Search Results (241)

Porcine circovirus 2 (PCV2) is a DNA virus that is classified in the genus Circovirus of the Circoviridae family, which is a causative agent of Porcine Circovirus-Associated disease (PCVAD). PCVAD continues to cause substantial losses in global pig farming, with PCV2d being the prevalent genotype worldwide, including in Vietnam. In this study, we focused on generating a recombinant PCV2d Cap protein fused to the GCN4pII motif (Cap2d-pII) in a plant-based system and evaluating its immunogenicity. The Cap2d-pII gene was cloned into a plant expression vector and introduced into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana leaves. Western blot analysis confirmed the high accumulation of the Cap2d-pII protein, which was purified by Immobilized affinity chromatography and used for immunizing mice. ELISA and immunoperoxidase monolayer assay results demonstrated that immunization with the recombinant protein elicited robust humoral and cellular immune responses. At 56 days after immunization, mice vaccinated with the Cap2d-pII protein generated PCV2d-specific IgG titers and IFN-γ responses that were consistent with those in mice receiving the commercial inactivated vaccine. These observations confirm that the plant-expressed Cap2d-pII antigen effectively activates both antibody- and T cell-mediated immune pathways. Collectively, this study identifies the Cap2d-pII protein as a promising plant-derived vaccine candidate for the development of effective and affordable PCV2d subunit vaccines. Full article

Antibodies labeled with gold nanoparticles are widely used in analytical systems. Consequently, the choice of methods for producing such conjugates requires a precise determination of the retained reactivity of the antibodies. Existing methods give highly variable results, necessitating new, simple, and accurate approaches. This study demonstrates how a fluorescein (FL) and anti-FL antibody pair can be used to comparatively evaluate conjugation conditions. The method is based on the quenching of fluorescein emission after binding to antibodies, enabling real-time monitoring of interactions in the reaction medium. Using this approach, we compared a series of conjugates of gold nanoparticles (average diameter 24 nm) and anti-FL antibodies that were obtained with antibody concentrations of 5, 10, and 20 μg/mL during the conjugation. The proportion of antibodies that retained their reactivity varied from 11% to 58%. We also estimated the blocking of the conjugates’ surfaces by two widely used reagents, bovine serum albumin and mercapto polyethylene glycol. It was found that the former provides better retention of antigen-binding activity of immobilized antibodies. The difference between these two kinds of preparations is most pronounced—reaching up to a twofold change—at lower antibody densities. Full article

An electrochemical immunosensor was fabricated to identify CA15-3, a biomarker for breast cancer (BC). A composite sensor substrate made of “zeolitic imidazolate framework-8” (ZIF-8) and “reduced graphene oxide” (rGO) was chosen and its conductivity was further improved by the addition of chitosan (CS)-doped gold nanoparticles (AuNPs). The CS@AuNPs are able to conjugate with antibodies via the strong Au-S interaction, which offers multiple active sites for antibody immobilization and enhances the sensor performance. This immunosensor is capable of ultrasensitive detection of CA15-3 by specific antigen–antibody –interactions. In healthy people, normal serum CA15-3 is up to 25 U/mL. Under optimized experimental conditions, the alteration in the signal intensity measured by the sensor was related to the CA15-3 activity. The quantitative relationship was linear over 0.001–400 U/mL with a limit of detection (LOD) of 0.0031 U/mL at a “signal-to-noise ratio” (S/N) of 3 and a “correlation coefficient” (r²) of 0.9983. The developed immunosensor showed great accuracy, stability, and selectivity, and was able to detect CA15-3 in human serum samples. These results validate its potential as a reliable analytical platform for BC diagnosis and early clinical screening. Full article

Backgroud/Objectives: Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern due to dogs serving as reservoirs for human infection. An accurate and rapid diagnostic method to distinguish symptomatic and asymptomatic CVL from healthy and vaccinated animals is essential for controlling canine and human disease. Developing innovative antibody detection techniques and exploring new antigens are essential for enhancing CVL testing efficiency. Our study focuses on a multiplex flow cytometry technique to detect Leishmania-specific antibodies in canine serum. This involved conjugating small peptides with carrier proteins or peptide tags, sequences designed to facilitate bead coupling. Methods: A peptide from the L. infantum A2 protein was coupled to beads in three forms: unconjugated, conjugated with BSA, and conjugated with a C-terminal β-alanine–lysine (x4)–cysteine TAG. This TAG was previously designed to enhance peptide solubility, improve binding efficiency, and provide functional groups for covalent attachment to the beads, ensuring stable immobilization in the multiplex assay. Results: Our results suggest that the multiplex approach shows promise as a rapid serological test for CVL, particularly with TAG-conjugated peptides, which optimize bead coupling. However, peptide/BSA conjugation revealed anti-BSA antibodies in samples from healthy and CVL dogs. Conclusions: In conclusion, our findings highlight the potential of multiplex methodologies to enhance CVL diagnostics and caution against using BSA as a bead coupling agent in serological tests for canine samples due to its impact on test specificity and sensitivity. Full article

Surface plasmon resonance (SPR) biosensors have been applied in various fields with the advantages of being label-free, having high specificity, having high sensitivity, and providing real-time monitoring. With the gradual improvement of SPR technology, SPR biosensors have been used for the detection of macromolecules such as proteins, peptides, and nucleic acids. Antibodies are generally used as the recognition component of SPR biosensors due to the high specificity of antibody–antigen binding. Recently, aptamers have become new choices instead of antibodies for their characteristic of high specificity with target molecules, high stability of chemical synthesis, convenience in storage, and ease of labeling. In this study, an aptamer-based SPR biosensor for chloramphenicol (CAP) detection was established through optimizing the conditions of CAP aptamer immobilization and analysis procedure, including biosensor type, signal enhancement, running buffer, sample diluent, and dissociation time. The results suggested that the optimal immobilization strategy of aptamers on the SPR biosensor was indirect immobilization based on the CM5 chip. The aptamer-based SPR biosensor had good specificity for CAP and could be used to detect CAP in real samples such as milk. Therefore, SPR biosensors have great application prospects in the food safety field, and aptamers deserve further study to improve the performance of the biosensor. Full article

Herein, we propose an ultrasensitive electrochemical immunosensor based on glucose oxidase labeling and enzyme-catalyzed Au staining. In brief, the primary antibody (Ab₁), bovine serum albumin, an antigen and then a bionanocomposite that contains a second antibody (Ab₂), poly(3-anilineboronic acid) (PABA), Au nanoparticles (AuNPs) and glucose oxidase (GOx) are modified on a glassy carbon electrode coated with multiwalled carbon nanotubes, yielding a corresponding sandwich-type immunoelectrode. In the presence of glucose, a chemical reduction of NaAuCl₄ by enzymatically generated H₂O₂ can precipitate a lot of gold on the Ab₂-PABA-AuNPs-GOx immobilized immunoelectrode. In situ anodic stripping voltammetry (ASV) detection of gold in 8 μL 1.0 M aqueous HBr-Br₂ is conducted for the antigen assay, and the ASV detection process takes approximately 6 min. This method is employed for the assay of human immunoglobulin G (IgG) and human carbohydrate antigen 125 (CA125), which demonstrates exceptional sensitivity, high selectivity and fewer required reagents/samples. The achieved limits of detection (S/N = 3) by the method are 0.25 fg mL⁻¹ for IgG (approximately equivalent to containing 1 IgG molecule in the 1 microlitre of the analytical solution) and 0.1 nU mL⁻¹ for CA125, which outperforms many previously reported results. Full article

This study presents a high-performance silicon nanowire (SiNW) array biosensor for the combined detection of two key colorectal cancer (CRC) biomarkers: circulating tumor DNA (ctDNA) and carcinoembryonic antigen (CEA). The device was fabricated using conventional micromachining techniques, enabling the integration of dual SiNW arrays on a single chip with precise control over structure and surface functionalization. Specific probe DNA and anti-CEA antibodies were immobilized on distinct array regions to facilitate targeted binding. The biosensor demonstrated exceptional performance, achieving an ultralow detection limit of 10 aM for ctDNA with a linear range from 0.1 fM to 10 pM, and a sensitivity of 1 fg/mL for CEA. It exhibited high selectivity against interfering substances, including single-base mismatched DNA and non-specific proteins, and maintained robust performance in human serum samples. The platform offers a scalable, label-free, and real-time detection solution with significant potential for application in early CRC screening and personalized medicine. Full article

Foodborne illnesses remain a global challenge, requiring rapid and sensitive detection platforms. We developed a magnetosome-based electrochemical immunosensor for lipopolysaccharide (LPS) antigens from Escherichia coli and Salmonella typhimurium. Magnetosomes isolated from Magnetospirillum sp. RJS1 were characterized by HR-TEM and functionalized with antibodies (2 CFU mL⁻¹), with FTIR confirming successful conjugation. The antibody–magnetosome complexes were immobilized on a chitosan/glutaraldehyde-modified glassy carbon electrode. AFM revealed globular (200–700 nm) and island-like (1–3 µm) features after antigen binding. Electrochemical impedance spectroscopy showed stepwise increases in charge-transfer resistance upon electrode modification and antigen interaction. The sensor exhibited high sensitivity toward E. coli (3–7 CFU mL⁻¹) and Salmonella (3–8 CFU mL⁻¹), achieving an immune sensitivity of 36.24 Ω/CFU mL⁻¹ and a detection limit of 1 CFU mL⁻¹. These results demonstrate the potential of magnetosome-based immunosensors as portable, efficient platforms for the rapid detection of foodborne pathogens in real samples. Full article

The ongoing COVID-19 pandemic has underscored the urgent need for rapid, sensitive, and versatile diagnostic tools. In this study, we developed a nanogold-based lateral flow immunoassay (LFIA) capable of detecting both SARS-CoV-2 nucleocapsid (N) protein antigens and anti-N IgG antibodies at the point of care. Following optimization of colloidal gold nanoparticle size, pH, and protein conjugation parameters, LFIA strips were assembled in two formats: a competitive assay for antigen detection and a sandwich assay for antibody detection. In the competitive format, gold nanoparticles (AuNPs)-conjugated N protein were used to detect varying concentrations of free N protein. The test line signal inversely correlated with antigen concentration, confirming the assay’s specificity and effectiveness. For antibody detection, the sandwich LFIA format employed immobilized anti-human IgG to capture anti-N antibodies in serum samples from COVID-19 patients. Strong test line signals were observed in samples collected ≥11 days post-symptom onset, indicating a time-dependent increase in IgG detectability. These results demonstrate that the AuNP-based LFIA platform provides a flexible, rapid, and low-cost diagnostic solution, suitable for both early antigen detection and serological monitoring during SARS-CoV-2 infection and recovery. Full article

Background: The accurate evaluation of anti-ABO antibodies is essential for risk stratification in ABO-incompatible (ABOi) transplantation. Historically, hemagglutination-based titration has been the cornerstone of such an assessment; however, different tools are being evaluated in this context. In recent years, several novel methods have been reported. Methods: A narrative review was conducted using PubMed, Scopus, and Google Scholar, focusing on recent studies evaluating anti-ABO antibody measurement techniques in the context of ABOi organ transplantation. Results: In addition to the conventional tube method, techniques such as column agglutination technology, flow cytometry, and enzyme-linked immunosorbent assay are utilized for anti-ABO antibody assessment. However, any particular technique, significant interinstitutional and interoperator variabilities have been reported due to differences in the detailed protocols and the inherently subjective nature of some techniques. Moreover, these assays are based on the antibody binding to ABO antigens expressed on red blood cells, which might not accurately reflect the clinical context of organ transplantation. In recent years, technological advances have enabled the development of novel assays evaluating antibody responses specifically against the ABO antigens expressed on vascular endothelial cells. These include glycan microarrays, which differentiate responses by ABO antigen subtypes, and CD31-based microarrays, wherein recombinant CD31 proteins expressing ABO antigens are immobilized. These approaches are applied to assess clinically relevant anti-ABO antibodies in the context of ABOi organ transplantation. Conclusions: The objective evaluation of antibody titers against ABO antigens on vascular endothelial cells might not only enable a more accurate risk assessment but also facilitate meaningful comparisons between institutions. Full article

7,12-Dimethylbenzo[a]anthracene (DMBA-7,12), a highly toxic and environmentally persistent polycyclic aromatic hydrocarbon (PAH), poses significant threats to marine biodiversity and human health due to its bioaccumulation through the food chain. Conventional chromatographic methods, while achieving comparable detection limits, are hindered by the need for expensive instrumentation and prolonged analysis times, rendering them unsuitable for rapid on-site monitoring of DMBA-7,12 in marine environments. Therefore, the development of novel, efficient detection techniques is imperative. In this study, we have successfully developed an electrochemical immunosensor based on a polydopamine (PDA)–chitosan (CTs) composite interface to overcome existing technical limitations. PDA provides a robust scaffold for antibody immobilization due to its strong adhesive properties, while CTs enhances signal amplification and biocompatibility. The synergistic integration of these materials combines the high efficiency of electrochemical detection with the specificity of antigen–antibody recognition, enabling precise qualitative and quantitative analysis of the target analyte through monitoring changes in the electrochemical properties at the electrode surface. By systematically optimizing key experimental parameters, including buffer pH, probe concentration, and antibody loading, we have constructed the first electrochemical immunosensor for detecting DMBA-7,12 in seawater. The sensor achieved a detection limit as low as 0.42 ng/mL. In spiked seawater samples, the recovery rates ranged from 95.53% to 99.44%, with relative standard deviations (RSDs) ≤ 4.6%, demonstrating excellent accuracy and reliability. This innovative approach offers a cost-effective and efficient solution for the in situ rapid monitoring of trace carcinogens in marine environments, potentially advancing the field of marine pollutant detection technologies. Full article

An electrochemical immunosensor for the quantification of prostate-specific antigens (PSAs) using silver nanocrystals (AgNCs) is reported. The silver nanocrystals were synthesized using a conventional citrate reduction protocol. The silver nanocrystals were characterized using scanning electron microscopy (SEM) and field effect scanning electron microscopy (FESEM), X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), Fourier-transform infrared spectroscopy (FTIR), UV-Vis spectroscopy, and small-angle X-ray scattering (SAXS). The proposed immunosensor was fabricated on a glassy carbon electrode (GCE), sequentially, by drop-coating AgNCs, the electro-deposition of EDC-NHS, the immobilization of anti-PSA antibody (Ab), and dropping of bovine serum albumin (BSA) to prevent non-specific binding sites. Each stage of the fabrication process was characterized by cyclic voltammetry (CV). Using square wave voltammetry (SWV), the proposed immunosensor displayed high sensitivity in detecting PSA over a concentration range of 1 to 10 ng/mL with a detection limit of 1.14 ng/mL and R² of 0.99%. The immunosensor was selective in the presence of interfering substances like glucose, urea, L-cysteine, and alpha-methylacyl-CoA racemase (AMACR) and it showed good stability and repeatability. These results compare favourably with some previously reported results on similar or related technologies for PSA detection. Full article

Norovirus, a foodborne pathogen, causes a significant economic and health burden globally. Although detection methods exist, they are expensive and non-field deployable. A flow-based dielectrophoretic biosensor was designed for the detection of foodborne pathogenic viruses and was tested using bacteriophage MS2 as a norovirus surrogate. The flow-based MS2 sensor comprises a concentrator and a detector. The concentrator is an interdigitated electrode array designed to impart dielectrophoretic effects to manipulate viral particles toward the detector in a fluidic channel. The detector is made of a silver electrode conjugated with anti-MS2 IgG to allow for antibody–antigen biorecognition events and is supplied with the electrical current for the purpose of measurement. Serially diluted MS2 suspensions were continuously injected into the fluidic channel at 0.1 mL/min. A cyclic voltammogram indicated that current measurements from single-walled carbon nanotube (SWCNT)-coated electrodes increased compared to uncoated electrodes. Additionally, a drop in the current measurements after antibody immobilization and MS2 capture was observed with the developed electrodes. Antibody immobilization at the biorecognition site provided greater current changes with the antibody-MS2 complexes vs. the assays without antibodies. The electric field applied to the fluidic channel at 10 V_pp and 1 MHz contributed to an increase in current changes in response to MS2 bound on the detector and was dependent on the MS2 concentrations in the sample. The developed biosensor was able to detect MS2 with a sensitivity of 10² PFU/mL within 15 min. Overall, this work demonstrates a proof of concept for a rapid and field-deployable strategy to detect foodborne pathogens. Full article

This study investigated the IgA antibodies targeting bovine serum albumin (BSA) in 27 adult celiac disease (CD) patients adhering to a gluten-free diet (GFD), compared to 123 controls (including individuals with autoimmune disorders, those with gastrointestinal cancers, and healthy donors). Serum samples were evaluated using a multiplex assay based on a microarray comprising 66 immobilized antigens, including autoantigens associated with autoimmune diseases, different albumins, cytokines, and inflammatory markers. Elevated IgA-BSA levels were detected in 22% of CD patients versus 3.25% of controls. IgA-BSA did not cross-react with milk proteins like casein, β-lactoglobulin, and γ-globulin, nor with autoantigens and human albumin, ruling out autoimmunity against self-proteins. The observed cross-reactivity with porcine albumin suggests that antibodies target epitopes shared by bovine and porcine albumin. Increased IgA-BSA levels may interfere with immunoassays performed using BSA as a stabilizer, necessitating protein-free buffers to avoid false results when testing CD patients. Elevated IgA-BSA levels may reflect ongoing gut barrier dysfunction in CD patients on a GFD, allowing dietary proteins like BSA to trigger immune responses. This study identifies a novel immune response in CD patients on a GFD, emphasizing the need for tailored diagnostic approaches (BSA-free assays) and further research into the clinical and dietary implications of IgA-BSA elevation. Full article

Photoelectrochemical (PEC) sensors have emerged as potential analysis techniques in recent years due to PEC’s benefits, which include straightforward operation, quick response times, and basic equipment. In this work, a new PEC sandwich immunoassay was fabricated, which was based on low-toxicity BiOI/BiOCl composites accompanied by enhanced signal detection via AgI-conjugated antibodies (Ab₂-AgI). Specifically, the low-toxicity inorganic semiconductor BiOI/BiOCl composites were first utilized in PEC bioanalysis. Owing to the unique configuration of energy levels between BiOI and BiOCl, the photoelectric response was more excellent than those of BiOI or BiOCl alone. Moreover, the Ab₂-AgI conjugates were utilized as signal amplification components through the specific antibody–antigen immunoreaction. In the presence of target Ag, the immobilized Ab₂-AgI conjugates clearly improve the steric hindrance of the sensing electrode and effectively hinder the transfer of photo-induced holes; meanwhile, AgI NPs can competitively absorb excitation light. A new PEC immunosensing platform for detecting tumor markers at 0 V under visible light excitation was developed, and using carcinoembryonic antigen (CEA) as a model analyte demonstrated an ultra-low detection limit of 4.9 fg·mL⁻¹. Meanwhile, it demonstrated excellent specificity and stability, potentially opening up a novel and promising platform for detecting other critical biomarkers. Full article