Borrelia burgdorferi sensu lato, a spirochete bacterium responsible for Lyme borreliosis-the most common tick-borne infection in North America and Europe-can trigger the production of antiphospholipid antibodies. These antibodies target host lipids such as cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS), which the spirochete incorporates into its membrane from the surrounding environment. Although antiphospholipid antibodies are typically associated with antiphospholipid syndrome (APS), they may also arise during infections, including Lyme borreliosis. This study aimed to develop and optimize several enzyme-linked immunosorbent assays (ELISAs) for measuring various antiphospholipid antibodies in patients with Lyme borreliosis. Thirty patients diagnosed with Lyme borreliosis were enrolled: ten with solitary erythema migrans (EM), ten with multiple EM (MEM), and ten with late manifestations known as acrodermatitis chronica atrophicans (ACA). Forty healthy blood donors served as controls. Four distinct antiphospholipid antibody ELISAs were developed, each using a different phospholipid coating: CL, PA, PC, and PS. Serum of APS patient was used as a positive control and for standard curve generation. All four ELISAs were successfully established and demonstrated good measurement precision. Significant differences in antiphospholipid antibody levels and positivity rates were observed between Lyme borreliosis patients and healthy blood donors. Notably, levels of antibodies directed against PA (aPA), PC (aPC), and PS (aPS), both IgG and IgM, were significantly higher in patients with late Lyme borreliosis, manifested as ACA, compared to healthy blood donors. In contrast, anti-CL (aCL) levels did not differ significantly between groups. Patients with ACA also showed the highest frequency of multiple antiphospholipid antibody positivity, with 7 out of 10 patients testing positive for three or more antiphospholipid antibodies. Accurate and precise in-house ELISAs for the detection of aCL, aPA, aPC, and aPS using APS sera as standard material were developed and validated for the analysis of samples of patients with Lyme borreliosis. Our data suggest that antiphospholipid antibody levels-specifically aPA, aPC, and aPS-differ across clinical manifestations of Lyme borreliosis, with the greatest increases observed in patients with ACA.
Background/Objectives: Chronic spontaneous urticaria (CSU) is characterized by almost daily wheals or angioedema lasting for more than six weeks and not attributable to a defined inducing factor. CSU reportedly affects 1-2% of the general population and may lead to a substantial impairment in patients' quality of life. Thus, developing methods that enable early diagnosis and assessment of disease activity is a major objective for scientists and clinicians. Methods: A significant proportion of CSU cases appears to be associated with autoimmune mechanisms, which mainly involve IgE autoantibodies (type I CSU), IgG autoantibodies (type IIb CSU), or both (type I and type IIb overlap). To this end, detection of specific IgE and/or IgG autoantibodies in CSU patients using biological or immunochemical assays can offer valuable information and enable further investigation and better management of the disease. Results: This review focuses on and presents various immunochemical assays, mainly ELISAs, for determining specific IgE and/or IgG autoantibodies, along with immunochemical methods for quantifying total IgE levels as an additional biomarker in CSU patients; the development and/or application of these assays has been reported in several papers published in the last decade on CSU. Conclusions: The methods presented have recently been applied and have substantially contributed to CSU diagnosis, endotyping and prediction of response to various treatments. Further validation of the existing immunochemical assays along with the development of reliable assays for novel autoantibodies and/or autoantigens will deepen our understanding of CSU pathogenesis and support the clinical diagnosis and treatment of CSU.
Type I interferons (IFN-I), including IFN-α, IFN-β, and IFN-ω, are central to antiviral defence and immune regulation. Autoantibodies targeting IFN-I (anti-IFN-I AAbs) have emerged as key pathogenic factors in severe coronavirus disease 2019 (COVID-19) and are detectable in systemic lupus erythematosus (SLE), a prototypic IFN-driven autoimmune disease. Here we compare the prevalence and clinical impact of anti-IFN-I autoantibodies (Aabs) in COVID-19 and SLE based on a structured review of 53 studies from 2014 to 2025 and highlight the clinical associations and therapeutic opportunities presented by these autoantibodies. In COVID-19, neutralising anti-IFN-α and/or anti-IFN-ω AAbs were consistently associated with severe disease and impaired antiviral responses, particularly in older male populations. In SLE, anti-IFN-α AAbs were variably detected; neutralising antibodies were associated with reduced interferon gene signatures in some cohorts but inconsistent correlations with disease activity. Therapeutically, anti-IFN-I AAbs in COVID-19 may inform risk stratification and early antiviral strategies, whereas in SLE, IFN-α blockade, including IFN-α kinoid vaccination, demonstrates modulation of IFN signatures but variable clinical benefit. Notably, these findings reveal an immunological paradox: the same neutralising mechanism that impairs antiviral defence in COVID-19 may attenuate chronic IFN-driven inflammation in SLE. Taken together, anti-IFN-I AAbs exert context-dependent effects: pathogenic in acute viral infection yet potentially modulatory in chronic IFN-driven autoimmunity. Prospective longitudinal studies are required to further clarify their translational utility and long-term clinical impact.
Ocrelizumab is a humanized monoclonal antibody targeting CD20, approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). The neutralizing activity of anti-drug antibodies (ADAs), especially neutralizing ADAs (nADAs) activity, should be examined considering that it can alter pharmacokinetic (PK) and pharmacodynamic (PD) profiles, reduce drug efficacy, and lead to life-threatening adverse events. This article presents data on the development and validation of an assay for neutralizing anti-drug antibodies (nADA) based on ADCC reporter cells for the analysis of patient sera in the context of ocrelizumab clinical studies. Critical steps and conditions to minimize assay variability were identified. The lower limit of detection was 549.6 ng/mL. The cutoff for nonspecific neutralization was determined as 19.7%. The presence of 0.37-3.0 μg/mL ocrelizumab in a biological sample enables the detection of 1.1-10.0 μg/mL polyclonal anti-ocrelizumab idiotype antibodies, respectively. The developed method can be used for immunogenicity studies of medicinal products containing ocrelizumab.
Monoclonal antibodies (mAbs) initially played a major role in outpatient COVID-19 management by providing rapid passive immunity and reducing progression to severe disease. However, continuous SARS-CoV-2 evolution progressively compromised the effectiveness of several anti-spike products. This narrative review summarizes the trajectory of COVID-19 mAbs across three phases: early clinical efficacy, loss of efficacy due to immune escape, and future directions. We conducted a narrative review focusing on mechanisms of action, pivotal clinical trials, and real-world effectiveness of neutralizing anti-spike mAbs and host-directed immunomodulatory mAbs. Emphasis was placed on the impact of variants-especially Omicron-on susceptibility and clinical use, as well as on emerging next-generation platforms. First-generation neutralizing mAbs substantially reduced the hospitalization rates during the Alpha and Delta waves, while immunomodulatory mAbs became standard options for the hyperinflammatory phase in hospitalized patients. With the emergence of Omicron and its sub-lineages, extensive immune escape led to marked reductions in neutralization for many earlier anti-spike agents and consequent restrictions in use. Later-generation approaches targeting more conserved epitopes provided temporary solutions but were also challenged by ongoing antigenic drift. Host-directed immunomodulators retained clinical relevance because their mechanism is independent of viral spike mutations. The clinical role of monoclonal antibodies in COVID-19 has been dynamic and increasingly constrained by viral evolution. Future strategies should prioritize broadly neutralizing antibodies targeting conserved epitopes, innovative delivery platforms, and integration with real-time surveillance to preserve clinical utility in the endemic phase and improve preparedness for future outbreaks.
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that has caused repeated epidemics across Africa and the Arabian Peninsula, posing a severe and growing threat to public health and livestock. Infection in ruminants causes high neonatal mortality and catastrophic abortion storms; human disease ranges from self-limiting febrile illness to hemorrhagic fever, encephalitis, and permanent blindness. No licensed human vaccines or specific antiviral therapeutics are available, creating an urgent unmet medical need. We systematically reviewed the peer-reviewed literature on RVFV neutralizing antibodies (NAbs), extracting and synthesizing data on antibody sources, epitope specificity, in vitro neutralizing potency, in vivo protective efficacy, and molecular mechanisms of action. A growing body of work has identified potent NAbs from immunized rodents, rabbits, alpacas, non-human primates, and convalescent patients. These NAbs predominantly target the Gn and Gc envelope glycoproteins. Their mechanisms include blocking host receptor (LRP1) binding, preventing the pH-dependent conformational rearrangement of the Gn-Gc complex, and directly inhibiting viral membrane fusion. Lead candidates, such as RVFV-268 and RVFV-140, achieve sub-nanogram neutralization and confer robust protection in rodent models against lethal challenge, aerosol exposure, and vertical transmission. Bispecific antibodies and combination strategies further enhance potency and the genetic barrier to viral escape. Substantial progress has illuminated the epitope landscape and neutralization mechanisms of RVFV, yielding promising clinical candidates. Translational challenges remain, including viral immune escape, antibody thermostability, and the need for rigorous preclinical evaluation. Future efforts should prioritize structure-guided engineering, rational antibody combinations, and testing in clinically predictive animal models.
Background/Objectives: The unprecedented structural and binding data for antibodies to the SARS-CoV-2 virus taken together with the mutations for the spike protein allows for a broad simulation study of antibody-spike protein binding. This provides an understanding of the co-evolution of human immunity and viral immunity escape. Methods: We utilized the YASARA molecular dynamics program to generate initial structures and simulate to equilibration for six SARS-CoV-2 variants and ten different antibodies sampling two different binding regions to the receptor binding domain of the spike (especially for the Class I antibodies in the same part of the spike that attaches to the ACE2 receptor protein) and one to the N-terminal domain of the spike. Starting structures for antibody binding to variant spike protein domains are perturbatively achieved through point mutations and insertions/deletions in the YASARA program. We employed YASARA to measure interfacial hydrogen bound counts between antibodies and variant spike proteins and the HawkDock MMGBSA program to characterize trends in binding energies with mutation for four of the antibodies. We utilized the VMD program to analyze the time course of hydrogen bond populations. Results: As seen in previous studies, interfacial hydrogen bond counts serve as an excellent proxy for binding energies without the large systematic error inherent in the latter. We find that there is generally a decline in antibody binding strength, as measured by interfacial hydrogen bond counts, with viral evolution, but that a modest re-entrance of binding strength is present for most antibodies studied. Generically, the antibody heavy chain binds more strongly to the spike protein, though for approximately half the antibodies the light chain binding strength converges to the heavy chain strength with viral evolution. Conclusions: The key conclusion is that the identified re-entrant immunity, speculatively arising from a balancing of maintenance of ACE2-spike binding while escaping antibodies through mutation, allows for some maintenance and even strengthening of immunity for later viral strains from early infection or vaccination.
Hemolytic Uremic Syndrome (HUS) is a severe clinical manifestation primarily triggered by Shiga toxin-producing Escherichia coli (STEC). While Shiga toxins (Stx) are central to the development of systemic endothelial damage, current recombinant antibody developments have overwhelmingly focused on neutralizing the Stx2 subtype. However, numerous STEC isolates produce Stx1 either independently or alongside Stx2, revealing a critical need to diversify the antibody repertoire for comprehensive antitoxin therapies. To address this, we characterized two novel, fully human recombinant Fabs targeting Stx1 (FabB6:Stx1 and FabC8:Stx1) selected from a synthetic library via phage display. We evaluated their binding specificity and neutralizing activity in Vero and human proximal tubular epithelial (HK-2) cells, as well as in primary human glomerular endothelial cells (HGEC exposed to HUS-derived STEC supernatants. Both Fabs exhibited high specificity and nanomolar affinity for Stx1. Notably, they displayed cell-type-dependent neutralization profiles, with FabC8:Stx1 demonstrating superior and more consistent neutralization in HK-2 cells. Crucially, when evaluated alongside previously characterized anti-Stx2 antibodies (FabC11:Stx1/Stx2 and FabF8:Stx2), the Stx1-specific Fabs conferred complementary protection against clinical STEC isolates. These findings support the inclusion of Stx1-targeting recombinant antibodies into broader multi-toxin neutralization strategies, thereby expanding the therapeutic potential against STEC-associated diseases.
Background/Objectives: The induction of anti-SARS-CoV-2 antibodies by COVID-19 vaccination reduces morbidity and mortality, but immune responses may be compromised in people living with HIV (PLWH). The aims of the current study were to determine whether viral suppression (VS) or immune reconstitution (IR) in PLWH directly affected their ability to produce effective levels of anti-SARS-CoV-2 antibodies in mucosal secretions or blood induced by vaccination. Methods: Anti-SARS-CoV-2 spike IgG, IgA and secretory IgA (SIgA) antibodies and their avidities were measured by ELISA in HIV-negative healthy controls (HC; n = 49) and PLWH (n = 94) using stimulated oral fluid (SOF) and serum. Frequencies of CD4/CD8 T cells and their expression of exhaustion/senescence were determined by flow cytometry. Cytokine levels were measured by cytokine bead arrays. Results: We showed that higher HIV burden negatively impacted the levels of systemic and mucosal anti-SARS-CoV-2 spike IgG antibodies produced. This differential IgG antibody production was unaffected by IR status, antiretroviral therapy duration or T cell exhaustion/senescence. PLWH elicited higher anti-SARS-CoV-2 spike IgA antibodies both in peripheral blood and oral mucosa and highr secretory IgA (SIgA) antibodies in the oral mucosa. PLWH with higher HIV RNA copies elicited lower IgG avidity but the IgA avidity indices remained unaffected. PLWH expressed higher levels of innate immunity cytokines in the oral mucosa, irrespective of the HIV RNA copies. Conclusions: Significantly fewer breakthrough infections in PLWH compared with HC, along with high IgA/SIgA antibodies and increased innate immunity cytokines in the SOF, suggest a potential role for mucosal immunity in the immunopathogenesis of COVID-19.
Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract infections in infants, young children, and older adults worldwide. With the approval of nirsevimab and HRSV vaccines, accurate measurement of neutralizing antibody levels has become essential for vaccine evaluation, immunization strategy design, and seroepidemiology. The plaque reduction neutralization test (PRNT) remains the gold standard, but it is slow, low-throughput, and requires high biosafety. In recent years, newer methods including focus reduction neutralization testing (FRNT), pseudovirus neutralization testing (PNT), and fluorescent/luminescent reporter virus systems (RVSs) have improved speed and throughput while maintaining high specificity. This review summarizes the principles, performance, applications, and standardization challenges of these assays, offering methodological guidance for HRSV research and prevention in China.
Objective: Living donor liver transplantation is a multifactorial process, and non-invasive serological parameters that may provide information about graft and patient status are still under investigation. However, time-dependent factors such as infection and rejection are often overlooked. Therefore, this study aimed to investigate anti-α-Gal1,3 levels and the expression of inflammatory and anti-inflammatory genes in peripheral blood samples obtained from 26 liver transplant patients before transplantation and at the first and sixth months post-transplantation. Additionally, 15 healthy volunteers were included as a control group, and patients were followed for two years to evaluate graft rejection. Method: A total of 26 living-donor liver transplant recipients and 15 healthy volunteers were included in the study. Peripheral blood samples were collected from patients before transplantation and at the first and sixth months after transplantation. Gene expression levels of IL2, IL4, IL6, IL10, TNF, IFNG, FOXP3, TREM1, CD14, and HLAG5 were analyzed by qRT-PCR, while anti-α-Gal IgM and IgG levels were measured by ELISA. In addition, biochemical parameters and microbiological culture analyses were evaluated, and bacterial identification was confirmed by MALDI-TOF MS when necessary. Analyses were performed across three time points with respect to infection status and graft rejection. Results: While the expression of IFNG, IL2, and HLAG5 significantly increased 6 months after transplantation, IL10 expression decreased significantly. In patients without rejection, IFNG expression increased significantly and IL10 expression decreased significantly at the sixth month. Microbiological evaluation showed that infections were more frequent, particularly during the first three months after transplantation. While anti-α-Gal IgG levels did not differ according to infection status, IgM levels decreased significantly at the sixth month, and this decline was associated with a reduced infection burden. Conclusions: In conclusion, IFNG and IL10 gene expression levels may serve as important indicators for predicting rejection after liver transplantation, whereas anti-α-Gal IgM levels may provide useful guidance in monitoring infection.
Background/Objectives: Hand, foot and mouth disease (HFMD) has recently emerged as a serious health threat, as certain serotypes can cause severe illness. Serotype distribution vary by region, and seroprevalence studies helps in developing preventive strategies. This study aimed to determine the seroprevalence of enterovirus type 71 (EV-A71), Coxsackievirus A16 (CV-A16), Coxsackievirus A10 (CV-A10), and Coxsackievirus A6 (CV-A6), the main causative agents of HFMD and to investigate risk factors for seropositivity. Methods: This multicenter, cross-sectional study was conducted across five major cities in Türkiye. Children (6 months-17 years) who presented to outpatient clinics for any reason were included between May 2024 and January 2025. Neutralizing antibodies were measured using a microneutralization assay. Statistical analyses included descriptive methods, appropriate group comparisons (Chi-square/Fisher's Exact), and backward logistic regression to identify factors associated with HFMD seropositivity. Results: The study included 998 participants (mean age: 8.6 ± 5.2 years; 51.3% male). CV-A6 antibodies were detected in 68.5%, EV-A71 in 66.5%, CV-A10 in 60.2%, and CV-A16 in 46.0% of samples. No viral antibodies were detected in 5.3% of serum samples (All-Negative group); antibodies against at least one HFMD agent were detected in 94.7% (Any-Positive group). HFMD seropositivity increased significantly with age. Handwashing habits did not differ between the groups. The any-positive group more often had a household member aged 12-18 years, a mother with lower education, and higher kindergarten attendance. In logistic regression analysis, age, average monthly household income, and mother's education level were the factors influencing seropositivity. Conclusions: The seroprevalence of HFMD-causing viruses in Türkiye is high from six months of age onward. Beyond promoting personal protective measures, the implementation of a vaccination program should also be considered.
Vestibular migraine (VM) is a common but underdiagnosed cause of episodic vertigo lacking evidence-based preventive treatments. Calcitonin gene-related peptide (CGRP) plays a central role in migraine pathogenesis and is expressed in vestibular structures, providing a rationale for CGRP-targeting therapies in VM. However, available evidence has not been systematically synthesized. We conducted a structured synthesis of studies evaluating CGRP-targeting therapies (monoclonal antibodies and gepants) in adults with definite/probable VM. We searched PubMed/MEDLINE, Embase, Scopus, and Web of Science. Eligible studies included randomized controlled trials, prospective/retrospective cohorts, and case series (≥10 patients) reporting quantitative outcomes. A two-tier synthesis was prespecified: quantitative meta-analysis where feasible, otherwise narrative synthesis with direction-of-effect analysis. Of 247 records, four observational studies met inclusion criteria (total N ≈ 103 patients). No RCTs were identified. All four studies evaluated CGRP monoclonal antibodies; no gepant studies met inclusion criteria. Outcome reporting was highly heterogeneous. Quantitative meta-analysis was not feasible. Direction-of-effect synthesis showed consistent improvement across all studies for vertigo frequency (4/4), Dizziness Handicap Inventory (2/2), and monthly migraine days (2/2). No serious adverse events were reported. CGRP monoclonal antibodies show a consistent direction of benefit for vestibular symptoms, migraine days, and dizziness handicap in observational VM studies, with a favorable safety profile. However, the absence of RCTs, small samples, lack of control groups, and heterogeneity preclude definitive conclusions. This synthesis highlights a critical evidence gap. Adequately powered, double-blind, placebo-controlled RCTs of CGRP-targeting therapies (both monoclonal antibodies and gepants) are urgently needed.
Gallbladder cancer (GBC) is an aggressive tumor that, together with the cholangiocarcinomas, constitutes the spectrum of biliary tract cancer (BTC). These tumors are characterized by a frequently late diagnosis, marked genomic heterogeneity, variable response to cytotoxic therapies, and poor overall survival in advanced stages. Nevertheless, the characterization of the tumor microenvironment (TME) and the identification of actionable molecular targets have driven the development of biological therapies. This review summarizes current and emerging evidence on monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs) in the management of GBC. The analysis addresses the early exploration of autoantibodies as potential diagnostic biomarkers, mechanistic hypotheses of immune evasion, and the clinical translation of targeted agents in the metastatic setting. Additionally, we critically discuss the extrapolation of data from global BTC trials to the specific GBC setting, the integration of population genetics into epidemiological studies such as the EULAT Eradicate GBC initiative, and the preliminary status of immunotherapy in perioperative scenarios.
Background: Chagas disease, caused by Trypanosoma cruzi, remains a major public health problem in Latin America and an emerging concern worldwide. Current chemotherapies show limited efficacy during chronic infection, and no licensed vaccine is currently available. We previously developed the chimeric antigen N-Tc52/TSKb20 as a vaccine candidate against T. cruzi infection. In a murine model, this vaccine induced robust antigen-specific immune response associated with protection shortly after vaccination. Objectives: Here, we investigated the long-term persistence and effector functions of the immune responses elicited by this vaccine candidate. Methods: Both female and male C57BL/6 mice were immunized with three doses of N-Tc52/TSKb20 formulated with QuilA adjuvant. Serum samples collected 170 days post-immunization were analyzed for antigen-specific antibodies by ELISA and for trypanolytic activity against cell-derived trypomastigotes using an in vitro functional assay. Cellular immune responses were evaluated by measuring cytokine production, T cell activation, and memory T cell responses following in vitro re-stimulation with the vaccine antigen or T. cruzi antigens. Results: N-Tc52/TSKb20 vaccination induced a sustained antigen-specific humoral response, characterized by long-lasting IgG2c antibodies and functional activity persisting for up to 170 days post-immunization. In parallel, vaccination promoted long-term activation of antigen-specific CD8+ T cells and production of TNF-α and IFN-γ upon antigen re-encounter. A sex-dependent tendency was observed for IL-10, with increased production in vaccinated female mice. Moreover, vaccinated animals exhibited increased frequencies of central and effector memory CD4+ and CD8+ T cells in response to T. cruzi antigens, with a predominant contribution of CD8+ T cells, indicating the establishment of parasite-specific T cell memory. Conclusions: Together, these findings demonstrate that vaccination with N-Tc52/TSKb20 induces a long-lasting Th1-biased immune response characterized by trypanolytic antibodies, functional and durable T cell responses, and parasite-specific memory T cells. This immunological profile supports the potential of N-Tc52/TSKb20 as a promising vaccine candidate for Chagas disease and highlights its capacity to elicit immune mechanisms that have been associated with protection against T. cruzi infection.
Background/Objectives: Genetic and environmental factors during early development contribute to autism pathogenesis. Maternal autoantibodies recognizing specific fetal brain proteins can predict autism risk in a subset of cases. These antibodies cross the placenta and bind to their target antigens, which play critical roles in neurodevelopment, thereby increasing autism risk in this mechanistically defined subtype, Maternal Autoantibody-Related Autism (MARA). A multi-ELISA assay that detects maternal autoantibody combinations associated with increased autism risk has been described in the literature. This study aimed to transfer the MARA related autoantibody component assays to a clinical development laboratory for optimization and performance characterization. Methods: Indirect ELISA assays for eight maternal autoantibodies targeting LDH-A, LDH-B, GAH, STI1, CRMP-1, CRMP-2, NSE, and YB-1 were transferred from an academic laboratory to a clinical development laboratory for optimization and determination of the analytical performance and preliminary assay cutoff values. Standard methodologies were used to assess linearity, sensitivity, specificity, precision, and stability. Predefined validation protocols based on professional guidelines with established acceptance criteria for each parameter were followed. Results: Optimized ELISAs met the acceptable analytical performance criteria. All assays except one demonstrated excellent linearity when diluted with buffer or non-reactive plasma. The sensitivity analysis showed the lower limit of quantification discretely above the limit of detection, and below the preliminary population-based threshold values. Coefficients of variation for within-lot reproducibility of positive samples were <15%, with two minor exceptions. Common interfering substances, except whole human IgG, did not affect assay performance. Microtiter assay plates were stable for at least six months without significant assay drift. Conclusions: These maternal autoantibody assays demonstrated high sensitivity, specificity, and robustness, supporting progression to validation in CLIA-certified clinical laboratories. These assays will enable rigorous clinical evaluation of the accuracy of specific antibody combinations previously reported in the peer reviewed literature to specifically correlate with autism.
Background: Antiphospholipid antibodies (aPLs) are essential for antiphospholipid syndrome (APS) diagnosis, which is based on clinical and laboratory parameters, including the detection of lupus-anticoagulant (LAC), anti-cardiolipin (aCL) and anti-β2-glycoprotein-I (aβ2-GPI) antibodies. The enzyme-linked immunosorbent assay (ELISA) is the reference methodology for classification criteria, although chemiluminescence immunoassays (CLIA) are more common in clinical practice. Methods: Since LAC reflects the functional activity of a large subset of antiphospholipids, through coagulation assays that enhance a phospholipid-dependent inhibitory effect, it has been used as a reference for assessing ELISA and CLIA reliability. Samples, separated into positive and negative LAC, were selected by CLIA detection in negative and positive IgG/IgM aCL and aβ2-GPI (cut-off > 20 U/mL). Results: The ELISA/CLIA comparison showed a 100% concordance in triple negative groups, highlighting an optimal analytical specificity; a higher concordance in the aβ2-GPI IgM-positive groups compared to positive aCL IgM (100% vs. 76% in LAC-positive groups; 82% vs. 71% in LAC-negative groups), as well as in aβ2-GPI IgM-negative groups compared to negative aCL IgM in LAC-positive groups (100% vs. 87.5%); and a massive concordance reduction in positive IgG aβ2-GPI and aCL groups (44% vs. 50% in LAC-positive groups; 4.8% vs. 4.5% in LAC-negative groups). Concordance increased in all groups with a higher CLIA cut-off (>50 U/mL). Conclusions: Although CLIA performances partly differed from ELISA, this does not preclude their use in APS diagnosis, which aims for higher sensitivity in detecting cases of disease. ELISA is confirmed to be more reliable for classification criteria that aim for high specificity to reduce false positives.
Epstein-Barr virus (EBV) has been implicated in the pathogenesis of multiple sclerosis (MS). Elevated immunoglobulin G (IgG) titers against EBV nuclear antigen 1 (EBNA1) represent the most consistent serological marker of MS risk, with levels remaining persistently elevated following disease onset. We conducted high-dimensional profiling of virus-specific antibody-associated immune features in 60 individuals with relapsing-onset MS followed over time and in 54 age- and sex-matched EBV-seropositive healthy controls. EBNA1-specific IgG in MS patients exhibited a distinct proinflammatory Fc signature, characterized by increased affinity for Fc γ receptors (FcγRs) and enhanced Fc-mediated effector functions, including antibody-dependent phagocytosis, complement activation, and natural killer cell engagement. This signature was not observed for antibodies against other common viral antigens and showed a strong association with MS. Increased FcγR binding by IgG, specific for both EBNA1 and the lytic EBV glycoprotein 350 (EBV-gp350) correlated with disease activity, defined as clinical relapse and/or presence of contrast-enhancing brain lesions (CELs). Both fragment antigen-binding and pro-inflammatory functional Fc-mediated features contribute to the association between EBNA1-specific antibody responses and MS. Fc effector functions of EBV-specific antibodies may actively participate in promoting focal disease activity in MS. ANN NEUROL 2026.
4-Chlorophenoxyacetic acid (4-CPA), a synthetic auxin analog, is employed in agriculture both as a plant growth regulator and as a constituent of herbicide formulations. Consequently, the establishment of simple and rapid detection methods is essential for effective environmental monitoring. This study reports the first development of a homogeneous fluorescence polarization immunoassay (FPIA) for the determination of 4-CPA. The monoclonal antibody (M1), raised against 4-CPA, was evaluated as a recognition element. Furthermore, two fluorescently labeled 4-CPA tracers-with ethylenediamine fluorescein thiocarbamate and aminohexylaminocarbonylfluorescein-were synthesized and purified, and their structures were unequivocally confirmed by high-performance liquid chromatography coupled with high-resolution mass spectrometric detection (HPLC-HRMS). Optimal concentrations of monoclonal antibodies and tracers were established, yielding a limit of detection of 1.2 ng/mL. The assay demonstrated a broad dynamic range of 2.3-300 ng/mL and a rapid analysis time of 15 min. Validation via the standard addition method in authentic open water samples resulted in recovery rates of 98-112%. To address the cross-reactivity with the prevalent herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), two novel strategies were devised and successfully implemented. The first approach involves the concurrent execution of two separate FPIAs-one for 2,4-D and one for 4-CPA-followed by the mathematical resolution of two analyte concentrations from the two measured binding values. The second strategy entails the preliminary selective removal of 2,4-D from sample matrices using affinity chromatography columns with immobilized anti-2,4-D antibodies prior to FPIA for 4-CPA. These proposed methodologies appear highly promising for overcoming the inherent limitations of traditional immunoassays when faced with significant cross-reactivity among structurally analogous compounds.
Background/Objectives: We report a case of severe dermatomyositis demonstrating characteristic widespread extraosseous uptake on 99mTc-methylene diphosphonate (99mTc-MDP) bone scintigraphy. This study highlights the diagnostic value of this modality in detecting active inflammatory myopathy when conventional muscle biopsy is inconclusive and introduces its novel use for intraoperative gamma-probe-guided biopsy to precisely target metabolically active muscle. This approach may help target metabolically active muscle in heterogeneous idiopathic inflammatory myopathies (IIMs). Case Presentation: A 49-year-old man developed progressive proximal muscle weakness (Medical Research Council grade 2/5 proximally, 5/5 distally) beginning in June 2025 following influenza infection, accompanied by dysphagia, classic dermatomyositis cutaneous manifestations, back pain, and difficulty standing. Laboratory evaluation revealed elevated inflammatory markers (ESR 55 mm/hr, CRP 20 mg/L), leukocytosis (16.58 × 109/L), markedly raised creatine kinase (19,937 IU/L), and troponin T levels. An initial quadriceps muscle biopsy performed on 29 July 2025 was non-diagnostic. Three-phase 99mTc-MDP scintigraphy (~1110 MBq) demonstrated intense, diffuse extraosseous uptake involving bilateral deltoids (symmetric), biceps and triceps (patchy), paraspinal muscles (longitudinal), gluteal muscles, thighs (quadriceps and hamstrings), and gastrocnemius muscles, with relative suppression of appendicular skeletal uptake on delayed images due to soft-tissue tracer dominance-findings consistent with severe inflammatory myopathy. Following reinjection (~1100 MBq), intraoperative gamma-probe-guided biopsy targeted areas of highest uptake (left quadriceps femoris and distal triceps brachii; intraoperative counts 1300-1400 versus background ~500). Histopathology revealed histiocyte-predominant inflammation with myofibre necrosis and regeneration, sparse CD4+ T-cell infiltrates, and absence of fibrosis, consistent with necrotising myopathy. Positive antinuclear antibodies and strong anti-Mi-2 antibodies confirmed the diagnosis of dermatomyositis. Treatment included pulse methylprednisolone followed by oral prednisone taper, methotrexate, azathioprine, intravenous immunoglobulin, and planned rituximab therapy. Discussion: Whole-body 99mTc-MDP scintigraphy provided a complementary whole-body functional assessment of disease extent, revealing widespread muscular involvement. The novel application of intraoperative gamma-probe-guided biopsy enabled real-time targeting of metabolically active muscle, facilitating targeted sampling after an initial non-diagnostic biopsy and yielding supportive histopathological findings. This dual diagnostic and interventional role demonstrates the technical feasibility of gamma-probe guidance in a diagnostically challenging case of dermatomyositis. Conclusions: In our case, the integration of 99mTc-MDP scintigraphy with gamma-probe-guided biopsy enabled precise targeting of metabolically active muscle following an initial non-diagnostic biopsy. This multimodal approach may be useful in selected diagnostically challenging cases of severe inflammatory myopathy. Larger studies are needed to evaluate its reproducibility and added value.