Detection of Viruses from Bioaerosols Using Anion Exchange Resin.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

High throughput determination of cleaning solutions to prevent the fouling of an anion exchange resin.

Effective cleaning of chromatography resin is required to prevent fouling and maximize the number of processing cycles which can be achieved. Optimization of resin cleaning procedures, however, can lead to prohibitive material, labor, and time requirements, even when using milliliter scale chromatography columns. In this work, high throughput (HT) techniques were used to evaluate cleaning agents for a monoclonal antibody (mAb) polishing step utilizing Fractogel(®) EMD TMAE HiCap (M) anion exchange (AEX) resin. For this particular mAb feed stream, the AEX resin could not be fully restored with traditional NaCl and NaOH cleaning solutions, resulting in a loss of impurity capacity with resin cycling. Miniaturized microliter scale chromatography columns and an automated liquid handling system (LHS) were employed to evaluate various experimental cleaning conditions. Cleaning agents were monitored for their ability to maintain resin impurity capacity over multiple processing cycles by analyzing the flowthrough material for turbidity and high molecular weight (HMW) content. HT experiments indicated that a 167 mM acetic acid strip solution followed by a 0.5 M NaOH, 2 M NaCl sanitization provided approximately 90% cleaning improvement over solutions containing solely NaCl and/or NaOH. Results from the microliter scale HT experiments were confirmed in subsequent evaluations at the milliliter scale. These results identify cleaning agents which may restore resin performance for applications involving fouling species in ion exchange systems. In addition, this work demonstrates the use of miniaturized columns operated with an automated LHS for HT evaluation of chromatographic cleaning procedures, effectively decreasing material requirements while simultaneously increasing throughput. Biotechnol. Bioeng. 2016;113: 1251-1259. © 2015 Wiley Periodicals, Inc.

Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies.

Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations. In this article, scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column studies were applied to characterize a case study where fouling had been observed during process development. The fouling was found to occur on an anion exchange (AEX) polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after successive batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate. Further studies were performed using CLSM to allow temporal and spatial measurements of protein adsorption within the resin, for clean, partially fouled and extensively fouled resin samples. These samples were packed within a miniaturized flowcell and challenged with fluorescently labeled albumin that enabled in situ measurements. The results indicated that the foulant has a significant impact on the kinetics of adsorption, severely decreasing the protein uptake rate, but only results in a minimal decrease in saturation capacity. The impact of the foulant on the kinetics of adsorption was further investigated by loading BSA onto fouled resin over an extended range of flow rates. By decreasing the flow rate during BSA loading, the capacity of the resin was recovered. These data support the hypothesis that the foulant is located on the particle surface, only penetrating the particle to a limited degree. The increased understanding into the nature of the fouling can help in the continued process development of this industrial example.

An anionic exchange stir rod sorptive extraction based on monolithic material for the extraction of non-steroidal anti-inflammatory drugs in environmental aqueous samples.

In this study, a stir rod sorptive extraction (SRSE) adsorbent material was prepared by coating poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) [poly(VP-co-EDMA)] monolithic polymer on stir rod, and then applied to the extraction of three non-steroidal anti-inflammatory drugs (NSAIDs) in environmental aqueous samples. The preparation conditions of monolithic material such as the amount of porogen and the ratio of functional monomer to cross-linker were investigated. To achieve the best extraction efficiency, several parameters, including pH value of sample solution, salt concentration in sample matrix, desorption solvent, extraction time, and desorption time, were optimized. By combining SRSE and high performance liquid chromatography with ultraviolet detector, a SRSE-HPLC/UV method for the determination of NSAIDs in environmental aqueous samples was proposed successfully. The limits of detection (LODs) of the developed method for three NSAIDs ranged between 0.09 and 0.25 ng/mL. Good method reproducibility presented as intra- and inter-day precisions were also obtained with the relative standard deviations (RSDs) less than 8.7% and 9.8%, respectively.

Selective preconcentration of uranyl ion by silica gel phases modified with chelating compounds as inorganic polymeric ion exchangers.

Four chemically modified chelating silica gel phases (I - IV) with ion exchange groups were tested for their potential capability to selectively bind, extract and preconcentrate uranyl ions (UO(2)(2+)) from different aqueous solutions as well as ore samples. Factors affecting such determination processes were studied and optimized. These included the pH of the contact solution, the mass of the silica gel phase extractant, the stirring time during the application of a static technique and the eluent concentration for desorption of the surface-bound uranyl ion and interfering anions and cations. All these factors were evaluated on the basis of determinations of the distribution coefficient value (K(d)) and the percent recovery (R%). Percent recovery values of 91% for silica phase (II) and 93% for silica phase (IV) were identified in the optimum conditions. The proposed preconcentration method was further applied to uranium ore samples as well as granite samples. The determined percentage and ppm values are in good agreement with the standard assigned ones. The structure of the synthesized silica gel phases (I - IV) and their uranyl bound complexes were identified and characterized by means of infrared analysis, thermal analysis (TGA) and potentiometric titration.

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up.

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.

SI-traceable certification of the amount content of cadium below the ng g(-1) level in blood samples by isotope dilution ICP-MS applied as a primary method of measurement.

The development and implementation of a method for the certification of cadmium in blood samples at low ng g(-1) and sub ng g(-1) levels is described. The analytical procedure is based on inductively coupled plasma isotope dilution mass spectrometry (ICP-IDMS) applied as a primary method of measurement. Two different sample digestion methods, an optimized microwave digestion procedure using HNO3 and H2O2 as oxidizing agents and a high-pressure asher digestion procedure, were developed and compared. The very high salt content of the digests and the high molybdenum content, which can cause oxide-based interferences with the Cd isotopes, were reduced by a chromatographic matrix separation step using an anion-exchange resin. All isotope ratio measurements were performed by a quadrupole ICP-MS equipped with an ultrasonic nebulizer with membrane desolvator. This sample introduction set-up was used to increase sensitivity and minimize the formation of oxides (less MoO+ interference with the Cd isotopes). Because of the very low Cd concentrations in the samples and the resulting need to minimize the procedural blank as much as possible, all sample-processing steps were performed in a clean room environment. Detection limits of 0.005 ng g(-1) Cd were achieved using sample weights of 2.7 g. The method described was used to recertify the cadmium content of three different blood reference materials from the Community Bureau of Reference (BCR) of the European Commission (BCR-194, BCR-195, BCR- 196). Cadmium concentrations ranged between approximately 0.2 ng g(-1) and approximately 12 ng g(-1). For these materials, SI-traceable certified values including total uncertainty budgets according to ISO and Eurachem guidelines were established.

The use of histological techniques for the demonstration of ion exchange resins.

AIM: To establish the staining characteristics of certain ion exchange resins in histological material, with a view to enabling confident differential identification. METHODS: Various histological staining procedures were applied to selected pathological material and prepared agar blocks containing the cation exchange resin calcium polystyrene sulphonate and the anion exchange resin cholestyramine. RESULTS: Calcium polystyrene sulphonate uniquely stained strongly by a direct Schiff's reagent procedure without any preoxidation and by the Ziehl-Neelsen method. Cholestyramine was negative by the former method but stained strongly with a standard Congo red technique. CONCLUSIONS: These staining results are consistent with the known structure and properties of polystyrene sulphonate and cholestyramine resins. Polystyrene sulphonate resins have the virtually pathognomonic feature of direct Schiff positivity, while morphology, location, and strong non-birefringent Congo red positivity facilitate the identification of cholestyramine. It is possible that the intrinsic staining characteristics of cholestyramine may be lost once it has bound to its target.

Identification of a 185-kDa band 3-related polypeptide in oxyntic cells.

Polyclonal antibodies to the purified mouse erythrocyte anion exchange protein (band 3) and to a conserved COOH-terminal peptide of mouse band 3 (alpha-Ct) recognized a single major 185-kDa polypeptide in immunoblots of a membrane fraction prepared from rabbit gastric glands. Competition studies revealed that the epitopes shared between the rabbit gastric 185-kDa antigen and the approximately 100-kDa mouse erythrocyte band 3 protein are restricted to the COOH-terminal domain of band 3, which is known to contain the catalytic site for anion exchange activity. Immunofluorescence microscopy was used to demonstrate that this band 3-related polypeptide is associated with the plasma membrane in a subpopulation of gastric gland cells composed exclusively of oxyntic cells, as judged by the coincidence of immunofluorescence with alpha-Ct and with a monoclonal antibody to the gastric H+-K+-ATPase. This alpha-Ct-reactive antigen was further localized to the cytoplasmic face of the basolateral membrane of oxyntic cells, which correlates well with the physiologically determined site of anion exchange activity. These data demonstrate the presence in gastric oxyntic cells of a novel member of the family of proteins related to the erythrocyte anion exchanger. The possibility that the 185-kDa polypeptide is an anion exchanger is discussed.

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane.

Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H+ (pHi) and Cl- (aCli) activities with double-barreled ion-selective microelectrodes. In a HCO3- -free solution of pH 7.4 (HEPES/Tris buffered), pHi was 7.25 and aCli was 33 mM. By an exposure to a HCO3- (25 mM HCO3-/5% CO2, pH 7.4) solution for 15 min, aCli was decreased to 25 mM, and pHi was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO3- concentration [HCO3-]i, calculated by the Henderson-Hasselbalch's equation, was 11 mM at 1 min after the exposure and then slowly increased to 15 mM. Readmission of the HCO3(-)-free solution reversed the changes in aCli and pHi. The intracellular buffering power was about 40 mM/pH. An addition of DIDS (0.2 mM) significantly inhibited the rates of change in aCli, pHi, and [HCO3-]i caused by admission/withdrawal of the HCO3- solution and decreased the buffer value. Replacement of all Cl- with gluconate in the HCO3- solution increased pHi, and readmission of Cl- decreased pHi. The rates of these changes in pHi were reduced by DIDS by 32-45% but not by amiloride (0.3 mM). In the HCO3- solution, a stimulation of intracellular HCO3- production by exposing the tissue to 25 mM NH4+ increased aCli significantly. While in the HCO3(-)-free solution or in the HCO3- solution containing DIDS, exposure to NH4+ had little effect on aCli.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of FAF anionite swelling on its sorption properties]. / Vliianie nebukhaniia anionita FAF na ego sorbtsionnye svoistva.

Experiments were carried out to clarify the effect of swelling coefficients of various samples of the polycondensation anion exchange resin FAF in the C1 form on its sorption properties. The anion exchange resin was used to eliminate contaminating proteins and low molecular weight agents from the allantois fluid and liver cultured cells of pigs. Thermal treatment of the anion exchange resin increased the coefficient of its swelling, leaving the structure unchanged. The resin structure was controlled by electron microscopy and X-ray scattering of low angle. When the resin samples with the swelling coefficients of 3.5 and 4.0 were used, 30-40% of proteins, 50% of reducing agents and neutral carbohydrates and a small quantity of phosphorus were adsorbed.