9) and 7.3 X 10(8) international units/mg of protein (determined by 125I radioactivity) for the 40,000- and 24,000-dalton species, respectively. The preparations at different steps of purification, including those of the highest purity, were tested for the anti-cell growth activity. Their activities were found to be indi...