Revolutionizing sexed sorting sperm using scFv antibodies combined with microbeads for porcine sexed semenThongkham, Hongsibsong, Mekchay
et alInt J Biol Macromol (2025) 308 (Pt 2), 142477
Abstract: This study aimed to evaluate porcine sperm sex selection using scFv antibodies combined with magnetic-activated cell sorting (MACS) to optimize X/Y sperm separation while maintaining essential sperm quality parameters, including viability, motility, membrane integrity, and apoptosis, with the goal of developing a novel method for swine sexing technology. The H4L4 scFv antibody, at a concentration of 1 mg/mL, effectively bound to PLA microbeads (HL-beads), achieving separation efficiencies of 75.4 ± 2.30 % for X-sperm and 78.6 ± 1.94 % for Y-sperm. FTIR confirmed the binding of H4L4 scFv to microbead carboxylic acid groups, and SEM verified sperm binding. In quality assessments, the X-enriched fraction (XF) presented a sperm quality comparable to that of conventional semen (CONV) and the negative control (NC). However, the Y-enriched fraction (YF) was lower in quality than the other groups were on Day 3 of storage. Apoptosis analysis revealed no significant difference in the number of necrotic cells among the XF, CONV, and NC groups. The XF group achieved a 76.1 % enriched X-sperm ratio, whereas the YF group achieved a 78.4 % enriched Y-sperm ratio. This method enhances the X-sperm proportion in the XF group with an acceptable quality for farm applications. These results demonstrate the potential of HL-beads for porcine sperm sexing, offering a promising approach for improving sex ratios in swine production.Copyright © 2025 Elsevier B.V. All rights reserved.
Visual Counting of Influenza A Viruses with Magnetic T4 Phage SPR ProbeHe, Liu, Zhou
et alACS Sens (2025)
Abstract: Influenza A virus (IAV) represents a considerable threat to both animal and human health, while current detection methods encounter challenges related to the spectrum, rapidity, and sensitivity of viral identification. Herein, we describe the development of a magnetic T4 phage surface plasmon resonance probe for universal, rapid, highly sensitive, and visually detectable IAV detection under dark field microscopy (DFM). Briefly, we initially fused the Soc protein of the T4 phage with a single-chain variable fragment (scFv) antibody that exhibits broad-spectrum affinity toward the hemagglutinins of group 1 and group 2 influenza viruses, resulting in the generation of the recombinant Soc-scFv protein. Additionally, we generated another recombinant protein called AviTag-Hoc by fusing the Hoc capsid protein of T4 phage with biotin receptor peptides (AviTag). These two recombinant proteins were assembled on the head region of the T4 phage lacking both Soc and Hoc proteins. Subsequently, the resulting assembly was covalently modified with biotin using biotin-protein ligase, enabling conjugation with streptavidin-modified magnetic nanoparticles (SA@MNPs) to generate the magnetic T4 phage probe (T4@scFv@MNPs). Binding experiments demonstrated that this magnetic phage probe specifically binds to a wide range of IAVs of group 1 and group 2. Furthermore, in the presence of influenza viruses, the magnetic T4 phage probe and antibodies functionalized chip can form a sandwich complex that appears as a distinct bright golden yellow fluorescence spot visible to the naked eye under DFM. The number of viruses in samples can be automatically counted using artificial intelligence-assisted software. Assay results from both pure and real virus samples show that our magnetic phage-based DFM strategy is highly time efficient, taking approximately 30 min to complete. The method also showed excellent virus binding efficiency (>85%) in both high and low concentration samples and an extremely low detection limit (1 PFU/μL).
SARS-CoV-2 nucleocapsid detection using a recombinant phage display-isolated single-chain fragment variableSalarifar, Rasaee
J Immunoassay Immunochem (2025)
Abstract: Diagnosis is an important factor in controlling disease. Single-chain fragment variables (scFvs) can be used for diagnosis; however, due to their immobilization issues, their application has been limited. Herein, we isolated a SARS-CoV-2 nucleocapsid phosphoprotein (NP)-specific scFv and propose it as a diagnostic tool in the scFv-displaying phage format to overcome the immobilization issue.Spleen from NP-immunized BALB/c mice was isolated, total RNA was extracted, and cDNA was synthesized. An scFv library was constructed, using the splicing by overlap extension (SOE) PCR technique, which was cloned into the pCANTAB5E phagemid. The phage library was panned against the NP antigen, and the output phages with the highest binding capability were screened for the most qualified scFv, which was later assessed in terms of sensitivity and specificity.The scFv-displaying phage library was panned against the recombinant NP in three rounds and 40 randomly selected colonies from the third round's outputs were screened. Alongside several clones, clone #31 was chosen as the most qualified scFv, which later exhibited favorable sensitivity and specificity against NP in further ELISA-based experiments.Clone #31 could be utilized to develop diagnostic tools and therapeutics against SARS-CoV-2.
Extracellular vesicles engineered to directly target encephalomyocarditis virus ameliorates multi-organ viremia in a lethal infection modelYue, Yang, Idris
et alVet Microbiol (2025) 304, 110448
Abstract: The outbreak and prevalence of encephalomyocarditis virus (EMCV) causes significant global mortality and morbidity to the pig industry. Though the current and most effective approach to control EMCV outbreak are done through inactivated vaccines, we have yet to see an effective antiviral agent that directly targets EMCV. Here, we present a molecular therapy consisting of extracellular vesicles (EVs) decorated with EMCV-specific single-chain variable fragment (scFv), engineered on the external loop of the EVS transmembrane domain CD63. These EMCV-scFv enriched EVs directly neutralizes infectious EMCV, thereby inhibiting viral proliferation in vitro. Importantly, we demonstrate that systemic delivery of these EVs reduced multi-organ viremia and clinically rescued EMCV infected mice in vivo. This is the first demonstration of the use of direct acting molecularly engineered EVs to target EMCV infection.Copyright © 2025 Elsevier B.V. All rights reserved.
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