Assay Reagents Research Articles

Isolation and characterization of polygalacturonase producing thermophilic Aspergillus niger isolated from decayed tomato fruits

This study aimed to isolate a fungal strain capable of producing acidophilic and thermostable polygalacturonase. In this study, the fungal isolate was isolated from decaying tomatoes. Based on the colony characteristics, microscopic and morphological observations, the isolated fungal pathogen has been identified as Aspergillus niger. The isolated fungus was used in solid-state fermentation to produce an acidic polygalacturonase enzyme. The enzyme was then purified using ammonium sulphate precipitation and column chromatography, and its activity was assayed by measuring the releasing sugar group from citrus pectin using a 3, 5-dinitrosalicylic acid (DNSA) reagent assay. The crude extract obtained from solid-state fermentation had an activity of 94.6 U/mL. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Sephadex G-200 was used to purify the enzyme 3.58 times, and its specific activity was determined to be 24.66 U/mg. The Sephacryl S-100 column achieved a final fold purification of 9.93 times and a specific activity of 68.41 U/mg. The purified enzyme performed best when polygalacturonic acid was used as a substrate. The enzyme's optimum temperature and pH were 55°C and 5, respectively. CaCl2 was found to be the best chelating ion for the enzyme. This enzyme is recommended for use in a variety of industrial applications as the enzyme was found to be stable at acidic pH and high temperature.

Designing silver nanoparticles impregnated acacia and tragacanth gum based copolymeric hydrogels for drug delivery applications

The current study investigates the utilization of gum acacia (GA) & tragacanth gum (TG) for the synthesis of silver nanoparticles (NPs) and their subsequent incorporation into hydrogels for potential applications in drug delivery (DD). Cefoperazone, an antibiotic drug, was encapsulated into hydrogels to enhance their antimicrobial activity. Copolymers were characterized by UV–Vis, TEM, FESEM, AFM, XPS, XRD, FTIR, TGA and DSC techniques. UV–Vis spectroscopy revealed the formation of NPs at a wavelength of 436 nm with an optical density of 2.65. TEM images depicted AgNPs with an average size of 37.29 ± 7.25 nm with their uniform distribution. XRD analysis demonstrated distinct crystalline peaks at 2θ = 38.34o, 43.80o, 64.60o and 77.42o attributed to AgNPs. XPS peaks observed at 368 & 374 eV, corresponding to the 3d5/2 and 3d3/2 modes of silver, respectively, confirmed the incorporation of AgNPs in the polymer matrix. Hydrogels demonstrated pH responsive sustained release of cefoperazone. The diffusion mechanism was non-Fickian and described by the Higuchi kinetic model. In the DPPH assay, copolymers exhibited a 32.36 % inhibition of free radicals, whereas in the F–C reagent assay, antioxidant activity was measured as 22.25 μg equivalent of gallic acid. The copolymers displayed blood compatible nature, as evidenced from haemolysis (2.11 ± 0.03 %) and thrombogenicity (74.71 ± 1.63 %) results. The interactions of copolymer with intestinal membrane attributed to mucoadhesive properties and required detachment force of 55.30 ± 2.80 mN to separate from the biological membrane. Growth of E. coli, P. aeruginosa and S. aureus bacteria was inhibited by copolymers with and without antibiotic drug. These characteristic properties of copolymers make them ideal materials for DD carriers.

An economical self-coalescing microfluidic device with an easily observable readout

Introduction: Self-coalescing microfluidic devices represent an exciting opportunity for leveraging viscous dominated flow phenomena to reconstitute a series of reagents with a single sample infusion. In a self-coalescing microfluidic device, spatially separated dried reagents are reconstituted using a single infusion. Due to the unique device geometry, the reagents remain spatially separated after infusion. As such, self-coalescing microfluidic devices have the potential to simplify point-of-care testing by simultaneously performing multiple colorimetric tests in one device. The current versions of these devices use standard, more costly microfabrication processes and are too small to be easily read by eye.Methods: Here, we created a low cost and scaled up version of a self-coalescing microfluidic device by using laser-cut-roll-based silicone tape. In addition to eliminating the need for cleanrooms, our approach simplifies the integration of assay reagents since they can be spotted onto a coverslip and covered with the tape microfluidic device. We empirically optimized our device, finding that flow rate significantly influenced the formation of self-coalescence as well as proper reagent reconstitution.Results and Discussion: When flow rates were too slow or fast, reagents would streak, leading to inadvertent mixing between different spatial locations. Our studies further revealed that geometry had a stronger influence on device operation in low-flow conditions. Our final optimized device exhibited a 100% success rate, demonstrated through self-coalescence with no reagent streaking at 100 μL/min, which enabled the isotropic diffusion of reagents. Furthermore, the spots are spatially separated and large enough to be visualized by the naked eye and captured by a smartphone camera for downstream analysis. Taken together, our innovative device leverages the advantages of sticker microfluidics and low-cost manufacturing methods to offer standalone functionality. This approach has the potential to significantly impact point-of-care diagnostics, particularly in resource-limited regions. By enabling multiplexed diagnostic assays through our novel technology, we aim to provide accessible and affordable healthcare solutions.

Development of a Smartphone-Integrated Handheld Automated Biochemical Analyzer for Point-of-Care Testing of Urinary Albumin

The level of urinary albumin is a critical indicator for the early diagnosis and management of chronic kidney disease (CKD). However, existing methods for detecting albumin are not conducive to point-of-care testing due to the complexity of reagent addition and incubation processes. This study presents a smartphone-integrated handheld automated biochemical analyzer (sHABA) designed for point-of-care testing of urinary albumin. The sHABA features a pre-loaded, disposable reagent cassette with reagents for the albumin assay arranged in the order of their addition within a hose. The smartphone-integrated analyzer can drive the reagents following a preset program, to enable automatic sequential addition. The sHABA has a detection limit for albumin of 5.9 mg/L and a linear detection range from 7 to 450 mg/L. The consistency of albumin level detection in 931 urine samples using sHABA with clinical tests indicates good sensitivity (95.78%) and specificity (90.16%). This research advances the field by providing an automated detection method for albumin in a portable device, allowing even untrained individuals to monitor CKD in real time at the patient's bedside. In the context of promoting tiered diagnosis and treatment, the sHABA has the potential to become an essential tool for the early diagnosis and comprehensive management of CKD and other chronic conditions.

Blood serum biomarkers in the dynamics of treatment for cerebrovascular pathology in post-COVID-19 patients

Neurological manifestations after COVID-19 infections can be attributed to chronic immune and inflammatory reactions induced by SARS-CoV-2, as well as endothelial dysfunction and other vascular issues. This study included 24 patients with cerebrovascular disease (CVD) who had contracted COVID-19 1 – 1.5 years before the examination (experimental group) and 20 patients with CVD who had not previously been infected with the coronavirus (comparison group). Both groups were representative in terms of age and gender. Blood serum biomarkers, including interleukin (IL)-6, endothelin-1 (ET-1), and vascular endothelial growth factor A (VEGF-A), were tested before and after a 14-day standard complex therapy in the hospital at our institute using appropriate enzyme-linked immunosorbent assay reagent kits. Increases in the levels of IL-6 (by 20%), ET-1 (by 200%), and VEGF-A (by 100%) were observed in patients with CVD who had contracted COVID-19 1 – 1.5 years earlier compared to the comparison group. The effectiveness of a 2-week therapeutic intervention in patients with a history of COVID-19 was found only for VEGF-A: its serum level decreased twofold, leading to the normalization of this indicator. The concentrations of IL-6 and ET-1 were not affected by the treatment. In addition, the serum levels of ET-1 and VEGF-A in unvaccinated individuals exceeded those in vaccinated patients.

Exploring new 5-Nitroimidazole Derivatives as Potent Acetylcholinesterase and Butyrylcholinesterase Enzyme Inhibitors.

Discovering new compounds capable of inhibiting physiologically and metabolically significant drug targets or enzymes is of paramount importance in biological chemistry. With this aim, new 5-nitroimidazole derivatives (1-4) were designed and synthesized, and their inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were discovered using acetyl (butyryl) thiocholine and Ellman's reagents for spectrophotometric assay. The inhibitory profiles of the synthesized compounds were assessed by comparing their IC50 and Ki values. Results demonstrate significant inhibitory activity of all synthesized compounds against both AChE and BuChE compared to the reference compound, donepezil. Notably, compound 4 exhibited dual inhibition of these enzymes, showing the highest activity against Electrophorus electricus AChE (EeAChE) with a Ki value of 0.024±0.009 nM and against equine BuChE (eqBuChE) with a Ki value of 0.087±0.017 nM. Furthermore, molecular modeling was conducted to study the interaction modes of the most potent compound (4) and donepezil in the active site of their related enzymes' crystal structures (PDB ID: 4EY7 and 4BDS, respectively). Additionally, drug-likeness, ADME, and toxicity profiles of the compounds and metronidazole were predicted. The above results indicated that the dual inhibition of these enzymes is considered as a promising strategy for the treatment of neurological disorder especially Alzheimer's disease.

Pulsed light processing of sugarcane juice: quality evaluation and microbial load assessment.

Freshly extracted sugarcane juice is an ideal substrate for microbial fermentation and browning reactions. The present study is the first report on the potential of pulsed light (PL) processing in improving microbial stability with the retention of major bioactive. PL processing at different levels of voltage (2.1-2.7 kV) and number of pulses (100-200) was explored. The present study aimed to investigate the impact of PL processing on the quality of sugarcane juice, bioactive composition and microbial load. The microbial load, such as aerobic mesophiles, yeast and mold, and total coliform, was reduced to below 1 log colony-forming units mL-1 in juice samples subjected to intense PL treatment at 2.7 kV. The maximum value of the total color difference of the sugarcane juice was below 4.0, even at extreme levels of PL process parameters. In comparison with the unprocessed juice, the reduction in total phenols (Folin ciocalteu reagent assay) and the total antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay) was limited to 6% and 16.7%, respectively, when treated at 2.7 kV/200 pulses. The pH and total soluble solids of the juice remained unaffected in all the processed samples. Among the process parameters considered, the treatment voltage was found to significantly affect the quality parameters and microbial load. PL processing at 2.1 kV/170 pulses gave an optimally processed juice with a microbial load below the permissible limit and desirability value of 0.77. The results suggest that the PL treatment is effective for enhancing the microbial stability and maintaining the bioactive components of the sugarcane juice. Furthermore, the outcomes from the present study are expected to pave the way for further in-depth investigation of the effect of PL treatment on the critical quality attributes and shelf life of sugarcane juice. The technology will be useful for adoption by different stakeholders, including manufacturers and retailers in the food processing sector. © 2024 Society of Chemical Industry.

Characterization of an expanded set of assays for immunomodulatory proteins using targeted mass spectrometry

Immunotherapies are revolutionizing cancer care, but many patients do not achieve durable responses and immune-related adverse events are difficult to predict. Quantifying the hundreds of proteins involved in cancer immunity has the potential to provide biomarkers to monitor and predict tumor response. We previously developed robust, multiplexed quantitative assays for immunomodulatory proteins using targeted mass spectrometry, providing measurements that can be performed reproducibly and harmonized across laboratories. Here, we expand upon those efforts in presenting data from a multiplexed immuno-oncology (IO)-3 assay panel targeting 43 peptides representing 39 immune- and inflammation-related proteins. A suite of novel monoclonal antibodies was generated as assay reagents, and the fully characterized antibodies are made available as a resource to the community. The publicly available dataset contains complete characterization of the assay performance, as well as the mass spectrometer parameters and reagent information necessary for implementation of the assay. Quantification of the proteins will provide benefit to correlative studies in clinical trials, identification of new biomarkers, and improve understanding of the immune response in cancer.

Strategies for Performing Factor Assays in the Presence of Emicizumab or Other Novel/Emerging Hemostatic Agents.

For several decades, therapeutic options for inherited deficiencies of factor VIII or IX (hemophilia A or B, respectively) have largely been the replacement of the missing clotting factor with plasma-derived or recombinant products. Hemostasis laboratories use standard activated partial thromboplastin time (aPTT)-based clotting or chromogenic assays to monitor plasma factor levels to guide therapy. The emergence in the past 10 years of extended half-life replacement products and other novel therapies for hemophilia has led to a reappraisal of assay suitability, with studies of product measurement showing some existing assay types or reagents to be unsuitable for some products. The hemostasis laboratory must adapt to the changing landscape by adding new assays or modifying existing assays to ensure accurate results for product measurement. These strategies include switching from a chromogenic assay to a clotting assay, or vice versa, changing an aPTT reagent brand, or introducing product specific calibrators. This article evaluates the effects of some of the newer treatment options on the laboratory testing of factor levels and related assays.

Creation of Single Cell Protein-Producing Mutants of Phaffia Rhodozyma

Recently, there has been an increasing demand for sustainable protein sources for aquaculture. In aquaculture, sustainable protein includes sources that can be produced with a minimal environmental footprint, such as single-cell proteins, proteins from algae, insects, and by-products from the agricultural industry. Single-cell proteins that are derived from microorganisms show a sustainable alternative to traditional protein sources due to their high protein content and rapid growth. This research focuses on creating mutants of the yeast Phaffia rhodozyma to increase single-cell protein production. This yeast has significant industrial potential and biological features. The yeast Phaffia rhodozyma is already known for its unique ability to synthesize astaxanthin, so this case will focus on its ability to produce protein. In the study, we used wild strain yeast and previously obtained white mutants on inorganic nitrogen media for further random mutagenesis methods to introduce specific mutations into the genome aimed at improving their ability to biosynthesize protein. Amino acid inhibitor was used to select potential protein-producing mutants. The protein content of the biomass obtained from the experiments was analyzed in detail using BCA (Protein Assay Reagent) analysis, which confirmed their potential as a source of nutrients for aquaculture. This method is based on the reaction between bicinchoninic acid and the peptide bonds of proteins, which results in the formation of a color whose intensity is proportional to the amount of protein in the sample. The results obtained allow further research into the development of cost-effective and environmentally friendly alternatives for feed in aquaculture.

Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 μL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Comparative antioxidant, antibacterial, and antidiabetic activities of in vitro-grown callus and wild-grown various parts of Piper longum L

BackgroundPiper longum L. is a tropical and subtropical medicinal plant that has been used as antidiabetic, diarrhea, stomachache, cough, asthma, and bronchitis since ancient times. The in vitro-grown callus from the leaf, however, has not been tested and utilized for any of these conditions. Thus, this research pursues to assess the comparative study of antioxidant, antibacterial, and antidiabetic activities of in vitro-grown callus and various parts of Piper longum-grown in the wild. MethodsThe antioxidant activity of crude extracts and the fractions were determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical scavenging assay, the antibacterial activity by agar-well diffusion method, and antidiabetic activity by α-amylase and α-glucosidase inhibition assay. In addition, the total phenol content (TPC) and total flavonoid contents (TFC) were calculated using Folin-Ciocalteau reagent and aluminum chloride complex-forming assay respectively. ResultsThe highest antioxidant activity (IC50=134.81±1.16 µg/mL), TPC (41.22±0.50 mg of GAE/g dry weight), and TFC (73.41±0.53 mg of QE/g dry weight) were obtained in the dichloromethane (DCM) fraction of root than other crude extracts and fractions. The DCM fractions of root exhibited the minimum inhibitory concentration (MIC) at 5.0 mg/mL and minimum bactericidal concentration (MBC) of 8.35 mg/mL to the Staphylococcus aureus. Moreover, the DCM fraction of root showed the highest α-amylase and α-glucosidase inhibition (IC50=365.21±31.02 and 489.07±27.96 µg/mL) than other crude extracts and fractions. To the best of our information, comparative antioxidant (IC50=206.61±0.64 µg/mL), antibacterial (suppressed 80 % of tested bacteria), and antidiabetic (IC50=1165.15±15.63 and 1304.76±12.43 µg/mL) properties of the in vitro-grown callus were recorded for the first time in P. longum, and were found to be comparative to those of the wild-grown parts. ConclusionsThese results showed that wild-grown P. longum roots, particularly the DCM fraction, could be an important source of natural antioxidants, antimicrobials, and antidiabetics for better curative uses than other wild-grown parts and in vitro-grown callus. In vitro-grown callus, however, could be used as a natural drug source and used to conserve P. longum in its natural habitats in the future.

Iron assay reagent recall over positive bias results: A critical analysis and potential solution

Iron assay reagent recall over positive bias results: A critical analysis and potential solution

BiSim Tool: a binding simulation tool to aid and simplify ligand-binding assay design and development.

Ligand-binding assays (LBAs) rely on the reversible, noncovalent binding between the analyte of interest and the assay reagents, and understanding their dynamic equilibrium is key to building robust LBA methods. Although the dynamic interplay of free and bound fractions can be calculated using mathematical models, these are not routinely applied. This approach is costly in terms of both assay development time and reagents, and can result in an under-explorationof the possible parameter combinations. Therefore, we have created a user-friendly simulation tool to facilitate LBA development (the BiSim Tool). We describe the models driving the mathematical simulations and the main features of our software solution by means of case studies, illustrating the tool's value in drug development. To support drug development for all patients worldwide, the BiSim Tool is now available as an open-source code project and as a free web-based tool at https://proteinbindingsimulation.shinyapps.io/BiSim-ProteinBindingSimulation [1].

Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides

ABSTRACT Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Diagnostic Value of Krebs von den Lungen (KL-6) for Interstitial Lung Disease: A European Prospective Cohort

IntroductionKrebs von den Lungen 6 (KL-6) is a mucin-1 glycoprotein produced by type II pneumocytes. High levels of KL-6 in blood may be found in patients with lung fibrosis. In Asia this biomarker is used for diagnosis and prognosis in interstitial lung diseases (ILD). There is a lack of information regarding KL-6 cut-off point for diagnosis and prognosis in European population. The aim of this study was to establish the cut-off point for serum KL-6 associated with the presence of ILD in the Spanish population. MethodsProspective study including subjects who underwent chest HRCT, PFTs and autoimmune blood analysis. Two groups were created: non-ILD subjects and ILD patients. Serum KL-6 concentrations were measured using a Lumipulse KL-6 reagent assay and the optimal cut-off value was evaluated by a ROC analysis. Data on demographics and smoking history was also collected. ResultsOne hundred seventy-nine patients were included, 102 with ILD. Median serum KL-6 values overall were 762U/mL, 1080 (±787)U/mL for the ILD group vs 340 (±152)U/mL for the non-ILD group (p<0.0001). The main radiological pattern was NSIP (43%). ROC analysis showed greater specificity (86%) and sensitivity (82%) for KL-6 465U/mL for detecting ILD patients. The multivariate logistic regression model pointed to the male sex, higher KL-6 values, lower FVC and low DLCO values as independent factors associated with ILD. ConclusionSerum KL-6 values greater than 465U/mL have excellent sensitivity and specificity for detecting ILD in our Spanish cohort. Multicentre studies are needed to validate our results.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 – 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Longitudinal analysis of 20Years of external quality assurance schemes for PCR/NAAT-based bacterial genome detection in diagnostic testing.

Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers. The study assesses the evolving diagnostic performance trends over 2decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains. The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance. Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.