Assay Technology Research Articles

Abstract IA007: Modulating cfDNA biology to enhance liquid biopsies

Abstract Liquid biopsies could transform cancer care but require higher sensitivity to detect early-stage tumors and minimal residual disease (MRD) and to enable tumor molecular profiling and therapy response monitoring for most patients. In this talk, I will focus on the significant biological bottlenecks upstream of cell-free DNA (cfDNA) testing. I will posit that in many circumstances, insufficient circulating tumor DNA (ctDNA) drawn in a blood tube cannot be overcome by assay technology alone, and that in vivo modulation of cfDNA biology may also be required to enhance liquid biopsies. Inspired by widespread use of diagnostic drugs in other areas of medicine, we sought to create the first “priming agents” for liquid biopsies that could be administered before a blood draw to recover more ctDNA. I will describe two such approaches—a monoclonal antibody against double-stranded DNA and a liposome nanoparticle—that briefly attenuate cfDNA clearance by shielding it from nuclease digestion and cellular uptake. These enabled >10x more ctDNA to be collected when administered within 1-2 hours of a blood draw in mice. This improved the analytical limit of detection for ctDNA testing by >10-fold, provided more complete representation of the tumor genome in each blood sample, and increased the sensitivity for detection of small tumors from <10% to >75% in mice. Envisioning a future where liquid biopsies are optionally enhanced with priming, I will also explore a unique potential pairing with our tumor-informed, whole-genome, mutation enrichment sequencing MRD test called MAESTRO, and how it stands to enable routine ctDNA detection below 1 part per million. Citation Format: Viktor Adalsteinsson. Modulating cfDNA biology to enhance liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA007.

Characterisation and Sensitivity of a Canine Mast Cell Tumour Line to Oncolytic Viruses

ABSTRACTCanine mast cell tumours (MCTs) are one of the most common skin cancers of dogs. Surgical removal is the primary treatment, but recurrence and metastasis can occur even with low‐grade tumours. As a result, new treatment strategies are being sought. We tested the potential of several oncolytic viruses (OVs) to infect and kill a cell line isolated from a canine MCT. Employing a resazurin‐based metabolic assay and flow cytometry technology, we used recombinant vesicular stomatitis virus (rVSV‐Δm51), avian orthoavulavirus‐1 (AOaV‐1), and Orf viruses in our assessment. Our study aimed to evaluate the potential of oncolytic virotherapy in treating canine cancers. We found that MCT‐1 cells showed different sensitivities to the OVs, with rVSV‐Δm51 showing the most promising results in vitro. These findings suggest that further investigation into using OVs for treating canine MCTs is needed, although clinical efficacy is yet to be determined.

Proteomics analysis of extracellular vesicles for biomarkers of autism spectrum disorder

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include social interaction deficits, language difficulties and restricted, repetitive behavior. Early intervention through medication and behavioral therapy can eliminate some ASD-related symptoms and significantly improve the life-quality of the affected individuals. Currently, the diagnosis of ASD is highly limited.MethodsTo investigate the feasibility of early diagnosis of ASD, we tested extracellular vesicles (EVs) proteins obtained from ASD cases. First, plasma EVs were isolated from healthy controls (HCs) and ASD individuals and were analyzed using proximity extension assay (PEA) technology to quantify 1,196 protein expression level. Second, machine learning analysis and bioinformatic approaches were applied to explore how a combination of EV proteins could serve as biomarkers for ASD diagnosis.ResultsNo significant differences in the EV morphology and EV size distribution between HCs and ASD were observed, but the EV number was slightly lower in ASD plasma. We identified the top five downregulated proteins in plasma EVs isolated from ASD individuals: WW domain-containing protein 2 (WWP2), Heat shock protein 27 (HSP27), C-type lectin domain family 1 member B (CLEC1B), Cluster of differentiation 40 (CD40), and folate receptor alpha (FRalpha). Machine learning analysis and correlation analysis support the idea that these five EV proteins can be potential biomarkers for ASD.ConclusionWe identified the top five downregulated proteins in ASD EVs and examined that a combination of EV proteins could serve as biomarkers for ASD diagnosis.

Compact Digital Immunoassay Platform Integrating ELISA with a Lateral Flow Strip

Background/Objectives: On-site diagnosis of infection in their early stages requires assays with high sensitivities that are compact and easy to operate out of the laboratory and hospital environments. However, current assay technologies fall short of these requirements and require highly skilled technicians to set up, operate, and interpret the results. Methods: To address these challenges, we developed and evaluated a Point-of-Care-Testing (PoCT) immunoassay platform called the D-strip. The D-strip platform combines the capabilities of a digital enzyme-linked immunoassay (ELISA) with a lateral flow assay (LFA). The D-strip sample flow cell is composed of the same components found in conventional LFAs, and its high sensitivity is due to its efficient implementation of ELISA. The fully integrated platform is simple and requires minimal user intervention to operate. Results: The D-strip exhibited a sample-to-result time of 15 min with a limit of detection (LOD) of 1.7 × 103 copies/mL for severe acute respiratory syndrome coronavirus 2 (SARS-2-CoV) antigen. The LOD of the D-strip is 488-fold higher than that for conventional LFAs and is comparable to a clinical laboratory test. Conclusions: The D-strip is a compact and highly sensitive immunoassay platform with a strong potential for application as a confirmatory assay outside the clinical laboratory.

The development of a novel high-throughput membrane potential assay and a solid-supported membrane (SSM)-based electrophysiological assay to study the pharmacological inhibition of GLUT9/SLC2A9 isoforms in a drug discovery program

GLUT9/SLC2A9 is a urate transporter and takes a fundamental role in the maintenance of normal serum urate levels. GLUT9 is the sole transporter of reabsorbed urate from renal epithelial cells to blood, thus making it an ideal pharmacological target for the development of urate-lowering drugs. None of the three currently available assays for studying GLUT9 pharmacological inhibition can support a high throughput drug discovery screening campaign. In this manuscript we present two novel assay technologies which can be used in a drug discovery screening cascade for GLUT9: a GLUT9 membrane potential assay for primary screening; and a solid-supported membrane (SSM)-based supported electrophysiological assay for secondary screening.

Squeezable Hydrogel Microparticles for Single Extracellular Vesicle Protein Profiling.

Extracellular vesicles (EVs) are promising for molecular diagnostics, but current analyses are limited by the rarity and compositional heterogeneity of EV protein expression. Therefore, single EV profiling methods require high sensitivity, multiplexing, and throughput to address these issues. Here a single EV analysis technique that utilizes squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) is described as a scaffold to immobilize EVs and perform an integrated rolling circle amplification (RCA) assay for an ultra-sensitive and multiplex analysis of single EV proteins. EVs are prepared into MHPs in a high-throughput manner with droplet microfluidics and optimally labeled with antibody-oligonucleotide conjugates in MHPs without steric limitations. By designing MHPs with high compressibility, single EV protein signals are amplified as RCA products that can be aligned on the same plane by physically squeezing MHPs and visualized with low magnification. This method provides a simple and scalable single EV imaging analysis pipeline for identifying multiplex marker expression patterns from single EVs. For validation, the single EV heterogeneity of highly expressed cancer cell markers is profiled across different cancer cell lines. These findings exemplify squeezable MHPs as a robust platform with high sensitivity, multiplexing, and scalability for resolving single EV heterogeneity and advancing molecular assay technologies.

Plasma proteomics of all-cause and cause-specific mortality in individuals with and without type 2 diabetes: Results from two population-based KORA cohorts

Abstract Background Protein biomarkers may contribute to the identification of vulnerable subgroups for premature mortality in middle-aged and older adults. Purpose Given the distinctive impact of type 2 diabetes (T2D) on mortality rates and protein levels, this study aimed to explore the association of proteins with all-cause and cause-specific mortality separately among individuals with and without baseline T2D and to assess their impact on the prediction of all-cause mortality. Methods Using proximity extension assay technology, we measured 233 cardiovascular (CV) disease- and inflammation-related proteins in two population-based KORA (Cooperative Health Research in the Region of Augsburg) cohorts. The discovery KORA S4 study, with 15.6 years of follow-up, included 1545 participants (T2D group: 116 total, 62 CV, 31 cancer-related and 23 other-cause deaths; non-T2D group: 321 total, 114 CV, 120 cancer-related and 87 other-cause deaths). The validation KORA-Age1 study, with 6.9 years of follow-up, included 1031 participants (T2D group: 76 total, 45 CV, 19 cancer-related and 12 other-cause deaths; non-T2D group: 169 total, 74 CV, 39 cancer-related and 56 other-cause deaths). Cox regression was used to examine associations of proteins with all-cause and cause-specific mortality. Furthermore, eXtreme Gradient Boosting was applied for identification of the most relevant proteins for the prediction of all-cause mortality stratifying by baseline T2D status. The predictive performance of the protein-based model and combined model including both proteins and clinical risk factors was compared to a clinical risk factor-based model using the C-index, category-free net reclassification index (cfNRI), and relative integrated discrimination improvement (IDI). Results After validation, we identified 48 proteins associated with all-cause mortality in the T2D group and 64 in the non-T2D group, respectively (37 overlapping proteins). Out of these, 45, eight, and 32 were associated with CV, cancer-related, and other-cause mortality in the T2D group, while 53, 42, and 48 were associated with the respective cause-specific mortality outcomes in the non-T2D group in the pooled analysis of both studies. The combined model improved the prediction of all-cause mortality in both the KORA S4 and KORA-Age1 cohorts among individuals with baseline T2D (KORA-Age1: ΔC-index: 0.065, 95%=0.009-0.129; cfNRI: 0.370, 95%=0.203-0.812; IDI: 0.129, 95%=0.059-0.253) as well as those without T2D (KORA-Age1: ΔC-index: 0.037, 95%=0.015-0.071; cfNRI: 0.370, 95%=0.236-0.632; IDI: 0.053, 95%=0.028-0.104), respectively, compared to the clinical model. Conclusion This study discovered shared and unique proteins in individuals with and without T2D, unveiling the complex dynamics of mortality. Moreover, our findings support the role of proteins in improving prediction of premature mortality beyond clinical risk factors in different T2D subgroups, providing insights for future prevention strategies.

Sex-biased proteomic regulation in obesity

Abstract Background Obesity is a major risk factor for heart failure with preserved ejection fraction, particularly in women. Obesity-associated inflammation is thought to contribute to this relationship. However, the role of biological sex in the regulation of circulating inflammatory proteins in obesity is not well understood. Purpose We aimed to explore sex differences in the plasma inflammatory protein profile in obese subjects. Methods Clinical data and samples were collected from 450 women and men with a body mass index (BMI) >27 kg/m² without known cardiovascular disease participating in the FAT associated CardiOvasculaR dysfunction study. Obesity was defined as BMI≥30 kg/m². Olink’s Target 96 inflammation panel, which profiles 92 proteins using proximity extension assay technology, was analysed in biobank samples. A comprehensive correlation analysis was performed to describe relationships between protein biomarkers and clinical variables. Gene enrichment analysis was performed to shed light on biological pathways associated with differentially expressed proteins (DEPs). To determine the functional significance of the DEPs, we used the repositories Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Pearson correlation coefficients were calculated to assess linear associations between the DEPs and relevant clinical variables. Results Women and men did not differ by age (49±9 vs 48±9 years), BMI (32.3±4.6 vs 31.6±3.7 kg/m²) or in prevalences of diabetes (both 11%) or obesity (65% vs 62%) (all p>0.05). However, hypertension was more common in men than women (74% vs 52%, p<0.001). In men (n=188), obesity was associated with downregulation of monocyte chemotactic protein 3, tumor necrosis factor (ligand) superfamily member 12, interleukin 10 and protein S100-A12 and with upregulation of neurotrophin-3 (all p<0.05), thereby suggesting a mechanism for the observed impaired inflammatory response and insulin resistance. In women (n=262), obesity was associated with the downregulation of interleukin 7, interleukin 12 subunit beta, interleukin 6, and C-X-C motif chemokine 11, suggesting a state of persistent low-grade inflammation. In addition, the upregulated sulfotransferase 1A1 and SIR2-like protein 2 proteins indicate a physiologic adaptation to oxidative stress and the metabolic excess endemic to obesity. Conclusion We found that the abundance of several circulating inflammatory proteins was altered in presence of obesity, and that the plasma proteomic profile differed by sex. Interestingly, there was no overlap in the DEPs between women and men in the presence of obesity. These findings support the hypothesis that the pathophysiological mechanisms driving obesity-associated inflammation and immune dysregulation differ by sex. In turn, this may aid in the pursuit of discovering sex-specific biomarkers to identify obese individuals at particular risk of heart failure.

Comparative Analysis of Quantitative Methods for Campylobacter spp. Quantification: ISO 10272-2:2017, Tempo® and Real-Time PCR in Refrigerated and Frozen Turkey Cuts

New technologies for more effective microbiological assays are being adopted by the food industry to intervene more rapidly in its production chain. The aim of this study was to evaluate the alternative methods of TEMPO® CAM and real-time PCR (rtPCR) Biotecon® in comparison with the ISO 10272-2:2017 reference method for Campylobacter spp. quantification in turkey meat, aiming to validate a quick and easily replicable method in these meat matrices. A total of 416 samples were analyzed over a one-year period. The TEMPO® methodology showed inadequate performance with a significant difference (p < 0.05) compared with the reference methodology; therefore, its use was not recommended for turkey meat matrices. However, the performance of the rtPCR Biotecon® methodology showed adequate performance with no significant difference (p > 0.05), and its use was recommended in turkey meat matrices. The study was limited to exclusive research in turkey meat matrices, and expansion of the research into other matrices is recommended to verify whether the behavior of alternative methodologies is similar. The findings of this study illustrate the necessity for a thorough and comprehensive evaluation during the implementation of alternative methodologies that may potentially supplant conventional approaches.

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

Machine Learning Approaches in Multi-Cancer Early Detection

Cancer is a prominent global cause of mortality, primarily due to delayed detection leading to limited treatment options. Current screening methods are mostly invasive and involve complex lengthy processes with high costs. Moreover, each screening typically focuses on a single type of cancer. This imposes a growing need for innovative, precise, and minimally invasive methods for early cancer detection. With the current advances in assay technologies and data science, multi-cancer early detection (MCED) tests are gaining increased interest in the research community as they offer potential for earlier diagnosis and improved patient outcomes. Different approaches are followed for MCED, and multiple machine learning methods are considered. In this paper, we systematically explore various MCED studies and their applied machine learning (ML) models for different types of biomarker data. We discuss the strengths and limitations of different study designs and compare their performance. Future directions are proposed, emphasizing the importance of integrating multi-omics data, enhancing model transparency, and fostering collaborative efforts to develop robust, cost effective and clinically applicable MCED tools.

Prevalence of ANA and Anti-GBM Antibodies among Patients with Renal Failure in Tikrit City

Background: End-stage renal disease (ESRD) people need regular hemodialysis because they have chronic renal disease, which is a major health issue. Aim: This study aimed to evaluate the prevalence and characteristics of ANA and Anti-GBM antibodies, associated infections, and bacterial isolates in hemodialysis patients, comparing the findings with a healthy control group. Materials and Methods: A cross-sectional hospital-based study was conducted from December 1, 2023, to May 31, 2024, at Tikrit Teaching Hospital. The study comprised a cohort of 240 patients, aged 15-75 years, who were diagnosed with chronic renal disease and were receiving hemodialysis. Additionally, a control group of 40 healthy persons was included. Enzyme-linked immunosorbent assay (ELISA) technology was utilized to collect blood samples for the purpose of detecting ANA and Anti-GBM antibodies. Results: There was a modest preponderance of men in the sex distribution of hemodialysis patients. The age distribution was adequately represented throughout several decades. The investigation revealed notable disparities in the occurrence of ANA and Anti-GBM antibodies between hemodialysis patients and the control group. Out of the patients undergoing hemodialysis, 32.29% were found to have ANA antibodies, and 29.17% were found to have Anti-GBM antibodies. In contrast, none of the individuals in the control group tested positive for either type of antibody. The prevalence of Hepatitis C and B viruses among hemodialysis patients was 8.33% and 5.21% respectively. 52.08% of the patients had diabetes, whereas 57.29% had hypertension. In addition, the bacteria obtained from the mouth exhibited a varied microbial composition, with the most common species being Staphylococcus epidermidis and Streptococcus viridans. Conclusion: This study emphasizes the notable occurrence of ANA and Anti-GBM antibodies in hemodialysis patients, which is associated with a heightened susceptibility to urinary tract infections (UTIs). Implementing efficient infection control techniques and enhancing oral hygiene practices are crucial for effectively controlling infections in hemodialysis patients. Additional investigation is required to examine the intricacies of the microbial composition in this particular group and to formulate thorough measures for preventing infections.

The PreS-Based Recombinant Vaccine VVX001 Induces Hepatitis B Virus Neutralizing Antibodies in a Low-Responder to HBsAg-Based HBV Vaccines

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

Using Spectral Flow Cytometry for CAR T-Cell Clinical Trials: Game Changing Technologies Enabling Novel Therapies

Monitoring chimeric antigen redirected (CAR) T-cells post-infusion in clinical trials is a specialized application of flow cytometry. Unlike the CAR T-cell monitoring for individual patients conducted in clinical laboratories, the data generated during a clinical trial will be used not only to monitor the therapeutic response of a single patient, but determine the success of the therapy itself, or even of an entire class of therapeutic compounds. The data, typically acquired at multiple testing laboratories, will be compiled into a single database. The data may also be used for mathematical modeling of cellular kinetics or to identify predictive biomarkers. With the expanded context of use, a robust, standardized assay is mandatory in order to generate a valuable and reliable data set. Hence, the requirements for assay validation, traceable calibration, technology transfer, cross-instrument standardization and regulatory compliance are high.

Localized surface plasmon resonance-enhanced photoelectrochemical immunoassay based on high-entropy yolk-shell Au@(ZnCdMnGaCu)xS

The yolk-shell structure has become a research hotspot in the field of photocatalysis in recent years due to its stability, movable yolk, and internal voids. However, yolk-shell has not been fully explored and applied in photoelectrochemical (PEC) immunoassays. In this work, a smartphone-based portable PEC immunoassay was constructed by using high-entropy yolk-shell Au@(ZnCdMnGaCu)xS as a photosensitive material, showing satisfactory detection with the assistance of localized surface plasmon resonance (LSPR). Specifically, a typical sandwich reaction introduces a detection antibody modified and labeled with gold nanoparticles (Au NPs) of alkaline phosphatase (ALP) into the system with the presence of prostate-specific antigen (PSA). Ascorbic acid (AA) content increased with increasing PSA and showed a linear enhancement of photocurrent at PSA concentrations of 0.01–50 ng mL−1 with a limit of detection of 8.94 pg mL−1. Additionally, the proposed assay technology not only provides a portable detection system for the immediate detection of tumor markers, but also offers a promising paradigm for the development of high-entropy materials.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Plasma Inflammatory Proteome Profile in a Cohort of Patients with Recurrent Vulvovaginal Candidiasis in Kenya.

Vulvovaginal candidiasis (VVC) affects up to 75% of women at least once during their lifetime, and up to 8% of women suffer from frequent recurrent episodes of VVC (RVVC). A lack of a protective host response underlies vaginal Candida infections, while a dysregulated hyperinflammatory response may drive RVVC. This study aimed to investigate the systemic inflammatory protein profile in women with RVVC in an African population, considering the potential influence of hormonal contraceptive use on systemic inflammation. Using multiplex Proximity Extension Assay technology, we measured 92 circulatory inflammatory proteins in plasma samples from 158 RVVC patients and 92 asymptomatic women (controls). Hormonal contraceptive use was not found to have a statistically significant correlation with a systemic inflammatory protein profile in either RVVC patients or the asymptomatic women. RVVC women had lower circulating Fibroblast Growth Factor 21 (FGF-21) concentrations compared with healthy controls (adjusted p value = 0.028). Reduced concentrations of FGF-21 may be linked to the immune pathology observed in RVVC cases through IL-1β. This study may help to identify new biomarkers for the diagnosis and future development of novel immunomodulatory treatments for RVVC.

Challenges and Approaches to Establishing Multi-Pathogen Serosurveillance: Findings from the 2023 Serosurveillance Summit.

Multiplex-based serological surveillance is a valuable but underutilized tool to understand gaps in population-level exposure, susceptibility, and immunity to infectious diseases. Assays for which blood samples can be tested for antibodies against several pathogens simultaneously, such as multiplex bead immunoassays, can more efficiently integrate public health surveillance in low- and middle-income countries. On March 7-8, 2023 a group of experts representing research institutions, multilateral organizations, private industry, and country partners met to discuss experiences, identify challenges and solutions, and create a community of practice for integrated, multi-pathogen serosurveillance using multiplex bead assay technologies. Participants were divided into six working groups: 1) supply chain; 2) laboratory assays; 3) seroepidemiology; 4) data analytics; 5) sustainable implementation; and 6) use case scenarios. These working groups discussed experiences, challenges, solutions, and research needs to facilitate integrated, multi-pathogen serosurveillance for public health. Several solutions were proposed to address challenges that cut across working groups.

The intestinal microbiome of infants with cow's milk-induced FPIES is enriched in taxa and genes of enterobacteria.

Food protein-induced enterocolitis syndrome (FPIES) is a severe type of non-IgE (immunoglobulin E)-mediated (NIM) food allergy, with cow's milk (CM) being the most common offending food. The relationship between the gut microbiota and its metabolites with the inflammatory process in infants with CM FPIES is unknown, although evidence suggests a microbial dysbiosis in NIM patients. This study was performed to contribute to the knowledge of the interaction between the gut microbiota and its derived metabolites with the local immune system in feces of infants with CM FPIES at diagnosis. Twelve infants with CM FPIES and a matched healthy control group were recruited and the gut microbiota was investigated by 16S amplicon and shotgun sequencing. Fatty acids (FAs) were measured by gas chromatography, while immune factors were determined by enzyme-linked immunosorbent assay and Luminex technology. A specific pattern of microbiota in the gut of CM FPIES patients was found, characterized by a high abundance of enterobacteria. Also, an intense excretion of FAs in the feces of these infants was observed. Furthermore, correlations were found between fecal bifidobacteria and immune factors. These fecal determinations may be useful to gain insight into the pathophysiology of this syndrome and should be taken in consideration for future studies of FPIES patients.

Simultaneous noninvasive ultrasensitive detection of prostate specific antigen and lncRNA PCA3 using multiplexed dual optical microfibers with strong plasmonic nanointerfaces

Low accuracy of diagnosing prostate cancer (PCa) was easily caused by only assaying single prostate specific antigen (PSA) biomarker. Although conventional reported methods for simultaneous detection of two specific PCa biomarkers could improve the diagnostic efficiency and accuracy, low detection sensitivity restrained their use in extreme early-stage PCa clinical assay applications. In order to overcome above drawbacks, this paper herein proposed a multiplexed dual optical microfibers separately functionalized with gold nanorods (GNRs) and Au nanobipyramids (Au NBPs) nanointerfaces with strong localized surface plasmon resonance (LSPR) effects. The sensors could simultaneously detect PSA protein biomarker and long noncoding RNA prostate cancer antigen 3 (lncRNA PCA3) with ultrahigh sensitivity and remarkable specificity. Consequently, the proposed dual optical microfibers multiplexed biosensors could detect the PSA protein and lncRNA PCA3 with ultra-low limit-of-detections (LODs) of 3.97 × 10−15 mol/L and 1.56 × 10−14 mol/L in pure phosphorus buffer solution (PBS), respectively, in which the obtained LODs were three orders of magnitude lower than existed state-of-the-art PCa assay technologies. Additionally, the sensors could discriminate target components from complicated physiological environment, that showing noticeable biosensing specificity of the sensors. With good performances of the sensors, they could successfully assay PSA and lncRNA PCA3 in undiluted human serum and urine simultaneously, respectively. Consequently, our proposed multiplexed sensors could real-time high-sensitivity simultaneously detect complicated human samples, that providing a novel valuable approach for the high-accurate diagnosis of early-stage PCa individuals.