Search Results (317)

Circulating tumor cells (CTCs) are valuable liquid-biopsy biomarkers, yet their extreme rarity makes high-purity, high-throughput enrichment challenging. In spiral inertial microfluidics, high cell loading induces long-range hydrodynamic interactions that broaden the focused blood-cell stream; consequently, a subpopulation completes the ~0.5 and ~1.0 Dean-cycle migrations with a phase delay, compressing the CTC–blood cell gap and degrading purity. Here we propose a Dean-cycle phase-regulated double-spiral design informed by this phenomenon. This design aims to mitigate the stream-broadening effect by boosting the Dean number during the first half-cycle to promote synchronized blood-cell migration and shifting the CTC equilibrium position near one full cycle to further widen the CTC–blood cell separation. We implement this strategy in a second-generation double-spiral microfluidic chip (SDMC) and scale it to a four-channel parallel array (ASDMC). Under optimized conditions, ASDMC processes diluted whole blood (hematocrit = 4%) without the need for red blood cell (RBC) lysis or antibody labeling, achieving a sample throughput of 1200 μL·min⁻¹. Specifically, it exhibits a mean recovery rate of 98.8% across three spiked tumor cell lines (MCF-7, PC-9, and Mahlavu) and a mean white blood cell (WBC) depletion efficiency of 93.3%. In a pilot clinical testing of 20 patients (NSCLC and HCC), enriched fractions enabled immunofluorescence identification of CK⁺CD45⁻DAPI⁺ CTCs, with an exploratory trend of increasing CTC counts with advanced disease stage (4–34 cells·mL⁻¹). These results describe a scalable, label-free platform, and the observed purification performance aligns with our proposed mechanism: Dean-cycle phase regulation to mitigate blood-cell migration lag. Our findings support further technical validation and clinical assessment in larger cohorts. Full article

Gangliosides containing O-acetyl-sialic acid or a sialic acid forming a lactone ring, classified as alkali-labile gangliosides (ALGs), were the focus of extensive research until the late 1990s. Their identification and isolation for structural characterization require strict avoidance of alkaline treatments, a common step in standard ganglioside purification protocols used to eliminate glycerophospholipid contamination. After 2000, scientific interest in alkali-labile gangliosides declined significantly, resulting in a dearth of new information and few updates on their biological significance. However, in recent years, new insights have emerged regarding the potential of anti-acetylated ganglioside antibodies for specific tumor treatments and how ganglioside structures can modulate the activity of membrane receptors through specific interactions. Further knowledge of alkali-labile gangliosides could be very useful for better understanding their significance in both normal and tumor cells. In this manuscript, we intend to revisit the existing knowledge on alkali-labile gangliosides and form hypotheses regarding their possible roles in cells. Full article

Native Staphylococcus aureus protein A exhibits strong affinity to the Fc and VH regions of human IgG1, IgG2, and IgG4, making it a valuable tool for monoclonal antibody (mAb) purification. However, its low stability under conditions such as increased alkaline concentrations during cleaning-in-place (CIP), protease exposure, thermal stress, and shear forces limits its usability for large-scale industrial applications. Recombinant Protein A (rProtein A) can be modified to improve key properties, including alkaline stability. In this study, we present targeted modifications to the C domain of native Protein A, evaluating multimeric variants for structural and functional improvements. The selected variant demonstrated extremely high stability after 60 h incubation at 0.5 M NaOH by maintaining more than >90% initial dynamic binding capacity (DBC) and up to 80% DBC after 40 h in 1.0 M NaOH. However, the most impressive result obtained was the stability of the ligand in 1.5 M NaOH, retaining 80% DBC after 22 h and 60% DBC after 40 h. To the best of our knowledge, this is the first time that such high alkaline stability is reported for a rProtein A. To assess its application in monoclonal antibody purification, the optimized rProtein A ligand was immobilized on agarose resin and tested in chromatography processes. The resulting chromatography resin functionalized with the CmZmb ligand (now commercialized by Sunresin, China under the name of rProtein A Seplife Suno) exhibited a high dynamic binding capacity of 70 mg/mL, minimal ligand leaching under operational conditions (~15 ppm), and extended lifecycle performance (88% DBC retained after 120 purification cycles with 0.5 M NaOH CIP), making it well-suited for industrial-scale applications. Full article

Background: Coxsackievirus B2 (CVB2) causes a range of diseases, including hand, foot, and mouth disease; myocarditis; acute flaccid paralysis; meningitis; and encephalitis. However, no specific antiviral drugs or vaccines are currently available for CVB2. Methods: We used plaque purification, virus titre determination, and serial passaging to screen and identify an inactivated CVB2 vaccine candidate strain, KM31-C05, which exhibited high viral titres and good genetic stability. Comprehensive biological characterization of this candidate strain was performed, including phylogenetic analysis, virulence assessment in BALB/c mice, one-step growth curve analysis, optimization of the multiplicity of infection, as well as determination of viral load, pathological evaluation, and immunohistochemical analysis in tissues of BALB/c suckling mice post-challenge. An experimental inactivated vaccine was prepared using KM31-C05 to evaluate its immunogenicity and protective efficacy. Results: The viral titres of KM31-C05 reached 10⁸ CCID50/mL. After 20 serial passages, only three amino acid mutations were identified (VP3-G165V, VP1-N84K, and VP1-D129N). Although the two VP1 mutations were located in surface-exposed loops, the strain maintained high neutralizing titres across passages, indicating good genetic stability. However, whether these sites affect virulence and replication requires further investigation. Phylogenetic analysis revealed that this strain belonged to genotype C, which is consistent with the strains circulating in mainland China in recent years. The experimental inactivated vaccine prepared from KM31-C05 induced effective neutralizing antibodies (1:128–1:256) in BALB/c mice and provided complete protection to suckling mice against lethal challenge with this CVB2 strain in maternal antibody protection experiments. Conclusions: KM31-C05 demonstrates potential as a CVB2 vaccine candidate in China and provides a theoretical basis for the development of a CVB2 vaccine. Full article

Background/Objectives: Multidrug-resistant (MDR) Acinetobacter baumannii (Ab) has emerged as a significant bacterial pathogen responsible for nosocomial infections. The most common clinical manifestations of Ab infection include ventilator-associated pneumonia and catheter-related bloodstream/urinary infections. Given the extensive MDR phenotype of Ab, preventive vaccination strategies are crucial for protecting susceptible populations. Methods: We utilized immunoinformatics to identify candidate peptides containing both putative B- and T-cell epitopes from proteins associated with Ab pathogenesis. Subsequently, we designed novel Acinetobacter Multi-Epitope Vaccines (AMEVs), each comprising an Ab thioredoxin A (TrxA) leader protein, five to seven of the identified peptide antigens, and a C-terminal His(6x)-tag to facilitate protein purification. Results: Subcutaneous vaccination of C57BL/6 mice with AMEV1 or AMEV2, formulated with TiterMax adjuvant, conferred 60% and 80% protection, respectively, against intraperitoneal Ab challenge. AMEV vaccination induced a robust antibody response to each corresponding whole protein and most of its component peptides. We then constructed an improved vaccine, AMEV5, which included the Ab TrxA protein and seven confirmed B-cell epitope peptides. Subcutaneous immunization of BALB/c mice (n = 10 per group) with rAMEV5 emulsified in Adda03 adjuvant activated antigen-specific IL-5-secreting T cells and antibody-producing B cells. Evaluation of vaccine efficacy demonstrated that AMEV2- and AMEV5-immunized mice were protected from a lethal intraperitoneal Ab challenge, with survival rates of 70% and 90%, respectively. Conclusions: These study results provide insights into the application of reverse vaccinology to combat the rise of MDR Acinetobacter infection. Full article

Circular RNA (circRNA) has emerged as an alternative RNA modality for vaccine development due to its covalently closed structure and enhanced molecular stability compared with linear messenger RNA (mRNA). Following the clinical success of mRNA vaccines, circRNA-based platforms have gained attention in both prophylactic and cancer immunization. Unlike linear mRNA, circRNA relies on cap-independent translation and is commonly produced through in vitro transcription coupled with ribozyme-mediated self-circularization. In prophylactic vaccination, circRNA vaccines have demonstrated sustained antigen availability, robust humoral and cellular immune responses, and flexibility in multivalent designs and targeted delivery strategies that support germinal center reactions and neutralizing antibody generation. In cancer vaccines, circRNA has been applied to tumor-associated antigens, neoantigens, and non-canonical peptides, with a primary focus on inducing potent antigen-specific CD8⁺ T cell immunity and enabling combination immunotherapy approaches. This review summarizes recent applications of circRNA-based vaccines in prophylactic and cancer settings, emphasizing in vitro transcription-compatible self-circularization strategies and discussing how methodological choices in RNA design, translation control, purification, and delivery shape immunological outcomes. Full article

Monoclonal antibodies represent one of the fastest-growing sectors of the biopharmaceutical industry. Their high therapeutic efficacy and reduced incidence of adverse effects compared to conventional therapies have led to an increasing demand for these products. The costliest stages of monoclonal antibody production are the separation and purification processes, which underscores the need for continuous development and optimization of applied methodologies. Active pharmaceutical ingredients must exhibit high purity and preserved biological activity in order to meet stringent regulatory requirements. Macromolecules such as monoclonal antibodies possess complex conformational structures that significantly influence their stability. The application of multi-step chromatographic processes during purification from cell culture harvests may induce structural alterations, including protein unfolding and aggregation, ultimately resulting in decreased product quality and therapeutic effectiveness. Such structural changes may also increase immunogenicity risk and reduce product shelf life, posing additional challenges for downstream processing. In addition, chromatographic media create microenvironments that differ markedly from bulk solution (e.g., high local protein concentration, confined pore spaces and heterogeneous surface chemistry). These effects can promote either self-association driven by colloidal interactions or partial unfolding followed by irreversible aggregation, depending on the unit operation and operating window. Practical mitigation is therefore rarely achieved by a single lever; instead, it requires an integrated view of resin selection, buffer composition (pH, salt type and ionic strength, and stabilizing additives), residence time and temperature, as well as an analytics strategy that combines orthogonal aggregation assays with structural probes. This work discusses the phenomena of unfolding and aggregation of therapeutic proteins, with particular emphasis on monoclonal antibodies occurring during chromatographic purification. Furthermore, key analytical methods, characterization techniques, and mitigation strategies aimed at improving product quality and reducing manufacturing costs are reviewed. Full article

Caprylic acid (CA) fractionation of serum is a simple and cost-effective method of producing high-quality immunoglobulins. While standardised procedures exist for CA purification of IgG for various animals, no published protocol exists for baboon IgG. This study aimed to optimise an efficient protocol for purifying IgG from baboon serum using CA through a stepwise one-factor-at-a-time (OFAT) approach. The effects of serum pH, CA concentration, stirring time and intensity, dialysis buffer, and lyophilisation were evaluated based on the protein content, with SDS-PAGE profiles and albumin–globulin ratios distinguishing IgG from residual albumin. Serum at pH 5.0 with 7% CA (v/v) produced the highest yield, minimising albumin while maximising IgG content. Lower pH (4.0–4.5) and CA (5–6%) reduced protein content, while a higher pH (5.5–6.0) and CA (8–15%) increased protein, but with elevated albumin and contaminants. Stirring serum vigorously at 1200 rpm for 60 min provided effective precipitation of non-IgG proteins. Lower intensities and shorter times resulted in higher albumin and residual proteins, while excessive stirring caused protein denaturation. Dialysis buffer composition had little impact, while lyophilisation significantly enhanced IgG concentration. The optimal protocol involved serum at pH 5.0, 7% CA (v/v), vigorous stirring (1200 rpm) for 60 min, and dialysis against sodium phosphate buffer (pH 7.4) followed by lyophilisation. The resulting IgG enrichment and purity were comparable to commercial-grade products. This study thus established optimal conditions for the purification of baboon IgG with CA, which could be used to support research in this animal model of immunology. Full article

Nanobodies (VHHs or single-domain antibodies) are powerful affinity reagents, but their routine use is often limited by production constraints and by the lack of a conserved Fc region for secondary detection. We describe a simplified strategy in which functional GST–nanobody fusion proteins are expressed directly in the cytoplasm of Escherichia coli Origami^TM 2 (DE3), a strain that supports disulfide bond formation through trxB/gor mutations. Using well-characterized nanobodies against GFP (Lag2) and mCherry (C11), we designed N-terminal GST fusions and confirmed by AlphaFold3-based modeling that both constructs preserve the GST fold and the VHH (Variable domain of the Heavy-chain antibody of Heavy-chain-only antibodies) β-sandwich with defined CDR loops and a predicted intradomain disulfide bond. Following IPTG induction and purification by glutathione affinity and size-exclusion chromatography, we obtained soluble GST-nb-GFP and GST-nb-mCherry at ~8–12 mg/L. Isothermal titration calorimetry showed nanomolar binding to their antigens (K_d ~123 nM for GFP and ~199 nM for mCherry). Consistent with conformational epitope recognition, GST-nanobodies were reactive in native-state dot blots but not in denaturing Western blots under the conditions tested. The GST moiety enabled indirect immunofluorescence via anti-GST antibodies, yielding specific labeling of GFP- or mCherry-tagged TGN38 in HeLa and H4 cells. Finally, we demonstrate “GST-nanobody pulldown” as a robust method for affinity capture from cell lysates. Together, this platform provides a low-cost, versatile route to functional nanobody reagents without requiring tag removal, and complements other nanobody designs (e.g., VHH-Fc fusions) in an application-dependent manner. Full article

Background: Tumor-lysate vaccines can capture tumor heterogeneity; however, their effectiveness may be reduced by antigen instability and short antigen presentation. Here, we aimed to improve antigen protection and prolong presentation by using a slow-degrading polymeric nanocarrier and an approved adjuvant. Methods: We encapsulated breast cancer cell lysates (MCF-7 and MDA-MB-231) in poly(ε-caprolactone) (PCL) nanoparticles using a double-emulsion (w/o/w) method and co-administered them with alum. We then characterized particle size, PDI, zeta potential, morphology, and in vitro release. Next, we evaluated nitric oxide (NO), TNF-α/IL-10 responses, and cytocompatibility in J774 macrophages. Finally, we quantified serum antibody titers in Balb/c mice after six biweekly immunizations, generated hybridomas, purified IgG, and tested antibody-mediated cytotoxicity alone and together with doxorubicin. Results: PCL nanoparticles were ~220–255 nm (PDI 0.10–0.19; ζ −2 to −3 mV) and released ~90–95% of encapsulated lysate by 800 h (~33 days). Encapsulated lysate (40 μg/mL) modestly increased NO versus control and increased further with alum (p < 0.05). TNF-α increased 7.4–9.72-fold, whereas IL-10 rose 2.82–3.11-fold. Importantly, encapsulated antigen + alum produced the highest ELISA responses after the sixth dose (6.36-fold for MCF-7 and 7.00-fold for MDA-MB-231 versus control; p < 0.05). Hybridoma-derived antibody signals increased through day 42, and Protein G purification yielded up to ~395 μg and ~318 μg IgG. Purified antibodies reduced cell viability, and viability decreased further when antibodies were combined with doxorubicin (to ~31.6% in MCF-7 and ~40.3% in MDA-MB-231). Conclusions: Overall, sustained PCL-mediated antigen release combined with alum strengthened humoral responses to tumor lysate and enabled recovery of functional antibodies with diagnostic capture and in vitro cytotoxic activity. In future work, key mechanistic steps such as lymph-node trafficking and cross-presentation should be tested directly. Full article

The detailed characterization of antigen-specific serum antibodies is hindered by the lack of efficient, gentle isolation methods. In this context, standard column affinity chromatography, although a powerful purification tool, presents practical challenges, including high antigen consumption and elution conditions that risk inducing antibody polyreactivity, while conventional acidic elution often compromises antibody integrity. This study introduces a novel microscale method for isolating specific immunoglobulins using anionic detergents as mild eluents. We employed antigen-functionalized hydrogel microarrays and magnetic beads as micro-immunosorbents. Among the tested detergents, sodium lauroyl glutamate (SLG) was optimal, achieving up to 78.3% recovery of functional antibodies. The optimized protocol, including recovery via G25-Sephadex gel filtration, effectively isolated specific antibodies from complex serum, retaining 58.5–85.3% of their functional bioactivity. Multiplex immunoassays confirmed the high specificity of the isolated antibodies and the lack of detergent-induced polyreactivity. The method was successfully adapted to isolate both specific antibodies (virus, dietary, and autoimmune) and total IgG, demonstrating versatility across platforms. This work establishes a robust, efficient, and gentle workflow for obtaining high-purity, bioactive antibodies, enabling their subsequent in-depth analysis for research applications. Full article

Background: The Fc region of immunoglobulin G (IgG) is a key target in therapeutic and analytical applications, such as antibody purification and site-specific bioconjugation. Although Protein A exhibits strong Fc-binding affinity, its large molecular weight and limited chemical flexibility pose challenges for use in compact or chemically defined systems. To address these limitations, we designed two α-helical peptides, SpA h1 and SpA h2, based on the Fc-binding helices of the Z34C domain from Staphylococcus aureus Protein A. Method: To enhance the structural stability and Fc-binding capability of these peptides, a lactam-based stapling strategy was employed by introducing lysine and glutamic acid residues at positions i and i + 4. Result: The resulting stapled peptides, (s)SpA h1 and (s)SpA h2, exhibited significantly improved α-helical content and IgG-binding performance, as demonstrated by circular dichroism (CD) spectroscopy and fluorescence-based IgG capture assays. Surface plasmon resonance (SPR) analysis confirmed specific, concentration-dependent interactions with the Fc region of human IgG, with (s)SpA h1 consistently showing the binding affinity and stability. Proteolytic resistance assays using α-chymotrypsin revealed that (s)SpA h1 maintained its structural integrity over time, exhibiting markedly enhanced resistance to enzymatic degradation compared to its linear counterpart. Furthermore, (s)SpA h1 exhibited strong Fc selectivity with minimal Fab affinity, confirming its suitability as a compact and Fc-specific binding ligand. Conclusions: These results confirm the successful design and development of structurally reinforced Fc-binding peptides that overcome the inherent limitations of short linear sequences through both high-affinity sequence optimization and lactam-based stapling. Among them, (s)SpA h1 demonstrates the most promising characteristics as a compact yet stable Fc-binding ligand, suitable for applications such as antibody purification and site-specific bioconjugation. Full article

Concurrent evaluation of the antiparasitic efficacy of synthetic and natural compounds can provide novel insights into the development of anti-Toxoplasma drugs. We assessed 16 synthetic compounds and two fractions derived from the leaves of Tabebuia rosea and Tabebuia chrysantha tree species for their in vitro activity against live parasites, employing strains that express green fluorescent protein and specific identification of bradyzoites using an anti-BAG1 monoclonal antibody. This study successfully identified several promising synthetic compounds with potent anti-Toxoplasma activity and favorable in vitro selectivity profiles, notably pyrazoline 2 and thiazolidinone 9. One thiazolidinone compound exhibited significant activity against extracellular tachyzoites, whereas one tree fraction demonstrated excellent activity against both tachyzoites and bradyzoites. Additionally, their in silico ADMET properties suggest their potential for good in vivo performance and CNS penetration. Although the natural extracts showed less potency in their crude form, they provide a basis for future purification efforts. The simultaneous evaluation of compounds sourced from diverse discovery pipelines can offer valuable insights into the development of drugs that target various biological pathways. Full article

Background/Objectives: The detection of anti-Coxiella antibodies using serological methods is essential for identifying exposed ruminants and preventing this important zoonotic disease in livestock. In recent years, numerous attempts have been made to increase diagnostic performance as well as simplify the production of serological assays. Commercially available tests often use whole-cell antigens, which can decrease specificity and require high-level biosafety facilities for manufacturing. The aim of this work was to produce three Coxiella burnetii (C. burnetii) antigens in recombinant form and assess them for the detection of anti-Coxiella antibodies in ruminants. Methods: Three recombinant C. burnetii antigens (Com-1, MceB, AdaA) were selected among immunodominant antigens and produced in a heterologous system (Escherichia coli). Following purification, the proteins were utilized to coat ELISA plates and evaluated for seroreactivity against sera from both negative and positive cattle. Results: Com-1 demonstrated the greatest agreement with the commercial test, albeit moderate. MceB exhibited nonspecific reactivity against a large number of sera, while the AdaA showed reactivity against only a few positive sera. Conclusions: Our findings are consistent with previous research, indicating that utilizing a single antigen to identify exposed animals is unfeasible with current knowledge, most likely due to the complex immunological response following C. burnetii infection in cattle. Consequently, it is critical to continue testing and identifying immunoreactive antigens in order to further investigate them and, potentially, select the most appropriate. Full article

Since the introduction of the DNA origami technology by Seeman and Rothemund, the integration of functional entities (nanoparticles, quantum dots, antibodies, etc.) has been of huge interest to broaden the area of applications for this technology. The possibility of precise functionalization of the DNA origami technology gives opportunity to build up complex novel structures, opening up endless opportunities in medicine, nanotechnology, photonics and many more. The main advantage of the DNA origami technology, namely the self-assembly mechanism, can represent a challenge in the construction of complex mixed-material structures. Commonly, DNA origami structures are purified post-assembly by filtration (either spin columns or membranes) to wash away excess staple strands. However, this purification step can be critical since these functionalized DNA origami structures tend to agglomerate during purification. Therefore, custom production and purification procedures need to be applied to produce purified functionalized DNA origami structures. In this paper, we present a workflow to produce functionalized DNA origami structures, as well as a method to qualify the successful hybridization of a quantum dot to a square frame DNA origami structure. Through the utilization of a FRET fluorophore–quencher pair as well as a subsequent assembly, successful hybridization can be performed and confirmed using photoluminescence measurements. Full article