Detection Reagent Research Articles

Portable 3D-Printed Paper Microfluidic System with a Smartphone Reader for Fast and Reliable Copper Ion Monitoring

Copper ions (Cu2+) are the third most essential transition metal ions critical to human health. Rapid detection of Cu2+ in water and biological fluids is of significant importance. In this study, we develop a sensitive multi-channel paper microfluidic device integrated with a 3D-printed smartphone-based colorimetric reader for the rapid detection of Cu2+. A novel rhodamine derivative, 1-(N,N-dichloromethine) amino-4-rhodamine B hydrazine-benzimide (RBCl), exhibiting high selectivity and sensitivity to Cu2+, was synthesized and applied as the detection reagent. The interaction mechanism between RBCl and Cu2+ was investigated, revealing a structural transition from a colorless spirolactam (closed-ring) to an open-ring amide structure, resulting in a pink color upon Cu2+ binding. A multi-channel paper microfluidic device with eight detection zones was fabricated, enabling the simultaneous analysis of eight samples. To enhance portability and quantification, a 3D-printed smartphone colorimetric reader was integrated, providing a rapid and efficient detection platform. The system achieved highly specific Cu2+ detection within 2 min, with a detection limit as low as 1.51 ng/mL, meeting water monitoring standards in most countries. Excellent recoveries were demonstrated in real samples, including tap water, river water, blood serum, and urine diluent. This integrated paper microfluidic system is highly sensitive and specific, offering a promising solution for water quality monitoring and health assessment through its rapid sample-to-answer capability.

A Highly Sensitive Immunoassay to Enable Quantification of a Nanobody-Based Imaging Agent in Human Serum.

Immuno-positron emission tomography (i-PET) is a non-invasive imaging technique that combines the specificity of monoclonal antibodies with the sensitivity of positron emission tomography to visualize and quantify the distribution of target antigens in vivo, providing detailed spatial information about the presence and localization of specific biomarkers. Due to the crucial role that cytotoxic T cells play in antitumor responses, a new tracer consisting of a humanized anti-CD8 nanobody labeled with fluorine-18 was developed to inform immuno-oncology treatments and support medical decision-making. Nanobodies are single-domain antibodies with low molecular weight and fast peripheral blood clearance, both of which are advantageous properties for same-day imaging. However, these unique characteristics pose bioanalytical challenges when developing clinical pharmacokinetic (PK) assays, including the need for high assay sensitivity. This manuscript focuses on overcoming bioanalytical challenges related to sensitivity and matrix interference during the development of a method to quantify this novel anti-CD8 nanobody tracer in human serum. Out of the three immunoassay platforms evaluated (ELISA, SMCxPRO™ and Gyrolab®), a Gyrolab method was ultimately selected due to its superior sensitivity, equal detectability of both conjugated and unconjugated forms of the nanobody and its ability to minimize matrix interference. By selecting the right assay format, along with the appropriate critical reagents for capture and detection, matrix interference was diminished. This novel PK method was successfully qualified demonstrating acceptable performance across all parameters. The acquired bioanalytical insights gained could be applied to nanobody-derived conjugates or other modalities that require high sensitivity in the clinical settings.

Plant-Based Antigen Production Strategy for SARS-CoV-2 Nucleoprotein and RBD and Its Application for Detection of Antibody Responses in COVID-19 Patients

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the development of efficient serological tests for monitoring the dynamics of the disease as well as the immune response after illness or vaccination was critical. In this regard, low-cost and fast production of immunogenic antigens is essential for the rapid development of diagnostic serological kits. This study assessed the plant-based production of nucleoprotein (N) of SARS-CoV-2 and chimeric receptor-binding domain (RBD) of SARS-CoV-2 presented by hepatitis E virus capsid (HEV/RBD) and validation of the plant-derived proteins as diagnostic antigens for serological tests. The target proteins were expressed in and purified from Nicotiana benthamiana plants. The resulting yield of chimeric HEV/RBD protein reached 100 mg/kg fresh weight and 30 mg/kg fresh weight for N protein. The purified N protein and HEV/RBD protein were used to develop an indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies to SARS-CoV-2 in human sera. To validate the iELISA tests, a panel of 84 sera from patients diagnosed with COVID-19 was used, and the results were compared to those obtained by another commercially available ELISA kit (Dia.Pro D. B., Sesto San Giovanni, Italy). The performance of an HEV/RBD in-house ELISA showed a sensitivity of 89.58% (95% Cl: 75.23–95.37) and a specificity of 94.44% (95% Cl: 76.94–98.2). Double Recognition iELISA based on HEV/RBD and N protein is characterized by a lower sensitivity of 85.42% (95% Cl: 72.24–93.93) and specificity of 94.44% (95% Cl: 81.34–99.32) at cut-off = 0.154, compared with iELISA based on HEV/RBD. Our study confirms that N and fusion HEV/RBD proteins, which are transiently expressed in plants, can be used to detect responses to SARS-CoV-2 in human sera reliably. Our research validates the commercial potential of using plants as an expression system for recombinant protein production and their application as diagnostic reagents for serological detection of infectious diseases, hence lowering the cost of diagnostic kits.

Monoclonal antibodies for diagnosis of viral diseases: a comprehensive review

Viruses cause infectious diseases in humans, ranging from mild to life-threatening. Early and accurate diagnosis is essential for timely treatment and prevention of complications. Diagnostic methods typically rely on detecting viral genetic material, proteins, or the host’s immune response, depending on the infection stage. Monoclonal antibodies, known for their specificity and high affinity, are widely used for diagnosing viral infections by enabling precise and sensitive detection of viral proteins. This review explores the applications of monoclonal antibody-based methods for detecting viral infections, emphasizing their pivotal role as sensitive detection reagents. These methods are crucial for developing robust immunodiagnostic assays capable of diagnosing various viral diseases in human, animal, and plant hosts, with special emphasis on SARS-CoV-2. The review also explores various assays that employ monoclonal antibodies for diagnostic purposes and the technologies used for their production. Understanding the principles and applications of monoclonal antibodies in diagnosing viral diseases is essential for implementing effective public health interventions and preventing future pandemics.

A phenylboronic-acid functionalized liquid photonic crystal reagent for convenient and reliable detection of glucose

A phenylboronic-acid functionalized liquid photonic crystal reagent for convenient and reliable detection of glucose

Activating Esterase D by PFD5 exerts antiviral effect through inhibiting glutathionization of LAMP1 during Senecavirus A infection

Seneca virus A (SVA) is a newly discovered small nucleic acid virus, which can cause swine blister disease (PVD). Currently, there is no drug or vaccine. Studies have shown that SVA relies on the endolysosomal pathway to accomplish intracellular transport and release, and can disrupt lysosomal homeostasis, but its specific mechanism has not been revealed. At present, there is limited research on vaccines and antiviral drugs for it. Esterase D (ESD), a serine hydrolase, is active against many substrates and plays a role in inhibiting viral replication. However, the specific mechanism of the thioesterase activity of ESD in the signaling pathway is unknown. In this study, we synthesized and screened a novel small-molecule ESD activator, FPD5 (4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro- 1h -pyrazol-3-yl)phenyl), capable of inhibiting the replication of SVA. To explore the mechanism of action of this process, we demonstrated the direct interaction between ESD and lysosomal membrane protein-1 (LAMP1) by CO-IP; Western blot revealed that FPD5 could activate ESD and exert a protective effect on LAMP1 and lysosomes. The deglutathionization function of ESD was verified by protein glutathione immunoblotting and detection reagents, and the affected cysteine residues were found by point mutation technique. The results showed that FPD5 activated ESD to exert thioesterase activity, reduced glutathionylation cysteine 375 in the LAMP1 and decreased SVA production. This study provides a theoretical basis for the development of small molecule drugs against SVA, and also suggests that the activation of the thioesterase activity of ESD is a new direction for future antiviral drug development.

PH-Activatable Molecular Probe for COX-2 Imaging in Human Oral Squamous Carcinoma Cells and Patient-Derived Tissues.

For developing a successful cancer therapeutic modality, the early precise detection of cancer cells in patient biopsies in oral squamous cell carcinoma (OSCC) is crucial. This could help researchers create new diagnostic and therapeutic tools and assist clinicians in recommending more effective treatment plans and improving patient survival. We have developed an SMPD, cyclooxygenase-2 (COX-2) targeting pH-activable fluorophore named CNP, combining a potent COX-2 inhibitor, celecoxib, linked to a naphthalimide fluorophore with an acidic microenvironment-responsive piperazine moiety for specific optical imaging of OSCC in cells and patient tissues. Compared to reference probe RNP lacking celecoxib, CNP selectively enters the COX-2 overexpressing oral cancer cells. Its acidity-responsive imaging response enhances selectivity over cancers with lower COX-2 expression levels and normal cells. Further, CNP is demonstrated in imaging OSCC cells in patient-derived biopsies. Thus, multifunctional CNP shows potential in exploring more reagents for fluorescence-based detection of OSCC cells in patient tissues with translational applications.

A Novel High-Throughput Sample-in-Result-Out Device for the Rapid Detection of Viral Nucleic Acids.

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

Synthesis and application of electrospun polyvinylidene fluoride/polyvinylidene glycol nanofibrous membrane for enhanced biomarker detection in immunochromatography assay

Immunochromatography assay (ICA) stands as a pioneering point-of-care testing (POCT) method, leveraging the simplicity, portability, and versatility of dry-strip testing method, chromatographic and antigen-antibody immunological reaction. Despite its numerous advantages, the shortcomings like low sensitivity, limited matrix, restricted matrix compatibility, and uncontrollable chromatographic rates, have hindered the ICA application. To overcome the aforementioned challenges, we proposed constructing a novel ICA strip based on homemade hydrophilic, flexible membranes to replace the commercially available nitrocellulose (NC). The key to the innovation lies in the spinning solution of a blend of poly (vinylidene fluoride) (PVDF) and polyethylene glycol (PEG), which is made into a nanofibrous membrane named EPVDF/PEG membrane by electrostatic spinning technique. This membrane, due to its high specific surface area and high porosity, can be loaded with more detection reagents than normal substrates, increasing the reaction time and achieving improved detection sensitivity. This membrane also has adjustable chromatographic rate, high protein loading capacity, good biocompatibility and excellent colouring ability. As a result, the established EPVDF/PEG-based ICA strips demonstrate an ultralow detection limit, as low as 0.5 mIU∙mL−1 when human chorionic gonadotropin (HCG) serves as the biomarker model, which is a 50-fold increase in detection sensitivity compared to commercial strips. Furthermore, EPVDF/PEG-based ICA strips can be produced on a large scale for the availability of efficient and mature electrospinning technologies. In summary, this work offers a transformative advancement in ICA development, potentially opening new horizons for sensitive biomarker detection.

Synthesis of 2-methyl-6-methoxy-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (MMQC-OSu) for streamlined and effective HPLC-based fluorescence detection of aliphatic amines in environmental samples

Aliphatic amines are widely distributed in the environment and food sources, posing potential health risks through skin and mucosal irritation. Consequently, their quantitative detection is crucial for assessing environmental health. Despite high reactivity and fluorescence properties of succinimidyl ester-based derivatization reagents, their application in the aliphatic amines detection is hampered by challenges such as limited detection sensitivity and fluorescence interference. We established an innovative synthetic approach to produce a series of succinimidyl esters with the desirable substituents. This advancement enabled the creation of efficient and highly sensitive reagents for the detection of aliphatic amines. Among them, 2-methyl-6-methoxy-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (MMQC-OSu) exhibited the best detection performance. MMQC-OSu reacted with aliphatic amines at 40 °C in pH 8.0 buffer for 20 min, which was subsequently separated in a C18 chromatographic column with the fluorescence detection wavelength of 336/432 nm. This detection approach featured a rapid and mild reaction process, minimal interference from corresponding hydrolysis products, and impressive sensitivity (0.05 nM). These characteristics indicate that MMQC-OSu significantly surpassing commercial aliphatic amines detection reagents. Finally, the detection strategy of MMQC-OSu was successfully applied in environmental sample analysis, with recovery rate of 93 %–108 % and RSDs between 1.4 % and 6.5 %.

Highly Sensitive and Specific Diagnosis of Enterovirus A71 by Reverse Transcription Multiple Cross-Displacement Amplification-Labeled Nanoparticles Biosensor.

Enterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia-Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription-multiple cross displacement amplification (RT-MCDA) combined with nanoparticle-based lateral flow biosensors (LFB). In the RT-MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT-MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71-RT-MCDA assay was 65℃ for 40 min. The EVA71-RT-MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross-reaction was found between EVA71 strains and non-EVA71 strains. For 125 clinical anal swab samples, with EVA71-RT-MCDA assay, 30 samples were positive, which was in consistent with the the conventional real-time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15-min specimen processing step, a 40-min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71-RT-MCDA-LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.

Development of bovine IgG3-specific assays using a novel recombinant single-domain binding reagent

Cattle express three subclasses of IgG antibody - IgG1, IgG2 and IgG3. Unlike IgG1 and IgG2, IgG3 was described relatively recently and the role of this subclass in immunity is unknown. Using recombinant bovine IgG1, IgG2 and IgG3 monoclonal antibodies (mAbs), we demonstrated that only one of the commercially available anti-bovine IgG mAbs tested was able to recognize IgG3, and no mAb exclusively bound IgG3. Here, we evaluated a small ankyrin repeat protein, called Ankyron™ AZS40101, that was generated to bind to bovine IgG3. Ankyron™ AZS40101 bound specifically to IgG3 with minimal reactivity to IgG1 and IgG2. Ankyron™ AZS40101 was shown to be useful in ELISA assays as either a capture or detection reagent. Utilisation of Ankyron™ AZS40101 alongside IgG1 and IgG2 specific mAbs to detect antigen-specific IgG subclasses in the serum of cattle sequentially vaccinated with heterologous foot-and-mouth disease virus capsid antigens revealed a low-level antigen-specific IgG3 response, in addition to IgG1 and IgG2 responses. Assessment of the total IgG1, IgG2 and IgG3 levels in healthy cattle plasma samples showed that IgG3 was measurable at a mean concentration of 0.19 mg/mL, although this was significantly lower than those of IgG1 (mean 3.28 mg/mL) and IgG2 (mean 3.41 mg/mL). Thus, Ankyron™ AZS40101 is a new reagent that provides utility for measurement of bovine IgG3 responses to infection and vaccination.

ALKBH5 promotes T-cell acute lymphoblastic leukemia growth via m6A-guided epigenetic inhibition of miR-20a-5p

This study investigates the role of ALKBH5-mediated m6A demethylation in T-cell acute lymphoblastic leukemia (T-ALL). T-ALL cell lines (HPB-ALL, MOLT4, Jurkat, CCRF-CEM) and human T cells were analyzed. CCRF-CEM and Jurkat cells were transfected with si-ALKBH5, miR-20a-5p-inhibitor, and pcDNA3.1-DDX5. The expression levels of ALKBH5, miR-20a-5p, and DDX5 in these cells were determined using qRT-PCR and Western blotting. Cell viability, proliferation, colony formation, and apoptosis were assessed using CCK-8, EdU staining, colony formation assay, and flow cytometry. mRNA m6A levels were quantified with an m6A RNA methylation detection reagent, and RNA immunoprecipitation was employed to measure the enrichment of DGCR8 and m6A on the primary transcript pri-miR-20a of miR-20a-5p. Dual-luciferase assay confirmed the binding relationship between miR-20a-5p and DDX5. Results showed that ALKBH5 and DDX5 were upregulated in T-ALL tissues and cells, whereas miR-20a-5p was downregulated. Silencing ALKBH5 inhibited T-ALL cell viability, colony formation, and proliferation, while promoting apoptosis. These effects were reversed by miR-20a-5p inhibition or DDX5 overexpression. ALKBH5 reduced the relative m6A level in T-ALL cells and decreased miR-20a-5p expression by reducing DGCR8 binding to pri-miR-20a-5p. miR-20a-5p suppressed DDX5 transcription. In conclusion, ALKBH5-mediated m6A demethylation decreases DGCR8 binding to pri-miR-20a, thereby repressing miR-20a-5p expression and enhancing DDX5 expression, ultimately inhibiting T-ALL cell apoptosis and promoting proliferation.

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

Re-appraising the evidence for the source, regulation and function of p53-family isoforms.

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

The Development and Application of Lyophilized LAMP Detection Reagent for Listeria monocytogenes.

Listeria monocytogenes, a zoonotic foodborne pathogen, presents a significant threat to global public health. Therefore, rapid and sensitive detection methods are crucial in mitigating the spread of L. monocytogenes induced diseases. This study introduced a loop-mediated isothermal amplification (LAMP) lyophilized powder detection reagent specifically designed for identifying Listeria monocytogenes. The reagent is user-friendly, quick, and can be easily transported and stored at room temperature. It exhibits no cross-reactivity with eight other types of bacteria and boasts a sensitivity of 101CFU/mL. In a comparative study of 30 samples, the LAMP lyophilized powder detection reagent demonstrated higher sensitivity than the commercial Listeria monocytogenes qPCR detection kit. Additionally, the experimental time was reduced by approximately 30min, making it highly suitable for rapid diagnosis. Preparation of lyophilized LAMP reagents may facilitate large-scale deployment, particularly in endemic areas or regions facing rapid outbreaks. This could greatly aid in controlling the transmission of pathogens, especially those transmitted through food.

Development of Donor–acceptor-type Molecules and Their Application as Analytical Reagents

π-Extended donor-acceptor (D-A)-type molecules, which bear both electron-donor and electron-acceptor substituents on the backbone, exhibit unique optical properties, such as bathochromic shifts in absorption and emission, large Stokes shifts, solvatochromic behavior, and fluorescence quenching in polar solvents. These unique properties are attributed to intramolecular charge transfer (ICT) or twisted intramolecular charge transfer (TICT) in the ground and excited states. This review article introduces three types of D-A-type molecules that are used as detection reagents for (1) methanol, (2) amino acids during solid-phase peptide synthesis (SPPS), and (3) amines present in the biological environment. For methanol detection, D-A-type fluorophores with basic guanidine moieties were developed to differentiate between methanol (MeOH) and ethanol (EtOH) based on the small difference in their pKa values (ΔpKa=0.4). Selective protonation of the guanidine moiety in methanol disrupts the D-A structure, allowing emission in the resultant polar environment. Similarly, an acid-base reaction between the hydrogen chloride (HCl) salts of the D-A-type molecules and amines is applied to detect amines during SPPS. In this method, a colorless solution of an HCl salt of the D-A-type molecule is deprotonated by amines, forming a yellow solution. This is the first reported quantitative and non-destructive colorimetric method for detecting amines. Finally, a turn-on-type amine-labeling reagent was developed for the nucleophilic aromatic substitution (SNAr) reaction. This new reagent enables protein staining of living cells with a large Stokes shift and without solvent-polarity-dependent fluorescence quenching.

A novel nucleic acid extraction system integrating a switching valve-assisted microfluidic cartridge and a constant pressure pump

BackgroundNucleic acid extraction (NAE) is essential in molecular diagnostics, genetic engineering, and DNA sequencing. While advances in switching valve-assisted, self-contained microfluidic (S2M) cartridges have improved the NAE processes, challenges persist in streamlining workflows, reducing processing times, and enhancing multi-target detection. Achieving these objectives requires precise control of on-chip fluid dynamics and efficient transfer of nucleic acid solutions (NAS) to detection areas. However, research on novel S2M cartridge designs and compatible fluid control systems is still limited. ResultThis study presented an innovative NAE system that integrated an S2M cartridge, a constant pressure (CP) pump, a metal heating bath, a rotating lever, and a magnet. The S2M cartridge contained the reagents for both NAE and detection, while the CP pump provided stable, precise, and rapid fluid delivery, outperforming traditional peristaltic and injection pumps, especially for handling small volumes and reducing pulsation. A custom rotating lever connects reagent chambers, facilitating mixing, cleaning, elution, and NAS transfer. This system showed advantages over manual methods, achieving all processes in 20 min. The CP pump-assisted S2M cartridge enabled rapid MBs re-suspension with a recovery rate of 80 %. The system's sensitivity, repeatability, and ease of use were also confirmed. The NAE system can extract Escherichia coli DNA (50 CFU mL^-1) from food and serum samples and viral DNA (HBV, HCV, HIV) at 200 copies mL^-1 from serum samples. SignificanceThis study highlighted the need for an efficient fluid system in the S2M cartridge for stable NAE. By streamlining NAE and facilitating on-chip reagent preparation, the system enhanced point-of-care diagnostics. Although manual steps remain, the CP pump effectively managed fluid processes, paving the way for automated pathogen detection. Moreover, when combined with visual detection reagents, the NAE system showed promise as a direct molecular diagnostic tool.

Evaluation of a New In Chemico Skin Corrosion Test

The purpose of this study is to evaluate a novel macromolecular test method for the identification of dermal corrosives. The simple in chemico test procedure involves allowing the material to be tested to interact with a skin biomarker for corrosivity and then adding a detection reagent. The corrosivity of the test substance is predicted based on the measured macromolecular damage, which results in reduced optical density of the detection reagent as compared with controls. This study aims to determine if such an extremely simple, cell-free test method can accurately identify dermal corrosives. To determine predictivity and repeatability, we tested 60 chemicals (30 in vivo dermal corrosives and 30 in vivo dermal noncorrosives; all tested in triplicate) representative of a broad range of chemical classes, functional groups, mixtures, and levels of toxicity. Validation results indicate the GHS multicategory and packing group assignment accuracy is on par with that of the Reconstructed Human Epidermis test method and the Membrane Barrier test method and for the global identification of corrosives, the method has a considerably higher accuracy (98 % vs. ∼80 %).

Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.