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At least 559 records · Page 31Linked to original sources
PubMed1996

The use of a microporous polyvinylidene fluoride (PVDF) membrane filter to separate contaminating viral particles from biologically important proteins.

Viral agents (influenza A virus, 80-120 nm; phage T1, 50 nm head, 150 nm head, 150 nm tail; phage PR772, 53 nm; poliovirus, 28-30 nm; and phage PP7, 25 nm) were used to determine the ability of a newly developed, modified polyvinylidene fluoride (PVDF) membrane filter to remove viruses from several fluids. These included ultrapure water, Dulbecco's modified Eagle minimum essential medium (DMEM) and DMEM with 10% fetal bovine serum (DMEM-10). Small volume (10 ml) filtration experiments were done with 47-mm disks while larger volumes (1 litre) were done with virus suspended in DMEM-10, using cartridge filters with a surface area of 1.63 m2. With 47-mm disks, influenza A virus and phage T1 were removed to below detectable limits in all fluids tested (titre reduction [Tr] > 2.0 x 10(6) and > 5.8 x 10(8), respectively). The retention of phage PP7 and poliovirus was consistent but fluid dependent. The greatest concentration of phage PP7 and poliovirus was removed from ultrapure water (phage PP7, Tr = 2.1 x 10(7); poliovirus, TR > 3.2 x 10(4), while the removal efficiency from DMEM-10 was substantially lower (phage PP7, Tr = 2.3; poliovirus, Tr = 2.1 x 10(2)). Results of cartridge challenges in DMEM-10 were comparable to the corresponding small disk challenges. These results demonstrate that this PVDF membrane filter was very effective (Tr > 10(6)) in removing viral particles (> 50 nm); smaller viruses (< 50 nm) were also consistently removed, but the level of removal depended on the virus and type of fluid tested. In separate experiments, the recovery of purified albumin (69,000 Da) and IgG (150,000 Da) in the filtrate was also determined at approx. 0.015 mg/ml and approx. 10 mg/ml. Recovery of albumin and IgG was > 90%. Efficient virus retention coupled with high recovery of protein < 150,000 Da suggest potential applications of this membrane filter, when protection against adventitious viral contaminants is desired.

Animals↗
PubMed1998

Clearance of murine leukaemia virus from monoclonal antibody solution by a hydrophilic PVDF microporous membrane filter.

Continuous cell lines (used in the manufacture of monoclonal antibodies and other therapeutic proteins) may harbour retroviral particles; nevertheless, they are deemed acceptable if the production system is validated for clearance of these contaminants. As part of a campaign to evaluate and validate appropriate virus removal methodologies for monoclonal immunoglobulin G (IgG) products, studies were undertaken with a hydrophilic polyvinylidene fluoride (PVDF) microporous membrane-the Ultipor(R) VF grade DV50 virus removal membrane. Testing conducted with murine leukaemia virus, a specific model virus used in viral validation studies to document removal of retroviruses, demonstrated a greater that 7 log clearance by the Ultipor(R) VR grade DV50 membrane. Filterability testing established the feasibility of positioning the filter at either of two different stages during manufacture. The results presented in this study demonstrate that membrane filtration through the Ultipor VF grade DV50 virus removal filter can constitute part of the purification regimen to effect removal of endogenous retrovirus-like contaminants from monoclonal IgG products.

Animals↗
PubMed2002

Towards proteome-wide production of monoclonal antibody by phage display.

Sequencing of the human genome reveals that there are approximately 30,000 genes that encode an even greater number of proteins which comprise the human proteome. Characterization of gene products at the genome-wide scale requires the development of high throughput methods to generate temporo-spatial information on each and every protein in the cell under normal and pathological conditions. Monoclonal antibodies are important reagents for these studies. We have developed a method to generate human monoclonal antibodies by selecting phage antibody libraries directly on antigen blotted onto poly(vinylidene fluoride) membranes. Cellular proteins are first separated by two dimensional (2D) gel electrophoresis, Western blotted onto poly(vinylidene fluoride) membranes, and used to select phage antibody libraries. Monoclonal antibodies can be generated against individual protein spots on a 2D gel. The antibodies are functional in Western blotting, ELISA, and immunohistochemistry. Automation of this process should allow high throughput production of monoclonal phage antibodies against cellular proteins as well as proteins that are uniquely expressed under pathological conditions.

Amino Acid Sequence↗
PubMed1995

Purification and identification of brain-derived neurotrophic factor from human serum.

Brain-derived neurotrophic factor (BDNF), a 27-kDa noncovalently linked homodimer with subunits of approximately 13.5 kDa as viewed by SDS-PAGE, is thought to be primarily produced in the central nervous system. We report here the isolation of BDNF from pooled normal human sera, using a two-step purification process followed by SDS-PAGE, transfer to a polyvinylidene difluoride membrane, and subsequent identification of the protein by sequence analysis of the appropriate band(s) from the membrane. The level of BDNF in pooled human sera was estimated to be approximately 15 ng/ml as determined by an enzyme-linked immunosorbant assay. The average for six individuals was 18.9 +/- 5.7 ng/ml. There is an approximately 200-fold increase in the levels of BDNF in serum relative to plasma. Results from experiments using differential centrifugation suggest that the source of this increase is due to release from platelets. The presence of high levels of BDNF in serum suggests a role for this neurotrophin either in nerve repair at sites of injured tissue or in nonneuronal functions.

Amino Acid Sequence↗
PubMed1995

Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive ligand spinach Rubisco. An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of approximately 60 kDa.

Chromatography, Affinity↗
PubMed1994

Reduction of acetaminophen interference in glucose sensors by a composite Nafion membrane: demonstration in rats and man.

Amperometric glucose sensors typically monitor the production of hydrogen peroxide generated in the course of the enzymatic oxidation of glucose. At the applied potential necessary to oxidize the peroxide produced, other species are also electroactive and contribute to the signal. Interference of ascorbate or urate has been effectively eliminated, but that resulting from the widely used analgesic acetaminophen is not. The aim of this work was to reduce this interference, which was found to be possible by introducing a membrane constructed of Nafion. We compared the in vitro sensitivity to acetaminophen of five Nafion sensors with that of five non-Nafion sensors with identical glucose sensitivity (2.0 +/- 0.4 vs 1.9 +/- 0.1 nA.mmol-1.l-1, NS): sensitivity to acetaminophen was 12.2 +/- 2.7 vs 30.8 +/- 6.3 nA.mmol-1.l-1, respectively (p < 0.05). These sensors were tested in rats by implanting in each animal one Nafion and one non-Nafion sensors. The in vivo sensitivity to glucose was similar (0.33 +/- 0.09 vs 0.30 +/- 0.05 nA.mmol-1.l-1, NS). The current generated by an acetaminophen infusion (plasma acetaminophen plateau = 140 +/- 10 mumol/l) was much decreased in the case of the Nafion sensor: 0.5 +/- 0.3 vs 2.0 +/- 0.7 nA, p < 0.05). Five Nafion sensors were implanted in the subcutaneous tissue of normal human volunteers who were given on oral dose of 500 mg acetaminophen.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗