SEARCH · Search PubMed

Results for “cross-reactivity”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed2025

Antibody responses in Klebsiella pneumoniae bloodstream infection: a prospective cohort study.

BACKGROUND: Klebsiella pneumoniae is a leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive K pneumoniae. We sought to determine whether the O-specific polysaccharide antigen is immunogenic in humans with K pneumoniae bloodstream infection. We also sought to define the cross-reactivity of human antibody responses among structurally related K pneumoniae O-specific polysaccharide subtypes and to assess the effect of capsule production on O-specific polysaccharide-targeted antibody binding and function. METHODS: In this prospective cohort study, we compared plasma antibody responses to O-specific polysaccharide in a cohort of consecutively enrolled patients with K pneumoniae bloodstream infection with controls, specifically a cohort of healthy individuals and a cohort of individuals with Enterococcus spp bloodstream infection. Patients were enrolled at the Massachusetts General Hospital, a tertiary hospital with affiliated clinics in the USA. We excluded patients whose isolates were not confirmed to be K pneumoniae by whole-genome sequencing. The primary outcome was the measurement of plasma IgG, IgM, and IgA antibody responses. We performed flow cytometry to measure the effects of K pneumoniae capsule production on O-specific polysaccharide antibody binding and O-specific polysaccharide antibody-mediated complement deposition, using patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. FINDINGS: We enrolled 129 consecutive patients with suspected K pneumoniae bloodstream infection between July 24, 2021, and August 4, 2022, of whom 69 patients (44 [64%] male and 25 [36%] female) with confirmed K pneumoniae bloodstream infection were eligible for immunological evaluation. Common O-specific polysaccharide serotypes (O1, O2, O3, and O5) accounted for 57 (83%) of 69 infections. O-specific polysaccharide was immunogenic in patients with K pneumoniae bloodstream infection, and peak O-specific polysaccharide-IgG antibody responses in patients were ten-fold to 30-fold higher than antibody responses detected in healthy controls, depending on the serotype. There was cross-reactivity among similar O-specific polysaccharide subtypes, including the O1v1 and O1v2, O2v1 and O2v2, and O3 and O3b subtypes, as well as between the O1 and O2 types. Capsule produced by both hyperencapsulated and non-hyperencapsulated K pneumoniae inhibited O-specific polysaccharide-targeted antibody binding and function. INTERPRETATION: O-specific polysaccharide was immunogenic in patients with K pneumoniae bloodstream infection, supporting its potential as a candidate vaccine antigen. The cross-reactivity observed between similar O-specific polysaccharide subtypes in patients with K pneumoniae bloodstream infection suggests that it might not be necessary to include all subtypes in an O-specific polysaccharide-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with O-specific polysaccharide antibody binding. This finding could limit the effectiveness of vaccines that exclusively target O-specific polysaccharide. FUNDING: National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

Humans↗

PubMed2026

Distinct HLA Associations for Antibody Multireactivity With Citrulline-Containing Type II Collagen Epitopes Versus More Limited Antibody Reactivity With Citrulline-Containing IgG Epitopes in Rheumatoid Arthritis.

OBJECTIVE: Anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) can be promiscuous, with cross-reactive binding to many antigens containing short motifs, or private with little cross-reactivity. Also, ACPA reactivity patterns differ among patients with RA, including for motif-containing epitopes in important self-antigens like collagen and IgG (bound by RA-associated rheumatoid factors [RFs]), with limited understanding of the underlying mechanism. The objective of this study was to determine if HLA alleles associate with ACPA reactivity patterns. METHODS: For 100 ACPA+RF+ participants with RA, serum IgG binding was quantified by enzyme-linked immunosorbent assay to 10 citrulline-containing peptides derived from Type II collagen and IgG1 (nine with motifs), and HLA loci were genotyped. Also, antibody and serum multireactivity were evaluated. HLA alleles present differentially in RA participants with high versus low IgG binding to specific peptides, as well as with multireactivity versus limited reactivity were identified by Fisher's exact test. RESULTS: Serum IgG multireactivity for citrulline-glycine motif-containing collagen peptides was high, at least partially due to promiscuous antibodies. HLA-DQA1*01:02 was present in more participants with anticitrullinated collagen antibodies and multireactive sera. In contrast, serum multireactivity was low for IgG1-derived peptides due at least in part to more private antibodies. Shared epitope-containing HLA-DRB1*04:01 was present more frequently in participants with RA-associated RFs irrespective of the citrulline-serine motif and less frequently in participants with anticitrullinated collagen antibodies. Several HLA alleles associated with specific antibody reactivities. CONCLUSION: Different HLA alleles may contribute to the different reactivity patterns of promiscuous anticitrullinated collagen antibodies and more private RA-associated RFs.

Humans↗

PubMed2025

RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus.

Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction- 1, ≥ 1.81 ag reaction-1, ≥ 62.5 fg reaction-1, and ≥ 2.5 pg reaction-1 for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction-1 for DENV-1 and DENV-4, and ≥ 0.5 ng reaction-1 for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.

Dengue Virus↗

PubMed2025

Seroprevalence of Chikungunya and O'nyong-nyong Viruses in Senegal, West Africa.

Arthritogenic alphaviruses such as chikungunya (CHIKV) and o'nyong-nyong (ONNV) viruses have shown capacity to cause widespread epidemics, with recurrent and sporadic outbreaks occurring throughout sub-Saharan Africa. We analyzed the seroprevalence for CHIKV and ONNV in 470 non-febrile subjects from three regions in Senegal (Sindia, 2018; Thies, 2018; and Kedougou, 2022/2023) using retrospective samples. We assessed the presence of anti-CHIKV IgG and neutralizing antibody titers against CHIKV and ONNV via enzyme-linked immunosorbent assay (ELISA) and microneutralization tests, respectively, and determined risk factors of CHIKV and ONNV exposure by binary logistic regression. The overall alphavirus seroprevalence based on an anti-CHIKV virus like particle (VLP) IgG ELISA was 38.5%, with rates varying geographically: Kedougou (48.6%), Thies (31.9%), and Sindia (14.9%). Neutralizing antibody titers revealed CHIKV and ONNV seroprevalence rates of 7.4% and 9.8%, respectively, with significant variations by region and age group. Cross-reactivity analysis showed that 82.9% of CHIKV cases exhibited a neutralizing response to ONNV, while 71.7% of ONNV cases cross-neutralized CHIKV. Residents of Thies had significantly higher odds of CHIKV infection (aOR, 3.147; 95% CI: 1.164-8.510) while ONNV infection was more likely in Kedougou (aOR, 3.888; 95% CI: 1.319-11.466). Furthermore, older age (> 40 years) was a significant risk factor both CHIKV (aOR, 2.094; 95% CI: 0.846-5.185) and ONNV infection (aOR, 2.745; 95% CI: 1.212-6.216). Our study confirms the co-circulation of CHIKV and ONNV in Senegal, highlighting their geographic and demographic distribution. These findings underscore the need for continued surveillance, alphavirus testing, and tailored public health strategies to mitigate their impact in Senegal.

Humans↗

PubMed2026

Parasites and allergies: a complex bidirectional relationship from evolutionary origins to modern therapeutics.

Parasites and allergic diseases are linked by a complex, bidirectional relationship shaped by long-term host-parasite coevolution. This review discusses how different parasites may either promote or attenuate allergic responses through immunological, epithelial, and microbiome-mediated mechanisms. IgE-mediated immunity, mast cell activation, eosinophilia, and pruritus may have evolved as protective responses against helminths and blood-feeding ectoparasites. In contrast, modern allergies may partly reflect misdirected responses to harmless environmental antigens. The effects of parasites on allergy are not uniform and depend on parasite type, infection site, exposure intensity and chronicity, host immune status, and the degree of host-parasite adaptation. Protozoa such as Giardia intestinalis may contribute to food allergy-related manifestations by disrupting the intestinal barrier, altering gut microbiota composition, and modifying mucosal immune responses, particularly in atopic individuals. In contrast, selected helminths may attenuate allergic inflammation by inducing regulatory T and B cells, anti-inflammatory cytokines, antigen-presenting cell modulation, and IgG4-associated mechanisms that can limit IgE-mediated effector responses. Molecular similarities between parasite-derived antigens and environmental allergens, including conserved protein families and carbohydrate epitopes, may contribute to cross-reactive IgE responses and complicate allergy diagnostics. Therefore, current research is shifting from live helminth therapy toward defined parasite-derived molecules and immunomodulatory pathways that may inspire safer and more controlled therapeutic strategies. A clearer understanding of parasite-allergy interactions may improve diagnostic interpretation and support the development of new approaches to the management of allergic disease.

Humans↗

PubMed2026

Identification of circulating parasite and host biomarkers in the serum proteome of Trypanosoma vivax-infected sheep under immunosuppression.

Bovine trypanosomiasis, caused by Trypanosoma vivax, presents a major threat to livestock health, primarily due to the lack of efficient field diagnostic tools. This study aimed to identify potential parasite and host-derived biomarkers through a longitudinal proteomic analysis of serum from sheep experimentally infected with a T. vivax isolate. Utilizing LC-MS/MS and bioinformatics, 154 proteins were identified, comprising 150 host (Ovis aries) and four pathogen proteins. Principal Component Analysis (PCA) demonstrated a clear separation of samples according to infection stages: Control, Infection (15 parasites/field), and Peak Infection (30-60 parasites/field) Among the parasite proteins, TvY486_0014340, TvY486_0040500, and TvY486_0042480 were identified as candidate antigens for future evaluation. In silico analysis revealed these proteins contain multiple B-cell epitopes with no cross-reactivity to related Trypanosoma species, supporting their potential for immunodiagnostic development. Additionally, three host proteins-folate receptor 3 (FOLR3) and complement components C1QA and C1QC-were significantly modulated across all infection phases. The upregulation of FOLR3 likely reflects a compensatory response to parasite-induced anemia, while the downregulation of C1Q components suggests immune evasion strategies. These findings highlight specific parasite antigens and host regulatory patterns that may serve as candidate biomarkers for the diagnosis and monitoring of T. vivax infection, facilitating the development of improved point-of-care assays.

Animals↗

PubMed2019

Development of a multiplex real-time RT-PCR assay for simultaneous detection and differentiation of influenza A, B, C, and D viruses.

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

Animals↗

PubMed2026

Development and evaluation of a one-pot RPA-Cas12a assay based on a primer-driven reverse screening strategy for preliminary screening of megalocytivirus-related viruses.

A primer-driven reverse-screening strategy was used to identify an RPA-Cas12a target suitable for the rapid preliminary screening of megalocytivirus-related viruses. The ISKNV reference genome NC_003494.1 was used as the initial template, and candidate amplification units were designed according to RPA primer-design requirements, primer physicochemical properties, and the availability of Cas12a protospacer-adjacent motif (PAM) sites and crRNA target sequences. Following preliminary amplification assessment, the retained candidate primers were aligned individually against 75 complete genome sequences of megalocytivirus-related viruses. Of these, 67 sequences met the predefined criteria for target-region integrity, primer-binding-site compatibility, and Cas12a recognition. Retrospective mapping to the reference genome located the candidate amplification region within ORF057L. Based on the resulting candidate detection unit, a one-pot RPA-Cas12a assay incorporating a commercially available lyophilized RPA amplification module was developed. Optimization showed that 400 nM reporter and 80 nM crRNA-1 provided relatively stable fluorescence output. A cut-off value of 1281.6 relative fluorescence units (RFU) was established as the mean plus three standard deviations of the endpoint fluorescence values obtained from 20 qPCR-negative samples. In analytical sensitivity testing, the assay generated fluorescence signals above the negative control at low plasmid copy numbers. However, because only a limited number of replicates were tested at these low template concentrations, these findings were not used to define a formal limit of detection. ISKNV, RSIV, and TRBIV samples tested positive, whereas the MRV sample produced an endpoint fluorescence value below the cut-off. Repeatability analysis of the same sample in six independent reactions yielded a coefficient of variation of 8.03%. Among the 39 samples examined, no discordant qualitative results were observed between the RPA-Cas12a assay and qPCR. These findings support the use of the ORF057L-targeted one-pot RPA-Cas12a assay as a rapid preliminary screening tool for megalocytivirus-related viruses. Nevertheless, its formal limit of detection, inter-batch stability, cross-reactivity with additional non-target pathogens, and clinical diagnostic performance require further evaluation.

Lyophilized RPA↗

PubMed2026

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of twenty-five steroids in equine serum.

Steroids are potential biomarkers for monitoring equine pregnancy. However, immunoassays currently used for their quantification suffer from cross-reactivity and limited specificity, thus requiring more accurate methods. This study reports the development and validation of a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of 25 steroids covering the main biosynthetic pathways of progestogens, corticosteroids, androgens, and estrogens. Steroids were extracted by protein precipitation followed by evaporation, derivatization, and reconstitution before LC-MS/MS analysis. A surrogate matrix was used for calibration and validation to avoid endogenous interference. Validation was performed according to and partly adapted from Clinical and Laboratory Standards Institute guidelines (CLSI), including linearity, trueness, precision, limits of detection and quantification, measurement uncertainty, recovery, matrix effects, carryover, selectivity, and stability. Calibration curves were fitted using the best-performing weighted linear or quadratic regression model, yielding excellent linearity (R2 > 0.990), trueness between -9.0% and 2.3%, and intra- and inter-day precision <6.3%. Lower limits of quantification ranged from 2.07 to 2250 pg/mL depending on physiological analytes concentration. Extraction recovery averaged 24.3-114.9%, matrix effects were acceptable, and accuracy ranged from 94.4% to 98.9%. No carryover or interferences were detected. Measurement uncertainty remained <15%. This study presents the first LC-MS/MS method partially validated per CLSI criteria for the quantification of 24 steroids in equine serum. The method offers a sensitive and specific alternative to immunoassays and provides a robust tool for equine steroid profiling with potential applications in pregnancy monitoring, placentitis diagnosis, and fetal sex determination.

Animals↗

PubMed2026

Surface shaving proteomics reveals a parasite-encoded protein embedded in the spore filaments of Ameson portunus.

The surfaces of microsporidian spores are frequently adorned with filamentous appendages of unknown origin and function. Although some studies suggest that these structures may be host-acquired, the absence of identified parasite-encoded components has hindered our understanding of their biogenesis and role in infection. Here, we applied surface shaving proteomics to profile the surface-exposed proteins of Ameson portunus -a microsporidian pathogen causing severe myopathy in portunid crabs. Our analysis identified 120 candidate surface proteins. Nineteen of these were highly enriched by both direct shaving and SDS-assisted methods, representing a high-confidence surfome. Among these, a previously uncharacterized protein, designated 8-2.11, was confirmed via immunofluorescence assay and immunoelectron microscopy. It was expressed early in development stage and specifically localized to the spore wall and hair-like projections (HLPs) of microsporidia. Notably, polyclonal antibodies against recombinant 8-2.11 recognized a native protein in spores, specifically labeled the HLP structures, and showed no cross-reactivity with host cells. Our results provide the first evidence of a parasite-encoded protein that is integral to HLP formation, challenging the prevailing hypothesis that these surface filaments are solely host-derived. This study establishes surface shaving as a powerful tool for microsporidian research and highlights 8-2.11 as a promising candidate for future functional studies on spore surface biology and host-parasite interactions.

Animals↗

PubMed2026

SCAN: A sample-to-answer cross-priming isothermal assay for on-site virus detection with RT-qPCR sensitivity and genomically similar virus differentiation specificity.

Genomically similar viruses often differ in pathogenicity and host tropism due to specific mutations, and failure to distinguish them risks misdiagnosis and ineffective control. Molecular methods can differentiate such viruses but require laboratory settings and skilled personnel, while field-deployable immunological methods suffer from cross-reactivity. To address this challenge, we developed SCAN (Sample-to-answer Cross-priming isothermal amplification Assay with Nucleic acid strip), a general framework for on-site detection of genomically similar viruses. Comparative bioinformatics of isolation and sequencing data identifies key conserved differential determinants for primer design, ensuring specificity and reducing non-specific amplification. A one-tube cross-priming isothermal amplification (CPA) enables rapid target amplification without thermal cycling, and the products are visually detected on a nucleic acid strip. All steps are integrated into a handheld, lightweight device (9.9 × 4.4 × 3.3 cm, <200 g) that also prevents aerosol contamination. Using transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), the latter a natural mutant of TGEV, as a model, SCAN achieves a detection limit of 102 copies/μL with sensitivity comparable to RT-qPCR and supports sample-to-answer testing within 80 min and simple operations. With verified high sensitivity, specificity, and accuracy, as well as field usability, SCAN provides a generalizable route for developing point-of-care tests (PoCT) that require precise field differentiation of closely related pathogens.

Cross-priming isothermal amplification↗

PubMed2026

Liquid biopsy for early detection of pancreatic ductal adenocarcinoma.

There is no clinically relevant blood-based assay for the detection of early-stage pancreatic ductal adenocarcinoma (PDAC), a solid malignancy characterized by poor outcomes. Here we developed, validated and tested a blood-based microRNA (miRNA) assay (which included hsa-miR-142-3p, hsa-miR-30c-5p, hsa-miR-335-5p, hsa-miR-340-5p, hsa-miR-200b-3p, hsa-miR-1260b, hsa-miR-145-3p, hsa-miR-145-5p, hsa-miR-429 and hsa-miR-200a-3p) and a composite score, PANXEON (PANcreatic cancer eXosome Early detectiON), that integrates the miRNA signature with carbohydrate antigen 19-9 for the detection of early-stage PDAC. We conducted an international, multicenter, observational, prospective biomarker study that involved 1,785 individuals with and without PDAC from four countries. The miRNA signature achieved an area under the receiver operating characteristic curve of 88.6% in the testing cohort, with a sensitivity of 83.8% for early-stage PDAC, while showing minimal cross-reactivity with other gastrointestinal cancers. In a cohort of 19 individuals, the miRNA signature levels decreased during neoadjuvant chemotherapy and after surgery and increased before disease recurrence. When combined with carbohydrate antigen 19-9 levels, this blood assay demonstrated a sensitivity of 86.8% for stage I-II PDAC, false-positive rates of 3.2% in low-risk controls and 15.6% in high-risk controls in the testing cohort. PANXEON demonstrates potential for detecting high-grade dysplasia in individuals with high-risk pancreatic cysts (64.3%). Collectively, we present a composite biomarker that may complement existing strategies for the detection of early-stage PDAC and warrants further large-scale prospective studies. ClinicalTrials.gov registration: NCT06388967 .

Journal Article↗

PubMed2026

A smartphone-integrated plasmonic biosensor for amplification-free detection of African swine fever virus.

African Swine Fever Virus (ASFV) poses a catastrophic threat to global swine production, with recent outbreaks across Europe, Asia, and the Caribbean, significantly elevating the biosecurity risk to the United States' billion-dollar pork industry. Current diagnostic gold standards are laboratory-dependent and introduce critical delays in outbreak response. To address this gap, a plasmonic biosensor based on functionalized gold nanoparticles (GNPs) was developed for the rapid, amplification-free detection of ASFV. GNPs were surface-functionalized with 11-mercaptoundecanoic acid (MUDA) and combined in situ with ASFV-specific oligonucleotide probes targeting a conserved region of the p72 (B646L) gene. The detection mechanism relies on acid-induced aggregation: hybridization of target ASFV DNA to the probe generates a rigid duplex that shields the nanoparticles from acid-induced destabilization, maintaining a ruby-red color, whereas in the absence of target DNA the GNPs aggregate, producing a visible red-to-blue color shift. The optimized plasmonic biosensor demonstrated 100% analytical specificity, with no cross-reactivity against a panel of 19 non-target bacterial genomic DNA samples representative of the swine environment. Detection limits determined by the IUPAC 3σ criterion were 285 copies per reaction for Probe 1 and 402 copies per reaction for Probe 2, within the same order of magnitude as the qPCR reference assay run on the same dilution series (approximately 312 copies per reaction) under the experimental conditions used here. A smartphone-based Bio-Analytics App employing an RGB color-conversion algorithm served as a quantitative reader, yielding signal-to-noise ratios (S/N) that strongly correlated with benchtop spectrophotometric readings (A520/A620 ratio, R2 = 0.96) and achieved diagnostic concordance with qPCR binary calls. This platform offers a robust and low-cost (∼$2 per test), amplification-free approach to ASFV screening with potential for point-of-need deployment, subject to future validation in clinical specimens.

Journal Article↗

PubMed2026

A homogeneous immunoassay based on AlphaLICA technology for detecting florfenicol residues in animal-derived foods.

Florfenicol (FF), a broad-spectrum amide antibiotic widely used in livestock, poultry, and aquaculture, poses potential threats to food safety and public health due to its residual accumulation. In this study, a novel homogeneous immunoassay based on Amplified Luminescent Proximity Homogeneous Assay (AlphaLICA) technology was developed for the first time for rapid screening of FF residues in milk and egg matrices. By covalently immobilizing the FF-BSA conjugate and goat anti-mouse IgG onto luminescent and photosensitive microspheres, respectively, the method achieved wash-free, homogeneous quantitative detection through a competitive immunoreaction. Under optimized conditions, the assay exhibited a linear range of 0.2-16.2 ng mL-1, with a limit of detection of 9.7 pg mL-1 and a limit of quantification of 183 pg mL-1. The intra- and inter-batch coefficients of variation ranged from 3.08% to 5.70% and 2.44% to 7.09%, respectively. Spike recovery rates in milk and egg matrices ranged from 93.18% to 107.17% (RSD ≤ 5.57%). Cross-reactivity with 11 other common antibiotics, including chloramphenicol and thiamphenicol, was below 0.1%, demonstrating excellent specificity. Comparative analysis with a commercial ELISA kit showed high consistency (r2 = 0.9332, p < 0.001). With high sensitivity, strong specificity, simple operation, and a detection time of only 10 min, this method provides a reliable technical platform for high-throughput, rapid monitoring of FF residues in milk and egg matrices.

Journal Article↗

PubMed2026

Enhancing Hemoglobin Bart's hydrops fetalis syndrome prevention: a single-tube multiplex real-time PCR assay for the comprehensive detection of four significant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA) found in Thailand.

BACKGROUND: Hemoglobin (Hb) Bart's hydrops fetalis is a major public health concern in Southeast Asia, particularly in Thailand. Current screening strategies target the two most common α0 -thalassemia deletions (--SEA and --THAI). METHOD: In this study, we developed a single-tube multiplex real-time PCR assay for the simultaneous detection of four clinically relevant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA). The assay was validated using 538 clinical samples with diverse thalassemia genotypes and compared against conventional gap-PCR as the reference method. Analytical performance, including sensitivity, specificity, and limit of detection (LOD), was evaluated. In addition, clinical utility was assessed in 22 prenatal diagnosis cases at risk of Hb Bart's hydrops fetalis. RESULTS: The study cohort demonstrated substantial genetic heterogeneity, comprising 43 distinct genotypes. The developed assay achieved 100% sensitivity and specificity for all targeted deletions, with complete concordance with gap-PCR results. No cross-reactivity was observed with α+-thalassemia. The assay demonstrated a high analytical sensitivity with a LOD of 9.76 × 10-3 ng per reaction. Whereas in prenatal diagnosis, all 22 fetal genotypes were accurately identified, including five cases of homozygous --SEA and one rare compound heterozygous --SEA/--CR fetus. CONCLUSIONS: This study presents a rapid, accurate, and cost-effective multiplex real-time PCR assay capable of detecting both common and rare α0-thalassemia deletions in a single reaction. The assay demonstrates strong potential for implementation in routine clinical laboratories and large-scale population screening, contributing to improved prevention and control of severe thalassemia syndromes in high-prevalence regions.

Humans↗

PubMed2011

Safety of liver gene transfer following peripheral intravascular delivery of adeno-associated virus (AAV)-5 and AAV-6 in a large animal model.

Intravascular delivery of adeno-associated virus (AAV) vector is commonly used for liver-directed gene therapy. In humans, the high prevalence of neutralizing antibodies to AAV-2 capsid and the wide cross-reactivity with other serotypes hamper vector transduction efficacy. Moreover, the safety of gene-based approaches depends on vector biodistribution, vector dose, and route of administration. Here we sought to characterize the safety of AAV-5 and AAV-6 for liver-mediated human factor IX (hFIX) expression in rabbits at doses of 1 × 10(12) or 1 × 10(13) viral genomes/kg. Circulating therapeutic levels of FIX were observed in both cohorts of AAV-6-hFIX, whereas for AAV-5-hFIX only the high dose was effective. Long-lasting inhibitory antibodies to hFIX were detected in three of the 10 AAV-6-injected animals but were absent in the AAV-5 group. Overall, vector shedding in the semen was transient and vector dose-dependent. However, the kinetics of clearance were remarkably faster for AAV-5 (3-5 weeks) compared with AAV-6 (10-13 weeks). AAV-6 vector sequences outside the liver were minimal at 20-30 weeks post-injection. In contrast, AAV-5 exhibited relatively high amounts of vector DNA in tissues other than the liver. Together these data are useful to further define the safety and potential for clinical translation of these AAV vectors.

Animals↗

PubMed2026

Interlaboratory Comparison of a Glucagon and Oxyntomodulin Immuno-LC-MS/MS Assay: Implications for Diabetes Research.

BACKGROUND: The quantification of plasma glucagon and oxyntomodulin is important in the assessment of α-cell function, which is impaired in patients with diabetes. We aimed to transfer between laboratories a novel assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific measurement of these highly homologous hormones. METHODS: Simultaneous measurement of glucagon and oxyntomodulin used immunoaffinity enrichment and LC-MS/MS. Immunoenrichment used monoclonal antibodies that are available at-cost to researchers (deposited at the Developmental Studies Hybridoma Bank). Pure synthetic glucagon, characterized for purity and concentration, was used as a calibrant and is available to others. A detailed standard operating procedure was shared between 3 laboratories and the performance of the method was evaluated with samples collected from patients with and without diabetes. Method comparison was made with 2 commercially available, FDA-registered glucagon immunoassays. RESULTS: The method was linear over the normal range (1-20 pM). When measured in duplicate, the median interlaboratory imprecision (%CV) of the measurement of 40 samples was 6.3% (IQR 4.5%) and 14.4% (IQR 12.4%) for glucagon and oxyntomodulin, respectively. Method comparison with commercially available immunoassays demonstrated good (Mercodia, R = 0.92) or fair (Ansh, R = 0.70) agreement. Multivariable linear regression using LC-MS/MS glucagon and oxyntomodulin concentrations to predict immunoassay results indicated significant cross-reactivity of each immunoassay with oxyntomodulin. CONCLUSION: We have validated a sensitive and specific assay for glucagon and oxyntomodulin that can be deployed in high-complexity clinical laboratories for research or the care of patients. Commercially available glucagon immunoassays have significant interference from molecules other than glucagon.

Journal Article↗

PubMed2026

A droplet digital PCR assay targeting 16 human papillomavirus genotypes.

Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.

PCR↗