Elution Buffer Research Articles

The suitability of using spectrophotometry to determine the concentration and purity of DNA extracted from processed food matrices

Species detection using DNA extracted from processed foods has become important in food authenticity testing. Extracting DNA from processed foods can be challenging, and it is considered essential to determine the concentration and purity of extracted DNA using spectrophotometry. While it has been found that chemicals from extraction reagents can interfere with readings at A260, the effect of chemical residue from a processed food matrix has not been investigated. The aim of this study was to determine whether spectrophotometry is suited to determine the concentration and purity of DNA extracted from processed foods. DNA was extracted from control DNA and six processed food matrices using 20 methods (including modifications) intended for processed foods. The concentration of DNA was measured using spectrophotometry, fluorometry and qPCR. Spectrophotometry was found to overestimate, whereas fluorometry underestimated the amount of extracted DNA. A novel approach was used to reduce the bias by calibrating the spectrophotometer using the extraction control instead of elution buffer. Finally, there was no relationship between the concentration, purity and amplifiability of DNA extracted from processed foods. Thus, spectrophotometry is not recommended as a suitable method to determine the concentration and purity of DNA extracted from processed foods.

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Membrane Chromatography and Fractionation of Proteins from Whey—A Review

Membrane chromatography (MC) is an emerging bioseparation technology combining the principles of membrane filtration and chromatography. In this process, one type of molecule is adsorbed in the stationary phase, whereas the other type of molecule is passed through the membrane pores without affecting the adsorbed molecule. In subsequent the step, the adsorbed molecule is recovered by an elution buffer with a unique ionic strength and pH. Functionalized microfiltration membranes are usually used in radial flow, axial flow, and lateral flow membrane modules in MC systems. In the MC process, the transport of a solute to a stationary phase is mainly achieved through convection and minimum pore diffusion. Therefore, mass transfer resistance and pressure drop become insignificant. Other characteristics of MC systems are a minimum clogging tendency in the stationary phase, the capability of operating with a high mobile phase flow rate, and the disposable (short term) application of stationary phase. The development and application of MC systems for the fractionation of individual proteins from whey for investigation and industrial-scale production are promising. A significant income from individual whey proteins together with the marketing of dairy foods may provide a new commercial outlook in dairy industry. In this review, information about the development of a MC system and its applications for the fractionation of individual protein from whey are presented in comprehensive manner.

Isolation and Quantification of Polystyrene Nanoplastics in Tissues by Low Pressure Size Exclusion Chromatography.

Ecotoxicity investigations of plastic nanoparticles (NPs) should pay more attention to their ability to pass barriers, accumulate, and initiate toxicity in cells. The purpose of this study was to develop a simple size exclusion chromatography (SEC) methodology to measure plastic NPs in biological tissues. A SEC column was prepared using a high-resolution gel for large macromolecules to separate plastic NPs from the protein/lipid pools in tissues. It was necessary to prepare the samples in high salt and non-ionic detergent (0.5 M NaCl and 0.2% Tween-20) and apply 0.2% Tween-20 containing 14 mM NaCl for the elution buffer to limit proteins adsorption to NPs. This methodology was able to resolve 50 and 100 nm polystyrene NPs from the protein/lipid pools in tissue homogenates. The fluorescent dye neutral red (NR) was also used for transparent NPs. Moreover, a sample fractionation step was also proposed for plastic NPs concentration using a salting-out methodology with saturated NaCl (5 M) and acetonitrile. Polystyrene NPs partition in acetonitrile, which were further analyzed by SEC. This methodology was tested in two case studies with clams collected in a high boat traffic (harbor) area and with caged freshwater mussels downstream of a large urban area. Although the present methodology was developed with polystyrene NPs it should be amenable to other plastic polymers that react with the NR fluorescent probe.

Multi‐omics Sample Preparation Workflow for Proteins and DNA Using the Reversible Protein Tag ProMTag

Sample preparation is a crucial first step for both genomics and proteomics workflows. Removal of contaminants such as salts, detergents, and other biologics while maintaining high yields of the desired product is key to reproducible and informative results from these analyses. More and more frequently, these ‐omics technologies are being used in tandem to gain deeper insights into biological processes. However, multi‐omics sample preparation remains tedious and usually requires many steps in multiple different sample preparation workflows.In this study, we present a new multi‐omics workflow for the simultaneous preparation of DNA and protein samples from a single starting cell lysate. We accomplished this using the ProMTag reversible click chemistry technology that allows for reversible modification of the surface of proteins. Using ProMTag we were able to tag proteins in a cell lysate, bind them to ProMTag capture resin, and then precipitate nucleic acids so they also stay with the resin. With the nucleic acids and proteins bound to the resin, we were then able to wash away detergents, salts, and other contaminants. We then eluted the nucleic acids by resolubilizing in a nucleic acid elution buffer. We then were able to reverse the ProMTag by adding the protein elution buffer and eluted the sample in a mass spectrometry (MS) compatible buffer ready for proteomic analysis. Using this workflow we got yields >75% for protein and >90% for DNA. Gel electrophoresis showed a genomic DNA band free of degradation and the 260/280 absorption ratio indicated a pure DNA sample. Whole genome sequencing and MS proteomics analysis were performed and compared to traditional separate sample preparation workflows for DNA and proteins. This work establishes a new, high yield, reproducible workflow for the simultaneous preparation of DNA and proteins for genomics and proteomic analysis from a single starting sample.

Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca2+-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca2+ ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.

Comparison of the impact of anion and cation selection onto cation exchange chromatography of model proteins

For a further improvement of ion exchange chromatography for protein purification, the optimization of mobile phase composition is an important factor. The effects of salt type selection on cation exchange chromatography (CEC) have been investigated in this work. Hereby, the significance of co-ion and displacing ion selection were compared. Therefore, the suitability of a constant cationic or anionic strength for multivalent salts for the determination of salt impact were evaluated. The retention and separation behavior of the three model proteins bovine serum albumin, lysozyme and α-chymotrypsin were investigated with varying salts in the elution buffer. For this, five different cations and five different anions were used. Hereby, the effects of anion and cation selection were analyzed independently. It was shown that a constant cationic strength is a suitable setup for the determination of salt effects on protein retention in CEC, as the resulting retention factors did not show a clear trend regarding ion valence. It was observed that for the used process conditions the anion selection could change the separation factor by up to 30% and the cation selection by up to 16%. These changes were attributed to protein specific salt effects of citrate on bovine serum albumin and magnesium on α-chymotrypsin. It was demonstrated that at constant cationic strength the co-ion selection can be of the same or even of higher importance for protein separation as the selection of the displacing ion in CEC.

Binary separation control in preparative gradient chromatography using iterative learning control

Purification of biopharmaceuticals has shifted toward continuous and integrated processes, in turn bringing along a need for monitoring and control to maintain a desired separation between the target pharmaceutical and any impurities it may carry. In this study, a cycle-to-cycle control of the retention volumes of two compounds in a chromatographic, ion exchange purification step was developed, allowing the process to maintain the desired retention volumes in the separation. The controller made use of a model-based, multivariate iterative learning control (ILC) algorithm that used a quadratic-criterion objective function for optimal set point control, along with feed-forward control based on direct model inversion for preemptive control of set point changes. The model was calibrated using 3 experiments, allowing for fast setup. The controller was tested by introducing three different disturbances to a sequence of otherwise identical ion exchange separation processes: a change in the salt concentration of the elution buffer, a change in set point, and a change in the pH of the elution buffer. It was capable of correcting for all disturbances within at most 3 cycles, proving its efficacy. The successful application of ILC for separation control in biopharmaceutical purification paves the way for the development of further ILC-based control strategies within the field, as well as combination with other control strategies.

Understanding the Significance of Sample Preparation in Studies of the Nanoparticle Metabolite Corona.

The adsorption of metabolites to the surface of nanomaterials is a growing area of interest in the field of bionanointeractions. Like its more-established protein counterpart, it is thought that the metabolite corona has a key role in the uptake, distribution, and toxicity of nanomaterials in organisms. Previous research has demonstrated that nanomaterials obtain a unique metabolite fingerprint when exposed to biological matrices; however, there have been some concerns raised over the reproducibility of bionanointeraction research due to challenges in dispersion of nanomaterials and their stability. As such, this work investigates a much-overlooked aspect of this field, i.e., sample preparation, which is vital to the accurate, reproducible, and informative analysis of the metabolite corona. The impact of elution buffer pH, volume, and ionic strength on the metabolite corona composition acquired by uncapped and polyvinylpyrrolidone (PVP)-capped TiO2 from mixtures of cationic and anionic metabolites was studied. We demonstrate the temporal evolution of the TiO2 metabolite corona and the recovery of the metabolite corona, which resulted from a complex biological matrix, in this case human plasma. This work also demonstrates that it is vital to optimize sample preparation for each nanomaterial being investigated, as the metabolite recovery from Fe3O4 and Dispex-capped TiO2 nanomaterials is significantly reduced compared to the aforementioned uncapped and PVP-capped TiO2 nanomaterials. These are important findings for future bionanointeraction studies, which is a rapidly emerging area of research in nanoscience.

Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies.

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Circulating Cell-Free DNA (ccfDNA) Isolation from Patients with Diffuse Large B-Cell Lymphoma (DLBCL): Comparative Analysis of Commercial Kits

Introduction: Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD) [1,2]. Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA.Methods: Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 ºC, followed by other centrifugation at 20.000 x g for 10 min at 4 ºC to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 ºC until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) [Table 1] according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 µL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany).Results: The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOOD (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL) [Figure 1]. The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360-380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb .Conclusion: We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rat-Bite Fever: Developing and testing a modified time-kill protocol based on Edberg and Edberg 1983 experiemnt in order to test S. moniliformis growth in 0.05% SPS and proposed growth curve

Abstract Introduction/Objective Streptobacillus moniliformis is the primary causative agent of rat-bite fever (RBF) and Haverhill fever (HF). Rat-bite fever and Haverhill fever are difficult to diagnose in a clinical setting and are likely severely under-represented and under-reported worldwide. Clinical presentation often includes fever, chills, myalgia, headache, and vomiting. Patients may also develop a maculopapular rash covering their extremities approximately 2-4 days after onset of fever followed by polyarthritis in roughly 50% of patients with a mortality rate of 10-13% if left untreated. This is further complicated by the fact that submission and processing guidelines of clinical samples for the recovery of the organism are based on ‘outdated’ techniques and clinical laboratory methods that are over 50 years removed from current procedures, instrumentation and guidance. Methods/Case Report DNA from collected frozen time-point samples, were collected (n=84) and extracted using an in-house custom protocol utilizing 180 µl of bacterial lysis buffer with 20 µl of PCR grade proteinase K, for a total of 200 µl. The blood culture time-point samples were then thawed on ice, and 200 µl of blood culture sample was then added to the lysis mix, vortexed and incubated at 65°C for 10 minutes (an additional 95°C inactivation step was omitted to avoid whole blood clotting). After incubation, each sample was vortexed briefly again and extracted using the Roche automated MagNA Pure Compact instrument with an initial input volume of 400 µl and stored in a final elution buffer volume of 100 µl. All quantatitive time kill data was colleced via a CDC in-house designed PCR assay developed for S. moniliformis. Results (if a Case Study enter NA) Experimenting with varying amounts of blood inoculum, 10 ml of blood was determined to provide the best results for detection and growth/viability as well as propose a theoretical growth curve for the organism. Conclusion We found that in 100% of the isolates tested (and all the variations of testing within), SPS (up to a concentration of 0.05 % w/v) in commercially available blood culture bottles appeared to be inactivated, allowing for the growth detection and culturing of S. moniliformis using an automated continuous blood culture system when 10 ml of blood was inoculated.

Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli

Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated.It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to ˂ 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.

Phi 6 recovery from inoculated fingerpads based on elution buffer and methodology

Phi 6 (Φ6) bacteriophage is a proposed surrogate to study pathogenic enveloped viruses including SARS-CoV-2—the causative agent of COVID-19—based on structural similarities, BSL-1 status, and ease of use. To determine the role of virus-contaminated hands in disease transmission, an enhanced understanding of buffer and method performance for Φ6 recovery needs to be determined. Four buffer types and three methodologies were investigated for the recovery of Φ6 from human fingerpads over a 30 min duration. Phosphate buffered saline (PBS), PBS + 0.1 % Tween, 0.1 M glycine + 3% beef extract, and viral transport medium were evaluated as buffers for recovery of Φ6 via a dish, modified glove juice, and vigorous swabbing method. Φ6 concentrations on fingerpads were determined at 0-, 5-, 10-, and 30-min post-inoculation. While there were observed differences in virus recovery across buffer and method types depending on the time point, log PFU recovery based on buffer type or methodology was not significantly different at any time point (P > 0.05). The results presented in this study will allow for future work on Φ6 persistence, transfer between hands and surfaces, and efficacy of hand hygiene methods to be performed using a well-characterized and validated recovery method.

CIRCULATING CELL-FREE DNA (CCFDNA) ISOLATION FROM PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL): COMPARATIVE ANALYSIS OF COMMERCIAL KITS

Introduction Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD). Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA. Methods Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 °C, followed by other centrifugation at 20.000 x g for 10 min at 4 °C to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 °C until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 μL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany). Results The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOO D (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL). The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360–380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb. Conclusion We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

The The Effect of Elution Volume for Immunoprecipitation on m6A-Seq Analysis

Objective: To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation. Method: Two volumes (125 ?L for Group C and 100 ?L for Group T) of elution buffer were used to explore whether smaller volume could save testing time. Result: Time consumption of elution in Group T was significantly shorter than that in Group C, while the efficiency of eluted m6A-containing fragments and the performance of m6A-Seq as indicated by m6A peak distributions showed no difference between the two groups. Conclusion: A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.

SARS-CoV-2 airborne transmission: A validated sampling and analytical method

The airborne transmission path for SARS-CoV-2 is of primary scientific and health-related interest as it could actually involve management, accessibility, use and functionality of many activities, including hospitals), schools, workplaces, factories, transport, sport venues and outdoor environment. It is necessary to develop a sampling and analytical method for virus-laden bioaerosol that could be considered reliable and validated according to ISO/IEC 17025 requirements.The present paper defines sample pretreatments aiming at recover SARS-CoV-2 from glass-fiber and PTFE filters employed by low and high-volume air samplers. Recovery test results focused on the sample concentration step carried out by means of ultracentrifugation are reported as well. Human coronavirus strain OC43 (a surrogate β-coronavirus with the same SARS-CoV-2 particle structure) was used to validate each step of the recovery tests.We found that the elution efficiency of coronavirus OC43 from glass-fiber and quartz filters could be strongly enhanced by using an elution buffer containing up to 40% of fetal calf serum. Moreover, the recovery from PTFE filters is much higher and easier than from glass-fiber filters: for glass-fiber filters a 3 h-shaking phase, followed by a 30 s-vortexing step, are necessary to elute viral infective particles; for PTFE, 60 min-shaking is enough. The effect of suction time on filters could be resumed as follows: sampling durations up to 20 min at a flow rate of 500 L/min do not affect recovery efficiencies from 10 cm glass-fiber filters, whereas the recovery efficiency of infectious virions from 4.7 cm PTFE filters decreases of a factor 2 after 3 h of sampling at a flow rate of 20 L/min. The recovery efficiency of ultracentrifugation turns out to be around 57%. The effect of storage temperature of filters immersed in a transport medium on coronavirus infectivity is assessed as well.Based on the sampling techniques and the analytical methods developed as described in the present study, many field tests were carried out reporting virus concentrations up to 50 genomic copies per cubic meter of air in domestic environment with poor ventilation condition, whereas in hospital wards the detectable concentrations of SARS-CoV-2 were generally lower than 10 genomic copies per cubic meter of air.

Melanin, a potential new matrix material for recombinant His-tag protein purification

Nickel-Sepharose (Ni-Sepharose) has been being applied as the most common matrix in purifying His-tag proteins based on the affinity interaction between histidine residues and Ni2+ ion. However, Sepharose still comes at high cost for this purification purpose, especially in developing countries as Vietnam. Here, we show for the first time that melanin from ink sacs of squids which is considered as biowaste in the food industry, can be used as a new potential matrix material instead of Sepharose. We utilized either melanin or melanin charged with metal ions as the stationary phase of affinity purification of His-tag proteins. The results showed that a recombinant His-tag protein VP28 in a protein pool was captured by melanin and Ni2+/Fe3+/Zn2+ chelated melanin. Experiments for releasing VP28 were performed only on the melanin and Ni2+-melanin matrices. The result showed that VP28 was quite selectively eluted when applying elution buffer of 250 mM imidazole overnight. The relative efficiency in releasing VP28 of melanin and Ni-melanin matrices roughly compared to Ni-Sepharose were about 38 and 18% respectively. Further optimization of this process may allow higher efficiency in the purification of His-tag proteins.

Cell Analysis from Dried Blood Spots: New Opportunities in Immunology, Hematology, and Infectious Diseases.

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50–70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS‐CoV‐2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.