Search Results (107)

Rapid blood type detection in point-of-care testing (POCT) scenarios is crucial for various clinical treatments. In this study, we present a sensitive, cost-effective, and straightforward biosensing approach for visual blood typing that notably simplifies the procedure by eliminating any need for blood sample pretreatment. Our technique achieves this by directly trapping and accumulating red blood cell (RBC) clusters within a photolithography-based microfluidic chip, thereby bypassing complex preprocessing. By employing an antigen–antibody assay involving isoagglutinins A, B, and/or D on the RBC surface and their corresponding antibodies, we effectively determine blood types. When antibodies are present, the corresponding RBCs bind to the antibody-conjugated RBC clusters, which are subsequently trapped within the microfluidic accumulation chip, resulting in the formation of a visible bar. The blood group can then be readily identified by observing this visual bar with the naked eye or under microscopy. Notably, we integrate two continuous mixing units (Z and S) at the entrance of the biochip to improve mixing efficiency and accelerate the antigen–antibody interaction. This method demonstrates high selectivity, accuracy, and stability across various clinical blood samples. Moreover, the sensor operates with minimal sample volume (as low as 10 μL) and delivers results within 5 min. The fabrication cost of the PDMS-based biochip is approximately $0.2 per chip, and the limit of detection (LOD) is determined to be 3 × 10⁶ cells/mL, indicating excellent sensitivity and affordability for practical use. Overall, this biochip provides a fast, low-cost, and reliable solution for emergency blood typing, particularly in resource-limited settings. Full article

Porcine circovirus type 3 (PCV3), initially identified in the United States in 2016, is associated with multisystemic inflammation, myocarditis, reproductive failure in sows, and growth retardation in piglets, posing a significant economic threat to the swine industry. In this study, prokaryotic-expressed recombinant PCV3 Cap protein was used to immunize mice and rabbits. A monoclonal antibody (mAb 4G1) was generated through hybridoma technology, targeting a novel linear epitope (³⁷DYYDKK⁴²) within the first β-sheet of the Cap structure. This epitope exhibits high conservation (99.35%, 1239/1247) based on sequence alignment analysis, and residues 39 and 42 are critical residues affecting mAb binding. Subsequently, using rabbit polyclonal antibody (pAb) as the capture antibody and mAb 4G1 as the detection antibody, a double antibody sandwich ELISA (DAS-ELISA) method was developed. The assay demonstrates a cut-off value of 0.271, a detection limit for positive pig serum is 1:800, and shows no cross-reactivity with other swine pathogens. Intra- and inter-assay coefficients of variation were <10%, with a linear detection range for Cap protein down to 3.4 ng/mL. The coincidence rate between the DAS-ELISA and qPCR was 93.33% (70/75) for PCV3 detection in serum, with a kappa value of 0.837. This study establishes a simple, sensitive, and operationally efficient DAS-ELISA and provides a reference for monitoring PCV3 infection in swine herds. Full article

Background: The concentration of antigen-specific antibodies in serum is usually measured in international units/mL. Therefore, the actual concentration of virus-specific antibodies in sera is unknown. Objectives: The aim of the study was to determine conversion factors for concentrations of IgG against hepatitis B surface antigen (HBs), SARS-CoV-2 receptor binding domain (RBD) and nucleoprotein (NP) as well as tetanus toxin (Ttx) in serum and to compare antigen-specific IgG concentrations in serum samples. Methods: Absorption equivalence ELISAs were used to determine conversion factors for international units (IU) for anti-HBs, anti-SARS-CoV-2-RBD and NP and for anti-Ttx immunoglobulin G. The antigen-specific IgG concentrations in serum samples were then measured in units/mL and the ratio of IgG concentrations in the sera was determined using the conversion factors. Results: One IU of anti-HBs IgG corresponded to 24.4 BAU of anti-CoV-2 RBD IgG, 6.87 BAU of anti-CoV-2 NP and 14 mIU of anti-Ttx IgG. One BAU anti-SARS-CoV-2 NP-specific IgG is equivalent to 3.5 BAU SARS-CoV-2 RBD-specific IgG. Conversion of international units showed that median serum anti-Ttx-IgG concentrations were 50 times higher and anti-CoV-2-RBD-IgG concentrations were 390 times higher than median anti-HBs-IgG concentrations. In addition, after SARS-CoV-2 infection, the concentration of NP-specific IgG in serum was generally higher than that of RBD-specific IgG. Conclusions: The study provides conversion factors for serum concentrations of IgG against HBs, SARS-CoV-2 RBD and NP, as well as Ttx-IgG. This offers new insights into serum IgG concentrations and allows conclusions to be drawn about plasma cell pools. Full article

Background: Gastric cancer (GC) is a malignant tumor of the gastrointestinal tract, characterized by high mortality rates and responsible for about one million new cases each year globally. Surgery is the main treatment, but achieving radical resection remains a relevant intraoperative challenge. Fluorescence-guided surgery offers clinicians greater capabilities for real-time detection of tumor nodules and visualization of tumor margins. In this field, the main challenge remains the development of fluorescent dyes that can selectively target tumor tissues. Methods: we examined the expression of the most suitable GC markers, including carcinoembryonic antigen cell adhesion molecule-5 (CEACAM5) and Claudin-4 (CLDN4), in GC cell lines. To further evaluate their expression, we performed immunohistochemistry (IHC) on tumor and healthy tissue samples from 30 GC patients who underwent partial gastrectomy at the Digestive System Surgery Unit, AOU Careggi, Florence. Additionally, we validated anti-CEACAM5 expression on patient-derived organoids. Furthermore, we developed a fluorescent molecule targeting CEACAM5 on the surface of GC cells and assessed its binding properties on patient tissue slices and fragments. Results: in this work, we first identified CEACAM5 as an optimal GC biomarker, and then we developed a fluorescent antibody specific for CEACAM5. We also evaluated its binding specificity for GC cell lines and patient-derived tumor tissue, achieving an optimal ability to discriminate tumor tissue from healthy mucosa. Conclusions: Overall, our results support the development of our fluorescent antibody as a promising tumor-specific imaging agent that, after further in vivo validation, could improve the accuracy of complete tumor resection. Full article

Bispecific antibodies (BsAbs) have shown potential in cancer treatment and have become a rapidly growing field in cancer immunotherapy. Unlike monoclonal antibodies with two identical binding sites, BsAbs simultaneously bind two distinct epitopes on the same or different antigens, allowing for a range of mechanisms of action, including engaging immune cells to kill cancer cells and blocking signaling pathways. Despite regulatory approvals for hematological malignancies in the last decade, their clinical success in solid malignancies has been lacking until recently. There are currently five BsAbs approved by the FDA in the United States for solid tumors—amivantamab, tarlatamab, tebentafusp, zanidatamab and zenocutuzumab—and two BsAbs approved in China—cadonilimab and ivonescimab. Currently, several BsAbs are under clinical development for solid tumors, but are mostly in early phase I and II trials. This review provides an overview of the basic mechanism of action of BsAbs, current FDA-approved BsAbs, and current BsAbs under clinical development, their challenges in clinical use, the management of toxicities, and future directions. Full article

SPR biosensors operate on the principle of evanescent wave propagation at metal–dielectric interfaces in total internal reflection conditions, with consequent photonic energy attenuation. This plasmonic excitation occurs in specific conditions of incident light wavelength, angle, and the dielectric refractive index. This principle has been the basis for SPR-based biosensor setups wherein mass/concentration-induced changes in the refractive indices of dielectric media reflect as plasmonic resonance condition changes quantitatively reported as arbitrary response units. SPR biosensors operating on this conceptual framework have been designed to study biomolecular interactions with real-time readout and in label-free setups, providing key kinetic characterization that has been valuable in various applications. SPR biosensors often feature antibodies as target affinity probes. Notably, the operational challenges encountered with antibodies have led to the development of aptamers—oligonucleotide biomolecules rationally designed to adopt tertiary structures, enabling high affinity and specific binding to a wide range of targets. Aptamers have been extensively adopted in SPR biosensor setups with promising clinical and industrial prospects. In this paper, we explore the growing literature on SPR setups featuring aptamers, specifically providing expert commentary on the current state and future implications of these SPR aptasensors for drug discovery as well as disease diagnosis and monitoring. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Many countries have established regulatory frameworks to monitor and mitigate Salmonella contamination in poultry products. The ability to rapidly quantify Salmonella is critical for poultry processors to facilitate early detection, implement corrective measures, and enhance product safety. This study aimed to develop an Immunomagnetic Chemiluminescent Assay (IMCA) for the quantification of Salmonella Typhimurium in ground chicken. Immunomagnetic microbeads functionalized with monoclonal antibodies were employed to selectively capture and concentrate Salmonella from ground chicken samples. A biotin-labeled monoclonal antibody, followed by an avidin-horseradish peroxidase conjugate, was used to bind the captured bacteria and initiate a chemiluminescent reaction catalyzed by peroxidase. Light emission was quantified in relative light units (RLUs) using two luminometers. Ground chicken samples were inoculated with a four-strain S. Typhimurium cocktail ranging from 0 to 3.5 Log CFU/g. Bacterial concentrations were confirmed using the Most Probable Number (MPN) method. Samples underwent enrichment in Buffered Peptone Water (BPW) supplemented with BAX MP Supplement at 42 °C for 6 and 8 h before analysis via IMCA. A linear regression analysis demonstrated that the optimal quantification of Salmonella was achieved at the 8 h enrichment period (R² ≥ 0.89), as compared to the 6 h enrichment. The limit of quantification (LOQ) was determined to be below 1 CFU/g. A strong positive correlation (R² ≥ 0.88) was observed between IMCA and MPN results, indicating methodological consistency. These findings support the application of IMCA as a rapid and reliable method for the detection and quantification of Salmonella in ground chicken. Full article

Background: The global SARS-CoV-2 pandemic revealed stark variability in clinical outcomes across populations, underscoring the need for region-tailored vaccination strategies. To inform standardised global immunisation efforts, this study compared longitudinal binding antibody responses and neutralisation capacities in mild COVID-19 cases from Uganda and the United Kingdom (UK). Methods: IgG responses to spike (S) and nucleocapsid (N) proteins, along with IgM responses to S and receptor-binding domain (RBD) proteins, were assessed in 29 Ugandan and 14 UK participants over 84 and 82 days, respectively. Antibody levels were quantified using a validated enzyme-linked immunosorbent assay (ELISA), alongside pseudovirus neutralisation assays targeting the D614G variant. Results: Ugandan participants exhibited higher early IgG and IgM levels, particularly against spike and RBD, with a rapid onset of responses that waned quickly. UK participants showed a slower but sustained increase in IgG and IgM levels. Neutralisation titres revealed elevated responses in 16.4% of Ugandan participants (>2000) compared to 4.5% of UK participants, suggesting a greater sensitivity to viral neutralisation. Conversely, 31.8% of UK participants exhibited low titres (<25) compared to 14.8% of Ugandan participants, indicating differences in resistance mechanisms. Neutralisation correlated strongly with spike and receptor-binding domain IgG in the UK cohort but showed weaker correlations in Ugandan participants. Conclusions: These findings highlight distinct population-level immune responses, suggesting that geographic factors shaped the quality and durability of SARS-CoV-2 immunity. Tailored vaccination strategies are essential to optimise immunity across diverse populations and improve global epidemic preparedness. Full article

Blood products, including apheresis platelets and plasma, are essential for medical use but pose risks of bacterial contamination and viral transmission. Platelets are prone to bacterial growth due to their storage conditions, while plasma requires extensive screening. This study explores rapid irradiation as an innovative pathogen reduction method. A clinical linear accelerator was configured to deliver ultra-high dose rate (6 kGy/min) irradiation to platelet and plasma components. Platelets spiked with Escherichia coli (E. coli; 10⁵ colony-forming units) were irradiated at 0.1–20 kGy, followed by bacterial growth and platelet count analysis. COVID-19 convalescent plasma (CCP) was irradiated at 25 kGy, and receptor-binding domain (RBD)-specific immunoglobulins (Ig) were assessed. Irradiation at 1 kGy reduced E. coli growth by 2.7-log without significant platelet loss, while 5 kGy achieved complete suppression. The estimated 6-log bacterial reduction dose (2.3 kGy) led to a 31% platelet count drop. Administering a 25 kGy virus-sterilizing dose to CCP resulted in a 9.2% decrease in RBD-specific IgG binding. This study demonstrates the proof-of-concept for rapid blood sterilization using a clinical linear accelerator. The method maintains platelet counts and CCP antibody binding at sterilizing doses, highlighting its potential as a point-of-care blood product sterilization solution. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Background/Objectives: The Ca²⁺-activated K⁺ channel K_Ca3.1 is not only involved in physiological processes such as immune reactions and control of vascular tone, but is highly expressed in various tumor entities. Thus, imaging of K_Ca3.1 channels comes into focus for the localization of high channel density, i.e., for tumor diagnosis. In particular, the physicochemical properties of the fluorescent probes should be improved compared to existing probes. Methods: The small molecule inhibitor of the K_Ca3.1 channel, senicapoc, was used as a warhead and was coupled with different fluorescent dyes. After synthesis of the novel probes, their physicochemical properties (lipophilicity, photophysical properties) and their ability to image K_Ca3.1 channels in A549-3R lung tumor cells were determined. Results: In order to increase the polarity and quantum yield of reported fluorescent probes, three strategies were followed: (1) An F-atom at the B-atom of bodipy-labeled senicapoc derivatives 9a, 9b, and 15a was replaced by a OCH₃ moiety, which decreased the logP value by one log-unit. (2) The p-phenylene moiety of the linker was replaced by an aliphatic tetramethylene linker decreasing the lipophilicity by 0.3–0.5 log-units. (3) Instead of bodipy dyes, fluorescein was coupled with the senicapoc warhead resulting in very polar probes 21a and 21b with low logP values of 1.5 and 1.3, respectively. Introduction of an ethyl moiety at the bodipy core increased the quantum yield, which resulted in the best punctate staining pattern of fixed and living A549-3R lung tumor cells with the ethylbodipy-labeled senicapoc derivative 10b. The specificity was shown by various control experiments. Co-staining with 10b and an antibody did not result in overlapping signals. Conclusions: The well-balanced lipophilicity and fluorescent quantum yield render the ethylbodipy-labeled senicapoc derivative 10b a very good probe to image selectively K_Ca3.1 ion channels in fixed and living tumor cells. It was hypothesized that the antibody binds selectively at the closed channel (58.5%), whereas the senicapoc–bodipy conjugate 10b binds selectively at the open channel (41.5%). The ratio 58.5:41.5 reflects the ratio of the ion channel in closed and open conformations. Full article

Background: Anthrax is a serious disease caused by Bacillus anthracis (B. anthracis) with a very high mortality when the spores of B. anthracis are inhaled (inhalational anthrax). Aerosolized B. anthracis spores can be used as a deadly bioweapon. Vaccination against anthrax is the only effective preventive measure and, hence, the anthrax vaccine was administered to United States (and other) troops during the 1990–91 Gulf War. However, the anthrax vaccine is not harmless, and the anthrax vaccination has been linked to the occurrence and severity of Gulf War Illness (GWI), a debilitating Chronic Multisymptom Illness (CMI). We hypothesized that this is partly due to the combination of two factors, namely (a) the cytotoxicity of the antigen (anthrax Protective Antigen, PA) contained in the vaccine, and (b) the Human Leukocyte Antigen (HLA) genotype of susceptible vaccinees, reducing their ability to make antibodies against the cytotoxic PA. Method: Here, we tested this hypothesis by determining the association between severity of GWI symptoms in 458 GW veterans and the overall strength of the binding affinity of the PA epitopes to the specific six Human Leukocyte Antigen (HLA) Class II alleles carried by each individual (two of each of the HLA-II genes: DPB1, DQB1, DRB1), responsible for initiating the process of antibody production in otherwise immunocompetent individuals, estimated in silico. Results: We found that the severity of GWI symptomatology was negatively and significantly correlated with the strength of the predicted binding affinity of PA peptides to HLA-II molecules ($r=-0.356, p<0.001)$; the stronger the overall binding affinity, the weaker the symptoms. Since the binding of a peptide to an HLA-II molecule is the first and necessary step in initiating the production of antibodies, the findings above support our hypothesis that the severity of GWI symptomatology is partly due to a lack of HLA-II protection. Conclusions: Reduced HLA protection against the toxic anthrax vaccine may underlie GWI. Full article

Background/Objectives: Since the World Health Organization declared COVID-19 a pandemic in March 2020, the virus has caused multiple waves of infection globally. Arizona State University (ASU), the largest four-year university in the United States, offers a uniquely diverse setting for assessing immunity within a large community. This study aimed to test our hypothesis that an increased number of exposures to SARS-CoV-2 RBD through vaccination/boosters/infection will increase SARS-CoV-2 antibody seroprevalence by increasing the longevity of anti-RBD and anti-RBD-neutralizing antibodies. Methods: A serosurvey was conducted at ASU from 30 January to 3 February 2023. Participants completed questionnaires about demographics, respiratory infection history, symptoms, and COVID-19 vaccination status. Blood samples were analyzed for anti-receptor binding domain (RBD) IgG and anti-nucleocapsid (NC) antibodies, offering a comprehensive view of immunity from both natural infection and vaccination. Results: The seroprevalence of anti-RBD IgG antibodies was 96.2% (95% CI: 94.8–97.2%), and 64.9% (95% CI: 61.9–67.8%) of participants had anti-NC antibodies. Anti-RBD IgG levels correlated strongly with neutralizing antibody levels, and participants who received more vaccine doses showed higher levels of both anti-RBD IgG and neutralizing antibodies. Increasing the number of exposures through vaccination and/or infection resulted in higher and long-lasting antibodies. Conclusions: The high levels of anti-RBD antibodies observed reflect substantial vaccine uptake within this population. Ongoing vaccination efforts, especially as new variants emerge, are essential to maintaining protective antibody levels. These findings underscore the importance of sustained public health initiatives to support broad-based immunity and protection. Full article

Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration. Full article