Paramagnetic Beads Research Articles

Total RNA Extraction from Rice Vegetative Tissues Using Magnetic Beads.

Ribonucleic acid (RNA) extraction is the first critical step in gene expression analysis. In this chapter, we describe a high-throughput RNA extraction method using guanidine thiocyanate and isopropyl alcohol (HighGI). The use of carboxyl-coated paramagnetic beads, instead of silica membrane columns, enables semi-automation using a liquid handling system and high-throughput RNA extraction for large-scale transcriptome studies. Homemade mixes of paramagnetic beads and buffers make HighGI inexpensive. In addition, HighGI-extracted RNA retains low molecular weight RNA molecules less than 200bp, which is typically lost in commercial column-based kits.

Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA.

Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.

A low-cost and versatile paramagnetic bead DNA extraction method for Mycobacterium ulcerans environmental surveillance.

Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

Development of a Combined Protein and Dye Extraction Approach for the Analysis of Keratin-Based Textiles.

Archaeological textiles represent precious remains from ancient culture; this is because of the historical and cultural importance of the information that can be obtained by such relics. However, the extremely complicated state of preservation of these textiles, which can be charred, partially or totally mineralized, with heavy soil or biological contamination, requires highly specialized and sensitive analytical tools to perform a comprehensive study. Starting from these considerations, the paper presents a combined workflow that provides the extraction of dyes and keratins and keratin-associated proteins in a single step, minimizing sampling while maximizing the amount of information gained. In the first phase, different approaches were tested and two different protocols were found suitable for the purpose of the unique workflow for dyes/keratin-proteins: a slightly modified urea protocol and a recently proposed new TCEP/CAA procedure. In the second step, after the extraction, different methods of cleanup and workflow for proteins and dyes were investigated to develop protocols that did not result in a loss of aliquots of the analytes of interest and to maximize the recovery of both components from the extracting solution. These protocols investigated the application of two types of paramagnetic beads, unmodified and carboxylate-coated hydrophilic magnetic beads, and dialysis and stage-tip protocols. The newly designed protocols have been applied to cochineal, weld, orchil, kermes, and indigo keratin-based dyed samples to evaluate the effectiveness of the protocols on several dye sources. These protocols, based on a single extraction step, show the possibility of investigating dyes and keratins from a unique sample of 1 mg or lesser, with respect to the thresholds of sensitivity and accuracy required in the study of textile artifacts of historical and artistic values.

Molecular identification of lactic acid bacteria from traditional fermented foods and screening exopolysaccharide production by using food wastes.

In this study, lactic acid bacteria (LAB) isolation from fermented foods and molecular identification using magnetic bead technology were performed. And then exopolysaccharide (EPS) production possibility was tested in agar medium, and the positive ones were selected for the next step. The bacteria that could produce higher carbohydrate level were grown in MRS medium fortified with whey and pumpkin waste. In our study, 19 different LAB species were identified from fermented products collected from different places in Hatay(Türkiye) province. In molecular identification, universal primer pairs, p806R/p8FPL, and PEU7/DG74 were used for PCR amplification. After that, PCR products purified using paramagnetic bead technology were sequenced by the Sanger sequencing method. The dominant species, 23.8% of the isolates, were identified as Lactiplantibacillus plantarum. As a technological property of LAB, exopolysaccharide production capability of forty-two LAB isolate was tested in agar medium, and after eleven isolates were selected as positive. Two LAB (Latilactobacillus curvatus SHA2-3B and Loigolactobacillus coryniformis SHA6-3B) had higher EPS production capability when they were grown in MRS broth fortified with pumpkin waste and whey. The highest EPS content (1750mg/L glucose equivalent) was determined in Loigolactobacillus coryniformis SHA6-3B grown in MRS broth fortified with 10% pumpkin waste. Besides the produced EPS samples were validated with FTIR and SEM methods.

Saliva-STAT: Sample-to-answer saliva test for COVID-19

Highly accessible and highly accurate diagnostics are necessary to combat rapidly-spreading infectious diseases, such as the recent COVID-19 pandemic. While lateral flow antigen tests have become pervasive, they are insufficiently sensitive to detect early or asymptomatic disease. Nucleic acid amplification tests provide the needed sensitivity, but accessibility of these tests continues to be a challenge due to the need for precise sample processing steps. Here we report a sample-to-answer test for saliva samples (saliva-STAT) that utilizes a battery-powered handheld instrument and a low-cost easily-manufacturable sample cassette to perform a nucleic acid amplification test for viral pathogens. To enable a completely automated assay, we leverage thermally responsive alkane partitions (TRAPs) and paramagnetic beads for virus purification and concentration, as well as reagent addition and mixing. Notably, the saliva STAT easily accommodates directly-dispensed saliva samples (in contrast to microfluidic devices), which is necessary for self-testing. Using the saliva-STAT platform, we demonstrate detection of down to 0.2 copies/μL of SARS-CoV-2 virus in saliva samples. We envision that the saliva-STAT could be used in walk-in clinics, mobile clinics, public testing locations, and in the home. With minor adjustments to the assay, the saliva-STAT platform can easily be adapted for other respiratory viruses, such as Influenza.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (μg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 μg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 μg of beads for sample preparation and long-term digestion (16h) with 0.15 μg Lys-C and 2.5 μg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18-29%, so improvement is needed. Peripheral blood mononuclear cells (PBMC) of CLL patients often contain high levels of CLL cells that can interfere with CAR T-cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. We previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. We aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR-T) from PBMC of CLL patients. We determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7+IL-15 had advantages over using IL-2 including greater accumulation of Hu19-CAR-T during in vitro proliferation assays. We determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T-cell purification because this method resulted in high T-cell purity. We determined that anti-CD3/CD28 paramagnetic beads were the optimal T-cell activation reagent. Finally, we developed a current good manufacturing practices (cGMP)-compliant clinical-scale protocol for producing Hu19-CAR-T from PBMC of CLL patients. These Hu19-CAR-T exhibited a full range of in vitro functions and eliminated leukemia from mice.

Zika Virus-Infected Monocyte Exosomes Mediate Cell-to-Cell Viral Transmission.

Zika fever is a reemerging arthropod-borne viral disease; however, Zika virus (ZIKV) can be transmitted by other, non-vector means. Severe Zika fever is characterized by neurological disorders, autoimmunity, or congenital Zika syndrome. Monocytes are primary ZIKV targets in humans and, in response to infection, release extracellular vesicles like exosomes. Exosomes mediate intercellular communication and are involved in the virus's ability to circumvent the immune response, promoting pathological processes. This study aimed to evaluate the role of monocyte exosomes in cell-to-cell viral transmission. We isolated exosomes from ZIKV-infected monocytes (Mø exo ZIKV) by differential ultracentrifugation and identified them by nanoparticle tracking analysis; transmission electron microscopy; and CD63, CD81, TSG101, and Alix detection by cytofluorometry. Purified exosome isolates were obtained by uncoupling from paramagnetic beads or by treatment with UV radiation and RNase A. We found that Mø exo ZIKV carry viral RNA and E/NS1 proteins and that their interaction with naïve cells favors viral transmission, infection, and cell differentiation/activation. These data suggest that Mø exo ZIKV are an efficient alternative pathway for ZIKV infection. Knowledge of these mechanisms contributes to understanding the pathogenesis of severe disease and to the development of new vaccines and therapies.

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.

An effective urobilin clearance strategy based on paramagnetic beads facilitates microscale proteomic analysis of urine.

Urine provides an ideal source for disease biomarker discovery. High-adhesion contaminants such as urobilin, which are difficult to remove from urine, can severely interfere with urinary proteomic analysis. Here, we aimed to establish a strategy based on single-pot, solid-phase-enhanced sample preparation (SP3) technology to prepare samples for urinary proteomics analysis that almost completely eliminates the impact of urobilin. A systematic evaluation of the effects of two urinary protein precipitation methods, two types of protein lysis buffers, and different ratios of magnetic digestion beads on the identification and quantification of the microscale urinary proteome was conducted. Our results indicate that methanol-chloroform precipitation, coupled with efficient lysis facilitated by urea, and subsequent enzymatic digestion using a mix of hydrophilic and hydrophobic magnetic beads offers the best performance. Further applying this strategy to the urine of patients with benign prostatic hyperplasia, prostate cancer and healthy individuals, combined with a narrow window of data-independent acquisition, FGFR4, MYLK, ORM2, GOLM1, SPP1, CD55, CSF1, DLD and TIMP3 were identified as potential biomarkers to discriminate benign prostatic hyperplasia and prostate cancer patients.

A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses

DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.

Benchmarking of Two Peptide Clean-Up Protocols: SP2 and Ethyl Acetate Extraction for Sodium Dodecyl Sulfate or Polyethylene Glycol Removal from Plant Samples before LC-MS/MS.

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.

Neuronal pentraxin‐2 by Simoa ‐ a potential synaptic biomarker in Alzheimer’s disease

AbstractBackgroundSynaptic dysfunction and degeneration are believed to be among the earliest and most reliable indicators of cognitive impairment in neurodegenerative disorders, including Alzheimer’s disease (AD). Discovering and verifying novel biomarkers that can quantify synaptic degeneration and serve as prognostic indicators would be valuable to track cognitive decline in clinical and drug trial settings. By using mass spectrometry, we have studied a panel of 17 synaptic proteins as potential biomarkers for AD and a broad range of neurodegenerative disorders. Of these proteins, neuronal pentraxin‐2 (NPTX2) has shown great promise as a synaptic biomarker, showing a decrease in symptomatic disease. Therefore, we aimed to develop a Single molecule array (Simoa) for NXPT2, a method more suited for routine clinical assessment than mass spectrometry‐based workflows.MethodWe developed an in‐house NPTX2 Simoa assay on the HD‐X platform (Quanterix) with paramagnetic beads coupled with a rabbit monoclonal capture antibody (EPR24020‐38, Abcam) and biotinylated rabbit monoclonal detector antibody (EPR15618, Abcam), both specifically targeting NPTX2. Full‐length recombinant NPTX2 (7816‐NP‐050, Biotechne) was used as the calibrator. Using the assay, we analyzed 55 healthy controls (78.0 (6.5 SD) years) and 54 AD patients (73.5 (7.0 SD) years) using 2 uL cerebrospinal fluid (CSF) in the BioFINDER pilot study. Group comparisons were performed using the Wilcoxon rank sum test and linear models adjusted for age and sex were used to assess associations of NPTX2 protein levels with cognitive function (mini mental status exam (MMSE)) and with neuroimaging, e.g., Aβ‐PET, tau‐PET (temporal ROI, Braak I‐IV) as well as cortical thickness in an AD signature composed of temporal regions in the AD group.ResultCSF NPTX2 concentration was found to be lower in AD compared with controls (p = 0.017, Figure). Lower NPTX2 concentration was associated with greater tau PET SUVR (β(SD) = ‐0.25(0.079), p‐value = 0.0027) and cortical thickness atrophy (β(SD) = 0.075(0.024), p‐value = 0.0034)) but not with amyloid‐β PET SUVR. Lower NPTX2 concentration was also associated with lower MMSE score (β(SD) = 2.62 (0.75), p‐value = 0.0010).ConclusionWe have successfully developed a Simoa assay of a novel CSF biomarker, NPTX2. This semi‐automated method allows for large‐scale evaluation of NPTX2, a leading candidate for synaptic degeneration in neurodegenerative disorders.

Development of simple, rapid, and sensitive methods for detection of hepatitis C virus RNA from whole blood using reverse transcription loop-mediated isothermal amplification.

Hepatitis C virus (HCV) infection is an underdiagnosed global health problem. Diagnosis of current HCV infections typically requires testing for HCV RNA using high-complexity laboratory tests. Methods for the detection of HCV RNA that are simple, inexpensive, rapid, and compatible with use outside of a laboratory setting are very important in order to improve access to hepatitis C diagnostic testing and facilitate accelerated linkage to care. We developed and evaluated three simple workflows for extracting HCV RNA from small volumes of whole blood for use in a sensitive, pan-genotypic RT-LAMP assay. The water workflow uses osmotic stress to release HCV RNA and has a limit of detection of 4.3 log10(IU/mL) (95% CI 4.0-4.9). The heat workflow uses a heating step to release HCV RNA and has a limit of detection of 4.2 log10(IU/mL) (95% CI 3.8-5.1). The bead workflow, which uses chemical lysis of the sample and a streamlined paramagnetic solid phase reversible immobilization bead procedure for nucleic acid purification, has a limit of detection of 2.8 log10(IU/mL) (95% CI 2.5-3.4). When used to test whole blood spiked with HCV RNA-positive plasma samples in which most HCV levels were below 5.0 log10(IU/mL), the water, heat, and bead workflows detected HCV RNA in 69%, 75%, and 94% of samples, respectively. These workflows are compatible with visual lateral flow dipsticks, and each takes less than 60 min from sample to result. Each workflow can be performed with minimal and inexpensive equipment. With further procedural simplifications, these workflows may form the basis of assays for the point-of-care diagnosis of HCV infections.

B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform

Abstract Background The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates. Methods Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype. Results In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution. Conclusion This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.

B-371 Two Cases of Multiple Myeloma Achieving Complete Remission But Presenting Residual M-protein by the EXENT® Solution

Abstract Background Disappearance of the monoclonal component is a condition for achieving complete response (CR), which requires confirmation by bone marrow biopsy for the absence of clonal plasma cells. Electrophoretic methods used for monitoring these patients lack sufficient sensitivity to identify low-level M-protein, potentially missing the presence of serological residual disease once the tests become negative. The EXENT® solution (in development by The Binding Site, part of Thermo Fisher scientific) uses matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification and quantification of M-proteins. The technique offers high sensitivity and specificity for identifying and tracking M-proteins. The aim of this study is to determine whether the superior analytical sensitivity of mass spectrometry conveys any clinical benefit to patient monitoring. Methods We present two multiple myeloma patients seen during routine practice. Baseline and longitudinal samples were available. Serum samples were retrospectively analysed with the EXENT® solution. Briefly, diluted samples were separately incubated with antisera specific for IgG, IgA, IgM, total-κ and total-λ conjugated to paramagnetic beads. Purified immunoglobulins were reduced and eluted from the beads, the solution mixed with matrix, spotted onto MALDI plates, and acquired by MALDI-TOF-MS to determine mass-to-charge (m/z), isotype and concentration of the M-proteins. Results were compared to serum protein electrophoresis (SPE) and free light chains (FLC; Freelite, The Binding Site). The EXENT solution responses were modelled on those from the International Myeloma Working Group. CR by MS refers to absence of the original monoclonal peak during follow-up. Results Case 1 is a 68-year-old white male presenting with 11.7 g/L monoclonal IgGκ on SPE and abnormal FLC ratio (3.06). After initiating therapy, FLC ratio normalized at month 2, whereas SPE became negative at month 8, at which time a CR was confirmed. The patient stayed in remission for over two-and-a-half years before relapsing at month 40, as determined by re-appearance of the M-protein on SPE (2.20 g/L). The EXENT solution also identified an IgGκ peak at baseline (m/z = 11629, 11.9 g/L). The peak remained present until month 19, disappeared for over a year, then reemerged at month 36 (0.39 g/L) to indicate progression. Thus, the EXENT solution identified the presence of residual M-protein (<1 g/L) for an additional eleven months after standard monitoring methods indicated CR; and recognized relapse from CR four months before SPE.Case 2 is a 76-year-old Asian male presenting with 32.9 g/L monoclonal IgGλ on SPE and normal FLC ratio (0.43). The EXENT solution reported an IgGλ peak (m/z = 11250, 44.3 g/L) at this time. SPE became negative ten months after starting treatment; and identified relapse from CR one year after. The EXENT solution remained positive throughout and was the only method detecting low-level M-protein (≤1.25 g/L) during the 1-year remission period. Conclusion EXENT demonstrates a potentially superior performance over electrophoretic methods for identifying residual serological disease during monitoring; and shows potential as a serum biomarker of early relapse in myeloma. The improved sensitivity of MS suggests it may delay and, in some cases, avoid the need for invasive methods to determine response to therapy.

DdRADseq kütüphanesi oluşturma işlemi fragman seçiminde uygun fiyatlı bir alternatif yöntem

Next generation sequencing (NGS) technologies constitute the most powerful scientific advance of 21st century with a promise of fast and cost effective data generation in biology. Yet, up to date NGS studies remain often limited to laboratories with established resources. In the present study, we employed construction of ddRADseq library by using routine lab consumables (agarose gel electrophoresis: AGE thereafter) compared to high-tech NGS consumables (paramagnetic beads) during size selection. The ddRADseq library was constructed for sequencing size selected based on universally used paramagnetic beads, while remaining aliquot was used as a template to assess the feasibility of ddRADseq library construction using AGE for labs with limited resources. Both libraries were optimised for 15 PCR cycles indicating similarity in template intensity. Post-PCR quantification of the libraries was comparable (~10 ng.µL-1). Size distribution assessment revealed a cleaner pick at the ddRADseq library size selected manually based on AGE. Similarly, intercalating agent of Qubit confirmed the quantity of libraries was similar (>3 ng.µL-1). Although being more time consuming due to pre-electrophoresis preparations, serial wash and staining steps, ddRADseq library construction is achievable using routine lab consumables provided to supply the adaptors and PCR primers for the initial wet-lab work. These results manifest the feasibility of ddRADseq library generation for labs with limited resources.

DNA concentration by solid phase reversible immobilization improves its yield and purity, and detection time of E. coli O157:H7 in foods by high resolution melt curve qPCR

For molecular detection of foodborne pathogens to be effective, an efficient technique for bacterial DNA concentration is required. Solid phase reversible immobilization (SPRI) paramagnetic beads can concentrate DNA from foods containing low amounts of bacteria because of their highly specific DNA-binding capacity. This study aimed to develop an upstream bacterial DNA concentration step using SPRI beads and compare its efficacy with immunomagnetic separation (IMS) for the detection of Shiga toxin producing E. coli (STEC) O157:H7 in food samples via a multiplex PCR assay. Food samples were inoculated with STEC O157:H7 at concentrations of 10, 100 and 1000 CFU/mL. The two different concentration methods were compared based on 1) DNA yield 2) DNA purity and 3) detection limit and time of the target pathogen using a high-resolution melt cure multiplex real-time PCR assay for the detection of the major virulence genes, eaeA, stx1 and stx2, in E. coli O157:H7. DNA samples isolated and concentrated by SPRI showed better purity and yield compared to IMS cell concentration. A detection limit of 10 CFU was attained for the majority of inoculated food samples utilizing DNA samples concentrated using SPRI in just 4 h as opposed to 6 or more hours using IMS. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect low concentrations of E. coli O157:H7 in food samples that cannot be detected by conventional culture techniques.

Nucleic Acid Quantification by Multi-Frequency Impedance Cytometry and Machine Learning

Determining nucleic acid concentrations in a sample is an important step prior to proceeding with downstream analysis in molecular diagnostics. Given the need for testing DNA amounts and its purity in many samples, including in samples with very small input DNA, there is utility of novel machine learning approaches for accurate and high-throughput DNA quantification. Here, we demonstrated the ability of a neural network to predict DNA amounts coupled to paramagnetic beads. To this end, a custom-made microfluidic chip is applied to detect DNA molecules bound to beads by measuring the impedance peak response (IPR) at multiple frequencies. We leveraged electrical measurements including the frequency and imaginary and real parts of the peak intensity within a microfluidic channel as the input of deep learning models to predict DNA concentration. Specifically, 10 different deep learning architectures are examined. The results of the proposed regression model indicate that an R_Squared of 97% with a slope of 0.68 is achievable. Consequently, machine learning models can be a suitable, fast, and accurate method to measure nucleic acid concentration in a sample. The results presented in this study demonstrate the ability of the proposed neural network to use the information embedded in raw impedance data to predict the amount of DNA concentration.