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PubMed1992

A simple method of concentrating monoclonal antibodies from culture supernatant by ultrafiltration.

How to obtain concentrated monoclonal antibodies from culture supernatant is of great importance to many scientists today. In this report, a simple, single-step technique is described to concentrate monoclonal antibodies directly from hybridoma-culture supernatant. The contaminants can be removed from the supernatant by using an ultrafiltration unit which allows macromolecules of less than 100,000 MW to pass through leaving behind monoclonal antibodies which is purified and concentrated more than 25-fold.

Antibodies, Monoclonal↗

PubMed1994

Bovine colostrum ultrafiltrate: an effective supplement for the culture of mouse-mouse hybridoma cells.

An ultrafiltrate fraction (UF) of bovine colostrum has been successfully used as a cell culture supplement for growth and monoclonal IgG antibody production of cultured mouse-mouse hybridomas derived from spleen cells. In this study we compared the ability of UF to support growth and antibody production of IgA hybridomas derived from Peyer's patch cells with that of an IgG hybridoma cell line. One IgG (LPC2) and two IgA hybridoma cell lines (RB3 and P2E7) were used as models. The optimal UF concentration for Ig production and cell growth for both the IgA hybridoma RB3 and the IgG hybridomas was 5-10%. Initial plating density was found to be a critical factor for IgA hybridoma cell growth: the IgA hybridomas required a seeding density of at least 70,000 cells/ml to grow compared to 15,000 IgG hybridoma cells/ml (Pakkanen et al., 1992). The addition of small amounts (up to 2%) of FBS in 10% UF supplemented medium did not enhance IgA production or cell growth. RB3 and LPC2 cells seeded at equal density and grown in 10% UF for 8 days attained maximum cell densities at 3-4 days that were 58% (RB3) or 34% (LPC2) lower than those in 10% FBS, but the total amounts of monoclonal antibody produced were 73% and 83%, respectively, of that in 10% FBS. Thus, Ig production per cell was 22-27% higher in 10% UF than in 10% FBS. Hybridoma cells could be cultured for at least 5 weeks without any reduction in growth rates, if medium was partially but not completely replaced twice a week. This suggests that hybridoma cells maintained in UF supplemented medium secrete growth promoting factors. Cells maintained in UF for up to 5 weeks sustained similar monoclonal antibody production rates as in short term culture. These results show that UF can be used as an economical and effective hybridoma culture supplement for the production of both IgG and IgA antibodies.

Animals↗

PubMed1984

Ultrafiltrable plasma testosterone and 5 alpha-dihydrotestosterone as measured across the human testes.

It has been confirmed that within the human plexus pampiniformis a venous-arterial transfer of testosterone (T) (5.95%) and of dihydrotestosterone (DHT) (16.58%) occurs. It could be established that in the testicular artery the percentage of ultrafiltrable DHT was significantly lower compared with the venous compartment. It is assumed that by this means a biological recycling of the active androgen DHT into the testis is achieved.

Aged↗

PubMed1983

Inhibitors of peripheral-type benzodiazepine receptors present in human urine and plasma ultrafiltrates.

Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.

Adsorption↗

PubMed1985

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose.

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3H-Water and 3H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3H-F and 3H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies.

Female↗

PubMed1990

Ultrafiltration membranes as carriers for lipase immobilization.

The feasibility of directly incorporating lipase from Rhizopus in the interior of poly(vinyl chloride) ultrafiltration membranes during the phase inversion process for their manufacturing has been demonstrated. The obtained membranes used for plant oil hydrolysis have shown better time stability as compared with those of lipase immobilized by adsorption. The specific activity of entrapped lipase achieves values higher than those of the soluble one. The activity of immobilized lipase is strongly affected by the rate of removing fatty acids from the interior of the membrane.

Enzymes, Immobilized↗

PubMed1995

The ultrafiltration and ESR study of Cd2+ binding to human erythrocyte spectrin.

The Cd2+ binding to human erythrocyte spectrin tetramer (SPT) was studied by the ultrafiltration method. The results indicate that the Cd2+ binding depends on Cd2+ concentration in a biphasic feature. Thus, it is different from the Tb(3+)-SPT reaction. The biphasic feature is in accordance with the results of fluorescence and circular dichroism (CD) studies on Cd(2+)-SPT complex. The data were analyzed by the Scatchard method. At the first stage (the mole ratio of Cd2+/SPD < 45), there are two types of Cd2+ binding sites, with the binding sites and corresponding binding constants determined as: n1 = 6, K1 = 3.3 x 10(5) M-1; n2 = 8, K2 = 9.1 x 10(4) M-1. At the second state (Cd2+/SPD > 45), the Cd2+ binding manifests a positive cooperative effect. The interaction between Cd2+ and maleimide spin labeled (MSL) SPT was studied by the ESR method. The results were fitted with the multiple equilibrium model, and one Cd2+ high-affinitive binding site with association constant 9.61(7) x 10(5) M-1 was obtained. The conjugation of maleimide with thiol group results in the decrease of Cd2+ high-affinitive binding sites from 6 to 1. This demonstrated that the thiol groups of SPT were involved in the high binding sites. The increase of mobile fraction of MSL in low Cd2+ concentration revealed that Cd2+ binding to SPT induces a significant conformation change of MSL-SPT. The rotation correlation time of MSL attached to SPT varied from 4.4 x 10(-9) to 6.5 x 10(-9) sec.

Binding Sites↗

PubMed1980

Separation of macromolecules by ultrafiltration: removal of poly(ethylene glycol) from human albumin.

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the 'permanent' effects were due to irreversible adsorption to the membrane. By measuring flux over a 10(6)-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (< 0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (> 0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.

Adsorption↗

PubMed1989

Ultrafiltration of human serum to determine the size of species that poison voltammetric electrodes.

Solutions containing the electroactive species tris(2,2'-bipyridine)-ruthenium dication and ferrocene carboxylate as well as bovine serum albumin (BSA), gamma globulin (IgG) or human serum were ultrafiltered. Cyclic voltammetry at a glassy carbon electrode showed that the deleterious effects of BSA and IgG were eliminated by passing the solution through a 30 kdalton filter. This was insufficient filtration of serum, however. Ultrafiltration with 3 or 5 kdalton filters was required for good voltammetry. This indicates that the removal of the preponderant proteins is necessary, but not sufficient, to protect electrochemical surfaces.

Biosensing Techniques↗

PubMed1978

A constant-volume ultrafiltration technique for the calculation of equilbrium binding data.

A constant-volume ultrafiltration technique is described, and details of its assessment presented. The retention characteristics of two membranes were evaluated using molecules of known molecular weight. The technique is rapid, precise, economical of material and yields equilibrium data. In these respects, it compares favourably with conventional techniques such as equilibrium dialysis.

Albumins↗

PubMed1984

Profiles of strongly polar and less polar acids obtained from human blood, plasma and serum by two-step ultrafiltration.

A two-step ultrafiltration method combined with anion-exchange chromatography is described for the separation of lipophilic and strongly lipophobic acids occurring in human blood, serum and plasma. After treatment with diazomethane, the acid fractions are separated further by gas chromatography. The acids were identified by their mass spectra. Profiles obtained from the strongly acidic fraction of blood samples of healthy individuals are characterized by main peaks corresponding to organic phosphoric acid esters. The peaks are absent in plasma and serum.

Acids↗

PubMed1993

Determination of acyclovir by ultrafiltration and high-performance liquid chromatography.

A simple, sensitive and rapid high-performance liquid chromatographic (HPLC) procedure to determine total serum acyclovir concentrations is described. The assay involves a heat inactivation step at 56 degrees C to prevent risk of infection, ultrafiltration as a pretreatment step prior to ion-pair reversed-phase liquid chromatography using guanosine as internal standard, and ultraviolet detection at 254 nm. This method has excellent recovery (97-100%), linearity (0.5-100 mg/l) and precision (1.2-8.0% coefficient of variation). The detection limit is 50 micrograms/l. The assay proved to be suitable for therapeutic drug monitoring of acyclovir.

Acyclovir↗

PubMed1995

Phenytoin free fraction determination: comparison of an improved direct serum injection high-performance liquid chromatographic method to ultrafiltration coupled with fluorescence polarization immunoassay.

Recent developments in restricted-access media (RAM) liquid chromatography make the simultaneous determination of total and free phenytoin concentrations possible by direct injection of drug-containing serum samples. A comparison of phenytoin free fraction determination by ultrafiltration coupled with fluorescence polarization immunoassay (TDX) to an improved direct injection RAM-HPLC method is presented. Our improved method differs from those previously reported with regard to column type, mobile-phase composition, and column temperature. Replicate samples analyzed by each method yielded similar values for serum phenytoin free fraction.

Anticonvulsants↗

PubMed1996

Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography.

An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. didanosine was analyzed using a 150 mm x 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20 degrees C, 4 degrees C and -20 degrees C.

Chromatography, High Pressure Liquid↗

PubMed1996

High-performance liquid chromatographic analysis of (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515) in brain and blood microdialysate (on-line) and in plasma ultrafiltrate of freely moving rats.

(S)-alpha-Amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515, I), a competitive antagonist of the N-methyl-D-aspartate receptor, has significant pharmacological activity in the central nervous system (CNS). An extremely sensitive and selective analytical method was developed for the simultaneous analysis of I and its hydroxylated analog (RDC, II) in the microdialysate (MD) and plasma ultrafiltrate (UF) of rats. Microdialysis was used for in vivo sampling of unbound drug in the CSF, cortical extracellular fluid and in the blood of freely moving rats. Compound II was used for retrodialysis-based in vivo calibration of microdialysis probes to estimate the recovery of I. Compound I, being extremely hydrophilic with a high degree of ionization at the physiological pH of 7.4, has limited access to the brain regions. This, combined with its low microdialysis recovery, made the estimation of low brain concentrations of I a challenge. The analytes in MD and UF were separated (within 5 min) by reversed-phase HPLC on a 250 x 4.6 mm I.D. Maxsil 5 microns RP-2 column, and fluorescence of the eluent was monitored at 255 nm (lambda ex) and 320 nm (lambda em). A 0.09% (v/v) aqueous solution of trifluoroacetic acid (1 ml/min) was used as the mobile phase. The response for I in MD and UF samples was linear from 5 to 2000 ng/ml and from 20 to 10,000 ng/ml, respectively. The between-run (n = 6) and within-run (n = 3) variability of the assay was < 15%. Plasma-protein binding of I (fu = 0.68) was determined to be linear from 0.1 to 10 micrograms/ml. The analytical sensitivity, precision and accuracy of this method was suitable for the characterization of the pharmacokinetics and the CNS distribution of I, following administration of intravenous (i.v.) infusion, single i.v. bolus and multiple i.v. bolus doses of I to freely moving rats, with continuous microdialysate sampling of multiple tissues and simultaneous on-line HPLC analysis. Pharmacokinetic parameters for I, as determined from concentrations in blood MD samples with on-line analysis, were in good agreement with those estimated from concentrations in the UF of plasma samples obtained by conventional sampling.

Animals↗

PubMed2004

Ultrafiltration-based assay for heparanase activity.

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.

Animals↗

PubMed2004

An analysis of oxygen consumption and oxygen delivery in euthermic infants after cardiopulmonary bypass with modified ultrafiltration.

BACKGROUND: The balance between systemic oxygen consumption (VO2) and delivery (DO2) is impaired after cardiopulmonary bypass (CPB) and is related to systemic inflammatory response syndrome. We sought to assess VO2 and DO2 and their relationship with proinflammatory cytokines after CPB with the use of modified ultrafiltration (MUF) in infants. METHODS: Sixteen infants, aged 1-11.5 months (median, 6.3 months), undergoing hypothermic CPB with MUF were studied during the first 12 hours after arrival in the intensive care unit (ICU). The central temperature was maintained at 36.8-37.1 degrees C using external cooling or warming. VO2 was continuously measured using respiratory mass spectrometry. Arterial blood samples for the tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-8 (IL-8) were taken and DO2 was calculated using the Fick principle on arrival at the ICU, and 2, 4, 8, and 12 hours postoperatively. Cytokines were additionally measured after induction of anesthesia and at the end of MUF. RESULTS: VO2 significantly decreased by 18.8% during the study period. DO2 was depressed throughout this period and reached a nadir at 8 hours (357.1 +/- 136.2 ml x min(-1) x m(-2)). The decrease in cytokines was accompanied with the decrease in VO2 despite varied relationships between the levels of each of the cytokines and VO2 measurements. CONCLUSIONS: Our data indicate an unusual continuous decrease in VO2 during the first 12 hours after CPB in infants. Control of body temperature to maintain euthermia in addition to the use of MUF may be beneficial to the balance between VO2 and DO2 in the early postoperative period.

Body Temperature↗

PubMed2005

Recovery of protein from poultry processing wastewater using membrane ultrafiltration.

The feasibility of using ultrafiltration (UF) to recover protein from poultry processing wastewater (PPW) after primary treatment was investigated. By using polysulfone membrane with 30,000 molecular-weight-cut-off, almost all crude proteins in PPW were retained, subsequently reducing the chemical oxygen demand (COD) in the effluent to less than 200 mg L(-1). Similar to the processing of proteinaceous materials, the average fluxes reached only 100 Lm(-2) h(-1). By identifying the optimal values of key operation parameters, including pH, volumetric flow rate, and transmembrane pressure (6.74, 683 mL min(-1) and 14 psi, respectively) using response surface methodology (RSM), the flux was improved to higher than 200 Lm(-2) h(-1). Although severe membrane fouling was still inevitable after processing, flushing the membrane with a cleaning reagent was found capable of effectively restoring membrane performance.

Animals↗