Wash Buffer Research Articles

Highly Sensitive Lab on a Chip (LOC) Immunoassay for Early Diagnosis of Respiratory Disease Caused by Respirable Crystalline Silica (RCS).

Respirable crystalline silica (RCS) produced in mining and construction industries can cause life-threatening diseases such as silicosis, lung cancer, and chronic obstructive pulmonary disease (COPD). These diseases could be more effectively treated and prevented if RCS-related biomarkers were identified and measured at an early stage of disease progression, which makes development of a point of care test (POCT) platform extremely desirable for early diagnosis. In this work, a new, highly sensitive lab on a chip (LOC) immunoassay has been designed, developed, and characterized for tumor necrosis factor α (TNF-α), a protein biomarker that causes lung inflammation due to RCS exposure. The designed LOC device is composed of four reservoirs for sample, enzyme conjugated detection antibody, wash buffer, and chemiluminescence substrate in liquid form, along with three spiral reaction chambers for test, positive control, and negative control. All reservoirs and spiral microchannels were connected in series and designed to perform sequential delivery of immunoassay reagents with minimal user intervention. The developed LOC measured TNF-α concentrations as low as 16 pg/mL in plasma from RCS-exposed rats and also had a limit of detection (LOD) of 0.5 pg/mL in spiked artificial serum. In addition, the analysis time was drastically reduced to about 30 min, as opposed to hours in conventional methods. Successful implementation of a highly sensitive, chemiluminescence-based immunoassay on a preloaded LOC with proper quality control, as reported in this work, can pave the way toward developing a new rapid POCT platform for in-field clinical diagnosis.

Improving electrocoagulation floatation for harvesting microalgae

Electro-coagulation floatation (ECF) is a foam-floatation dewatering method that has been shown to be a highly effective, rapid, and scalable separation methodology. In this manuscript, an in-depth analysis of the gas and flocculant levels observed during the process is provided, with microbubbles observed in the 5–80 μm size range at a concentration of 102–103 bubbles mL−1. Electrolysis of microalgae culture was then observed, demonstrating both effective separation using aluminium electrodes (nine microalgal species tested, 1–40 μm size range, motile and non-motile, marine and freshwater), and sterilisation of culture through bleaching with inert titanium electrodes. Atomic force microscopy was used to visualise floc formation in the presence and absence of algae, showing nanoscale structures on the magnitude of 40–400 nm and entrapped microalgal cells. Improvements to aid industrial biotechnology processing were investigated: protein-doping was found to improve foam stability without inducing cell lysis, and an oxalate buffer wash regime was found to dissolve the flocculant whilst producing no observable difference in the final algal lipid or pigment profiles, leaving the cells viable at the end of the process. ECF separated microalgal culture had an algal biomass loading of 13% and as such was ideal for direct down-stream processing through hydrothermal liquefaction. High bio-crude yields were achieved, though this was reduced slightly on addition of the Al(OH)3 after ECF, with carbon being distributed away to the aqueous and solid residue phases. The amenability and compatibility of ECF to integration with, or replacement of, existing centrifugation and settling processes suggests this process may be of significant interest to the biotechnology industry.

A method for improving protein A chromatography's aggregate removal capability

Protein A chromatography is generally less effective at removing antibody aggregates under typical conditions. We recently developed a method that can significantly improve Protein A's aggregate removal capability. This method involves adding calcium chloride/polyethylene glycol (PEG) or sodium chloride/PEG combination to wash and elution buffers. Each salt alone showed some resolution-enhancing effect when its concentration reaches a certain level, and this effect was significantly enhanced by the presence of PEG, which itself had no effect on resolution. The synergistic effect of salt and PEG results in almost complete separation of monomer from aggregates. For the particular case used for method development and demonstration, in comparison with the control run the optimized procedure reduces aggregates in elution pool from 20% to 3–4%. This new method, by facilitating aggregate removal at the capture step, improves the overall robustness of downstream process.

The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance

Post-load column wash in Protein A chromatography can effectively improve host cell protein (HCP) clearance. A commonly used wash additive for this purpose is sodium chloride. However, the adequate amount of sodium chloride required for effective HCP clearance is less consistent in literature. In this study we investigated the impact of different amounts of sodium chloride on HCP clearance with five monoclonal antibodies (mAbs). For each mAb, elution pool HCP levels from runs under different wash conditions are compared. For all five mAbs, the data suggested that 250 mM would be an adequate amount for the salt to largely achieve its HCP reducing effect. The same conclusion is also reached for calcium chloride, a less commonly used but equally effective Protein A wash additive for HCP clearance.

MESIA: Magnetic force-assisted electrochemical sandwich immunoassays for quantification of prostate-specific antigen in human serum

We propose a new immunoassay technique, called magnetic-force assisted electrochemical sandwich immunoassay (MESIA), where serum biomarkers can be determined by magnetic actuation and electrochemical detection of gold-coated iron oxide nanoparticles as probes for immunocomplex formation. In MESIA, neither washing buffer nor fluidic parts are necessary, because the formation of immunocomplexes and the removal of unbound probes are controlled by magnetic forces. Electrochemical pretreatment and measurement of the gold-coated magnetic probes allows highly sensitive, precise, and robust system for quantification of target analytes. Using MESIA, the concentration of prostate-specific antigen (PSA) in 10 μl of human serum is determined within 5 min. The limit of detection is 0.085 ng/mL, and the average coefficient of variance is 8.85% for five different PSA concentrations ranging from 0 to 25 ng/mL. This method shows good precision and reproducibility (<10%) and high correlation with cobas e 801 (r = 0.997) for clinical patient samples. We believe this technique to be useful in the development of a point-of-care testing platform for diagnosis and prognosis of various diseases, such as cancer, based on quantification of biomarkers in a drop of blood.

Heterogeneous Immunoassay Using Channels and Droplets in a Digital Microfluidic Platform.

This work presents a heterogeneous immunoassay using the integrated functionalities of a channel and droplets in a digital microfluidic (DMF) platform. Droplet functionality in DMF allows for the programmable manipulation of discrete sample and reagent droplets in the range of nanoliters. Pressure-driven channels become advantageous over droplets when sample must be washed, as the supernatant can be thoroughly removed in a convenient and rapid manner while the sample is immobilized. Herein, we demonstrate a magnetic bead-based, enzyme-linked immunosorbent assay (ELISA) using ~60 nL of human interleukin-6 (IL-6) sample. The wash buffer was introduced in the form of a wall-less virtual electrowetting channel by a syringe pump at the flow rate of 10 μL/min with ~100% bead retention rate. Critical parameters such as sample wash flow rate and bead retention rate were optimized for reliable assay results. A colorimetric readout was analyzed in the International Commission on Illumination (CIE) color space without the need for costly equipment. The concepts presented in this work are potentially applicable in rapid neonatal disease screening using a finger prick blood sample in a DMF platform.

Wash Buffer for TAP

Wash Buffer for TAP

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two‐stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre‐existing high‐molecular‐weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter‐current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow‐through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

Urinary circulating DNA and circulating antigen for diagnosis of schistosomiasis mansoni: a field study.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

A Magnetic Bead-Based DNA Extraction Protocol Suitable for High-Throughput Genotyping in Shrimp Breeding Programs

Due to their convenience, magnetic bead-based nucleic acid extraction kits are commonly used in shrimp genotyping and pathogen screening applications. However, in advanced breeding programs requiring the testing of many thousands of shrimp, their cost can be prohibitive. Various permutations of different Proteinase K digestion, tissue lysis and bead washing buffers as well as magnetic bead types were thus evaluated to devise a high-throughput shrimp DNA extraction (SDE) protocol capable of recovering high-purity DNA using a KingFisher™ Flex Magnetic Particle processor. When genotyped using a MassARRAY® platform (Agena Bioscience) requiring 60-61 genome regions to be co-amplified in a single multiplexed PCR, DNA extracted from shrimp muscle tissue using either the SDE protocol or a commercial kit generated comparable single-nucleotide polymorphism (SNP) call data. The SDE protocol also extracted high-purity DNA from salmon fin clips. It thus offers potential to markedly reduce the costs of large-scale genotyping in shrimp and salmon breeding programs.

Low-Salt Wash Buffer for CLIP

Low-Salt Wash Buffer for CLIP

High-Salt Wash Buffer for CLIP

High-Salt Wash Buffer for CLIP

Capture and Storage of Plant Genomic DNA on a Readily Available Cellulose Matrix

Whatman FTA® Cards are a fast and efficient method for capturing and storing nucleic acids but can be cost-prohibitive for large numbers of samples. We developed a method that substitutes a readily-available cellulose matrix and homemade washing buffer for commercial FTA® Cards and FTA® Purification Reagent. This method is suitable for long-term storage of DNA from many plant species prior to PCR.

DNA retention on depth filters

Depth filtration is a commonly-used bioprocessing unit operation for harvest clarification that reduces the levels of process- and product-related impurities such as cell debris, host-cell proteins, nucleic acids and protein aggregates. Since depth filters comprise multiple components, different functionalities may contribute to such retention, making the mechanisms by which different impurities are removed difficult to decouple. Here we probe the mechanisms by which double-stranded DNA (dsDNA) is retained on depth filter media by visualizing the distribution of fluorescently-labeled retained DNA on spent depth filter discs using confocal fluorescence microscopy. The extent of DNA displacement into the depth filter was found to increase with decreasing DNA length and with increasing operational parameters such as wash volume and buffer ionic strength. In addition, using 5-ethynyl-2′-deoxyuridine (EdU) to label DNA in dividing CHO cells, we showed that Chinese hamster ovary (CHO) cellular DNA in the lysate supernatant migrates deeper into the depth filter than the lysate re-suspended pellet, elucidating the role of the size of the DNA in its form as an impurity. Apart from aiding DNA purification and removal, our experimental approaches and findings can be leveraged in studying the transport and retention of nucleic acids and other impurities on depth filters at a small scale.

P105 An accelerated method for reverse sequence-specific oligonucleotide probe-based HLA typing

Turnaround time (TAT) for HLA typing is critical for transplantation. We sought to establish a faster protocol for low-resolution HLA typing using the rSSOP methodology. The LABType™ rSSOP method (One Lambda) was altered to reduce hybridization time. Modifications included centrifugation and incubation duration reductions, and reagent volume adjustments. Single parameters were first changed and tested. Successful modifications were used in the next round of tests. All modifications were tested with a set of PCR amplicons prepared from three samples, which contained previously HLA-typed high quality DNA. Following a series of rSSOP typings for these samples at HLA-A, B, C, DRB1, and DQB1/DQA1 loci, an accelerated protocol was established. Discovery phase concluded with protocol validation at all loci including HLA-DRB3/4/5 and DPB1/DPA1. Full validation confirmed intra-operator, inter- and intra-assay, as well as multiple DNA source reproducibility. DNA was extracted from different samples including ACD blood (n = 4), EDTA blood (n = 4), cord segment (n = 5), buccal swab (n = 5) and mouth-wash samples (n = 4). The original centrifugation of 3000 rpm for 2 min (total 2 min 22 s) was changed to a quick spin (up to 3000 rpm; 33 s), reducing each centrifugation step by 1 min 50 s. Additionally, one centrifugation and one transfer of plate step were eliminated. The duration of incubation time was changed for denaturation (from 10 min to 2.5 min), hybridization (from 15 min to 5 min), and streptavidin-PE (SAPE, from 5 min to 2.5 min). The reagent volume modifications includes the reduction of washing buffer (from 100 μl to 50 μl per well) and increase in SAPE (from 25 μl to 50 μl per well). Compared to the original protocol, the accelerated protocol reduced the hybridization time approximately 30 min. This method allowed us to obtain consistent and accurate typing results for all tested HLA loci among different operators, intra- and inter-assays, and a variety of DNA samples. Fulfilling requests for faster TAT may be achieved by adaptation of current methodologies, which may help improving patient management. The accelerated rSSOP protocol is an effective method for reducing testing time, while still giving robust results for HLA class I and class II typing. (RLU and JJX contributed equally).

Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics

We present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vertical microfluidic probe containing a microstructure designed for rapid switching between probe solution and nonfluorescent imaging buffer. The FISH signal is monitored in real time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description of the method as well as a demonstration of its applicability using a model system of centromeric probes (Cen17). We demonstrate the applicability of the method for characterization of FISH kinetics under conditions of varying probe concentration, destabilizing agent (formamide) content, volume exclusion agent (dextran sulfate) content, and ionic strength. We show that our method can be used to investigate the effect of each of these variables and provide insight into processes affecting in situ hybridization, facilitating the design of new assays.

A Novel, Automated Dispersive Pipette Extraction Technology Greatly Simplifies Catecholamine Sample Preparation for Downstream LC-MS/MS Analysis.

Catecholamines are integral neurotransmitters in the central and peripheral nervous system. Clinically, catecholamine levels are determined to help diagnose disease and measure corresponding therapeutic effectiveness. However, manual extraction of catecholamines and their metabolites may be labor-intensive and user-variable and require a variety of peripheral laboratory devices, especially at low sample concentrations. Here, we propose a novel solid-phase extraction (SPE) method using patented dispersive pipette extraction (DPX) tip technology. The tips are readily integrated into an automated workflow to extract these compounds from urine, which increases analytical throughput while removing human variability and error. Diphenylboronic acid (DPBA) forms a stable, negatively charged complex with catecholamines in the samples, and when aspirated into the DPX tip, the complexed analytes are retained on a styrene divinyl benzene sorbent. Wash buffers remove interfering compounds, after which the complex is eluted from the tip using an acidic aqueous solution and subsequently measured via liquid chromatography with tandem mass spectrometry (LC-MS/MS). The automated DPX method for catecholamine sample preparation from urine has excellent linearity over more than three orders of magnitude with concentrations ranging from 0.5 to 1000 ng/mL, with replicate analyses resulting in coefficients of variation of less than 8%. This high-throughput workflow is appropriate for use in regulated laboratories.

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions.

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Binding and Washing (B&amp;W) Buffer (2×)

Binding and Washing (B&amp;W) Buffer (2×)

Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants

The need for fast and convenient protein purification is enormous, not only on the industrial scale for the production of therapeutics but also in life science research. Most protein purification strategies require multiple steps, which are time consuming, cost and resource intensive. Selective release of the intracellular target product can considerably facilitate downstream processing by minimizing the HCP concentration before subsequent purification processes. Here, we present a simple method to selectively release soluble overexpressed proteins from E. coli with minimal laboratory equipment requirements. This method is based on the creation of pores in the cell envelope by a single freeze-thaw cycle. Proteins of various sizes were released by several 50 mM Tris pH 9.5 buffer washes with high yields. Consequently, the need for detergents and other additives were circumvented. Green fluorescent protein (GFP), was tagged with peptides of various charges and polarity. Releasing these GFP variants with our method enabled the successful one-step chromatographic purification on a quaternary amine resin despite the different isoelectric points of the target proteins. Up to 90% of the target protein elution peak during anion exchange chromatography was of >95% purity. This optimized process is more productive compared to multi-step purification procedures currently available in literature.