Electrochemiluminescence Assay Research Articles

Normative Data on Dehydroepiandrosterone Sulphate (DHEAS) and its Attributes in Healthy Indian Children and Adolescents.

Dehydroepiandrosterone sulphate (DHEAS), the biochemical indicator of adrenarche and pubarche, is of paramount importance in the evaluation of puberty-related disorders. The reference range of DHEAS should be ethnicity, age, sex, pubarche and Tanner stage specific. Anthropometry, puberty assessment and hormonal parameters were estimated using electrochemiluminescence assay. Bone age was estimated using the BoneXpert software. Of 2191 healthy Indian children aged 5-18 y screened at Chandigarh, 1919 were included in the final analysis (994 boys). The median DHEAS levels at pubarche stage P2 were 82.10 (55.0-129.0) g/dl in girls and 132.50 (95.12-205.50) g/dl in boys. By ROC analysis, the level of DHEAS at pubarche was 63.7g/dl (sensitivity 72.6%, specificity 64.4%) in girls and 82.2g/dl (sensitivity 81.8%, specificity 68.8%) in boys. The median age at adrenarche was 9.5 y in both sexes. On multivariate regression analysis; bone age, body mass index (BMI), gonadal steroids, and insulin-like growth factor-1(IGF-1) significantly correlated with serum DHEAS levels in either sex.

Reference Intervals of Serum Iodine Concentration in Chinese Pregnant Women.

The study aims to establish trimester-specific reference ranges for serum iodine (SI) in Chinese pregnant women and explore its associations with maternal and infantile thyroid function. Apparently healthy pregnant women were enrolled during their first antenatal visit. Fasting venous and spot urine samples were collected for determining serum and urinary iodine (UI) levels by a validated inductively coupled plasma mass spectrometry. Serum free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin (TSH), and neonatal TSH levels were tested by electro-chemiluminescent assay. The reference ranges of SI were established by percentile method and reported as 2.5-97.5%. ROC analysis was applied to compare the discriminative ability of SI, UI, and UI to urinary creatine ratio (UI /UCr) in early pregnancy for various thyroid conditions. The trimester-specific reference ranges of SI for Chinese pregnant women were 60.91-114.53 μg/L for the first trimester (T1, n = 1029), 54.57-103.42 μg/L for the second trimester (T2, n = 379), and 52.03-110.40 μg/L for the third trimester (T3, n = 455). Maternal SI at T1 but not UI and UI/UCr was significantly correlated with FT3 (r = 0.393, P < 0.001), FT4 (r = 0.637, P < 0.001), and TSH (r = -0.299, P<0.001). Maternal SI change% from T1 to T2 (but not SI change% from T1 to T3) had marginal correlation with neonatal TSH (r=-0.106, P=0.046). ROC analysis showed that maternal SI at T1 had better predictability for several thyroid conditions than UIC and UI/UCr.

The free cortisol calculated: correlation with the free cortisol concentrations measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis and establishment of reference intervals

Background Changes in cortisol binding globulin (CBG) impact the total serum cortisol concentration and affect the accurate assessment of adrenal function. Free biologically cortisol can be calculated using different equations or directly measured after complicated procedures. Methods The free cortisol index (FCI) obtained using the Bonte formula as well as the free cortisol concentration calculated (Coolens equation) were first estimated for 45 healthy workers. The CBG level was determined by a competitive radioimmunoassay and the total cortisol concentration, was measured with an electrochemiluminescent assay. The correlations between FCI, the free cortisol concentrations calculated and the free cortisol levels measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis were studied for those 45 samples. Reference limits were established on 158 healthy hospital workers and patients with serum samples collected between 7:30 am and 10 am. Results The FCI as well as the free cortisol concentrations calculated obtained for the 45 samples correlated significantly with the free cortisol levels measured. Although the cortisol and CBG levels were statistically higher in women using contraceptives compared with women not taking them as well as men, the calculated FCI and free cortisol concentrations did not differ between these groups. The medians (P2.5-P97.5) obtained for the 158 healthy workers were respectively 26.4% (12.3-51.6%) and 10.6 nmol/L (4.3-26.7 nmol/L). Conclusions This study highlighted a significant correlation between the FCI, the free cortisol concentrations calculated and the free cortisol levels measured with LC-MS/MS, it has also allowed the establishment of reference intervals for calculated FCI and free cortisol.

Quantification of anti-drug antibodies against E6011, an anti-fractalkine monoclonal antibody, in monkey and human serum, by an electrochemiluminescence assay

E6011, a humanized anti-fractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. Therapeutic antibodies may induce production of anti-drug antibodies (ADA) that may deteriorate efficacy and/or enhance immunogenic reaction. It is important to have an ADA assay to understand the characteristics of biotherapeutics under development. A simple and reproducible assay has thus been developed for the determination of ADA against E6011 in monkey and human serum by electrochemiluminescence (ECL) detection. An immune-complex of biotinylated E6011, ADA, and ruthenium-labeled E6011 was attached to avidin-coated wells for ECL signal detection. Screening and confirmatory cutpoints were determined to judge negative or positive ADA. Sensitivity of ADA was 1.61 and 1.34 ng/mL in monkey and human serum, respectively. Accuracy and precision of the assay were within ±20% and 20%, respectively. Drug tolerance of the assay in monkey and human sera was ensured up to 100 and 1000 μg/mL E6011 at the surrogate ADA levels of 1 and 4 μg/mL, respectively. The developed assay was successfully applied to ADA quantification in monkeys and humans in support of immunogenicity assessments.

Low-Background Signal-On Homogeneous Electrochemiluminescence Biosensor for Hepatitis B Virus Detection Based on the Regulation of the Length of DNA Modified on the Nanoparticles by CRISPR/Cas12a and Hybridization Chain Reaction.

In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction. The efficiency of the nanoparticles reaching the negatively charged working electrode can be regulated and realize the change of the ECL signal. In addition, long DNA on the surface of the luminescent body may prevent the coreactant from entering the pore to react with ruthenium bipyridine. These factors combine to produce a low-background system. The presence of the target can activate the CRISPR/Cas12a system and make trigger DNA disappear from the nanoparticle surface, and strong ECL can be detected. The sensor does not require a complex electrode modification; therefore, it has better reproducibility. Additionally, due to dual signal amplification, the sensor has a high sensitivity. In the range of 10 fM to 10 nM, the ECL intensity exhibits a strong linear relationship with the logarithm of the target concentration, and the detection limit is 7.41 fM. This sensor has shown high accuracy in detecting clinical samples, which holds significant potential for application in clinical testing.

Identification of Autoantibodies to a Hybrid Insulin Peptide in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that attacks the insulin-producing b cells of the pancreatic islets. Autoantibodies to b cell proteins typically appear in the circulation years before disease onset, and serve as the most accurate biomarkers of T1D risk. Our laboratory has recently discovered novel b cell proteins comprising hybrid proinsulin:islet amyloid polypeptide peptides (IAPP). T cells from a diabetic mouse model and T1D patients are activated by these hybrid peptides. In this study, we asked whether these hybrid molecules could serve as antigens for autoantibodies in T1D and prediabetic patients. We analyzed sera from T1D patients, prediabetics and healthy age-matched donors. Using a highly sensitive electrochemiluminescence assay, sera were screened for binding to recombinant proinsulin:IAPP probes or truncated derivatives. Our results show that sera from T1D patients contain antibodies that bind larger hybrid proinsulin:IAPP probes, but not proinsulin or insulin, at significantly increased frequencies compared to normal donors. Examination of sera from prediabetic patients confirms titers of antibodies to these hybrid probes in more than 80% of individuals, often before seroconversion. These results suggest that hybrid insulin peptides are common autoantigens in T1D and prediabetic patients, and that antibodies to these peptides may serve as valuable early biomarkers of the disease.

Erythrocytic alpha-synuclein as potential biomarker for the differentiation between essential tremor and Parkinson's disease.

The differentiation between essential tremor (ET) and Parkinson's disease (PD) can be difficult because of the symptom overlaps. Erythrocytes are the major source of peripheral α-synuclein (α-syn), which is the most studied pathological molecular of PD. We have reported that erythrocytic α-syn levels in PD patients are significantly increased compared to those in healthy controls (HCs). However, little is known about the levels of erythrocytic α-syn species in ET patients. This study includes 15 patients with ET, 64 patients with PD, and 49 age and sex matched HCs. A well-established electrochemiluminescence assay was used to measure the erythrocytic total and aggregated α-syn levels. The receiver operating characteristic (ROC) curve analysis was applied to evaluate the diagnostic values of erythrocytic α-syn for ET diagnosis and differentiation. The correlations of erythrocytic α-syn levels with disease durations were tested using Spearman's Rank Correlation analysis. We found that both erythrocytic total and aggregated α-syn concentrations are significantly increased in PD and ET patients compared to those in HCs. Erythrocytic total α-syn levels are significantly higher in ET patients than those in PD group. Furthermore, the ratios of erythrocytic aggregated to total α-syn levels in ET patients are significantly decreased than those in PD and HC subjects. We also found a significant association of erythrocytic aggregated α-syn levels with the disease duration of ET patients. Our findings suggest new insight into the changes of erythrocytic total and aggregated α-syn levels as potential biomarkers for ET patients.

SERUM TESTOSTERONE AS A MARKER OF RESPONSE TO ANDROGEN DEPRIVATION THERAPY IN METASTATIC PROSTATE CANCER

Introduction: Prostate cancer (PCa) is the second most widespread cause of cancer and the sixth foremost cause of cancer death among men worldwide. The sustained presence of particular hormones and growth factors stimulates the prostate, such as other sex accessory tissues, in its growth, maintenance, and secretory function. In the current study, we investigated the association of serum testosterone level and prostate-specific antigen (PSA) level before androgen deprivation therapy in metastatic PCa (mPCa) treated with hormone therapy. Methods: This retrospective cohort was conducted from July 2017 to December 2019 in the Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh on patients diagnosed with mPCa of age &gt;40 years. Total serum testosterone was measured by Electro chemiluminescent immune assay before the treatment. PSA in serum specimens was evaluated. Results: The mean age was 67.74±8.12 (55–80) years. Serum PSA levels at baseline, 3 months were 530.98±617.28 (ng/dL), and 56.31±89.04 (ng/dL), respectively. The baseline S. testosterone was 288.89±246.53 (ng/mL). Weak negative correlation between the two (p=0.051) As the serum testosterone levels decrease the PSA levels increase and vice-versa. Positive correlation between baseline testosterone and PSA decline (p&lt;0.05). On histopathology, 58.7% (n=71) of the patients had perineural involvement, while 41.3% (n=50) did not have perineural involvement. Conclusion: We conclude that the effect of testosterone might have a possible role in the assessment of treatment response as assessed by PSA. However, the exact implication and its role in disease assessment need to be examined in a larger prospective cohort.

Serum Concentrations of Infliximab and IL-6 for Predicting One-Year Discontinuation of Infliximab Treatment Owing to Secondary Non-response in Patients with Rheumatoid Arthritis.

Secondary non-response to infliximab (IFX) occurs in some patients with rheumatoid arthritis (RA). Although therapeutic drug monitoring (TDM) is a useful tool to optimize IFX therapy, it is unclear whether it can help to identify the risk of secondary non-response. This study aimed to explore the utility of serum levels of IFX or other biomarkers to predict IFX discontinuation owing to secondary non-response. A single-center, retrospective study was conducted using the Kyoto University Rheumatoid Arthritis Management Alliance cohort database between 2011 and 2020. Serum IFX levels were measured using liquid chromatography-tandem mass spectrometry. An electrochemiluminescence assay was used to quantify serum levels of tumor necrosis factor-α and interleukin-6 and detect anti-drug antibodies. Eighty-four out of 310 patients were eligible for this study. The cutoff levels of biomarkers were determined by receiver operating characteristic analysis. IFX persistence was similar between groups stratified using IFX levels, tumor necrosis factor-α levels, interleukin-6 levels, and anti-drug antibodies positivity. The group with lower IFX and higher interleukin-6 levels had the worst therapy persistence (p = 0.017) and the most frequent disease worsening (90.0%, p < 0.001). Evaluating both interleukin-6 and IFX levels, not just IFX alone, enabled us to identify patients at risk of discontinuing IFX treatment. These findings support the utility of measuring IFX and interleukin-6 levels for successful maintenance therapy for RA.

Filgotinib Modulates Inflammation-Associated Peripheral Blood Protein Biomarkers in Adults with Active Rheumatoid Arthritis and Prior Inadequate Response to Methotrexate.

Our aim was to evaluate protein biomarker changes related to the administration of filgotinib, a Janus kinase (JAK) 1 preferential inhibitor, in patients with moderately to severely active rheumatoid arthritis (RA) with inadequate response to methotrexate. Plasma and serum samples were collected from patients enrolled in FINCH 1 (NCT02889796), a Phase 3 trial. Patients with stable backgrounds of methotrexate were randomly assigned once-daily oral filgotinib 200 or 100mg, subcutaneous adalimumab 40mg every 2weeks (W), or placebo. Up to 35 biomarkers were analyzed at baseline, W4, and W12 with enzyme-linked immunosorbent assays and chemiluminescence and electrochemiluminescence assays. At baseline, four distinct biomarker clusters were identified. The strongest intragroup correlations were in bone-cartilage resorption/inflammation and JAK/signal transducer and activator of transcription (STAT) signaling activity. At baseline, significant positive correlations were identified for cytokines with patient-reported pain and with patient measures of fatigue. Filgotinib reduced levels of cytokines associated with inflammation and cell migration as early as W4 and through W12. Compared to adalimumab, filgotinib induced significant reductions in bone-related turnover biomarkers, N-telopeptide of type 1 collagen and C-telopeptide 1, as well as biomarkers associated with baseline disease activity. No baseline predictors of therapeutic response to filgotinib were identified. Filgotinib reduced peripheral protein biomarkers associated with JAK/STAT signaling, inflammatory signaling, immune cell migration, and bone resorption as soon as W4 in FINCH 1. Effects were dose-dependent and consistent with the clinical efficacy of filgotinib observed in FINCH 1. The changes in peripheral biomarkers associated with filgotinib treatment in methotrexate-experienced patients are consistent with changes observed in both methotrexate-naïve and biologic disease-modifying antirheumatic drug-experienced RA populations. These data demonstrate dose-dependent effects of preferential JAK1 inhibition by filgotinib on peripheral blood protein biomarkers in methotrexate-experienced patients with RA. ClinicalTrials.gov, NCT02889796.

NPTX2 in Cerebrospinal Fluid Predicts the Progression From Normal Cognition to Mild Cognitive Impairment.

This study examined whether cerebrospinal fluid (CSF) baseline levels of the synaptic protein NPTX2 predict time to onset of symptoms of mild cognitive impairment (MCI), both alone and when accounting for traditional CSF Alzheimer's disease (AD) biomarker levels. Longitudinal NPTX2 levels were also examined. CSF was collected longitudinally from 269 cognitively normal BIOCARD Study participants (mean baseline age = 57.7 years; mean follow-up = 16.3 years; n = 77 progressed to MCI/dementia). NPTX2 levels were measured from 3 correlated peptides using quantitative parallel reaction monitoring mass spectrometry. Levels of Aβ42 /Aβ40 , p-tau181 , and t-tau were measured from the same CSF specimens using Lumipulse automated electrochemiluminescence assays. In Cox regression models, lower baseline NPTX2 levels were associated with an earlier time to MCI symptom onset (hazard ratio [HR] = 0.76, SE = 0.09, p = 0.023). This association was significant for progression within 7 years (p = 0.036) and after 7 years from baseline (p = 0.001). Baseline NPTX2 levels improved prediction of time to MCI symptom onset after accounting for baseline AD biomarker levels (p < 0.01), and NPTX2 did not interact with the CSF AD biomarkers or APOE-ε4 genetic status. In linear mixed effects models, higher baseline p-tau181 and t-tau levels were associated with higher baseline levels of NPTX2 (both p < 0.001) and greater rates of NPTX2 declines over time. NPTX2 may be a valuable prognostic biomarker during preclinical AD that provides additive and independent prediction of MCI onset among individuals who are cognitively normal. We hypothesize that NPTX2-mediated circuit homeostasis confers resilience during the early phase of AD. ANN NEUROL 2023;94:620-631.

Potential role between inflammatory cytokines and Tie-2 receptor levels and clinical symptoms in patients with first-episode schizophrenia

BackgroundSchizophrenia (SCZ) is associated with chronic low-grade inflammation, which may be involved in the underlying pathological mechanism of the disease and may influence patient prognosis. We evaluated the differences in serum cytokine and Tie-2 receptor levels between patients with first-episode SCZ and healthy controls and explored the correlation thereof with clinical symptoms.MethodsSeventy-six participants were recruited for the present study, including 40 patients with first-episode SCZ and 36 healthy controls. Positive and Negative Syndrome Scale (PANSS) and Brief Psychiatric Rating Scale (BPRS) scores, demographic data, and blood samples were collected at baseline. A hypersensitive Meso Scale Discovery (MSD) electrochemiluminescence assay system was used to measure cytokine and Tie-2 receptor levels. Spearman’s correlation and stepwise linear regression were used to analyze the data.ResultsSerum interleukin-1β and -4 levels were significantly increased, and Tie-2 levels were significantly decreased, in first-episode SCZ patients as compared to healthy controls. IL-1β levels were positively correlated with total BPRS scores, resistance subscores, and PANSS positive subscores. Furthermore, IL-1β levels were negatively correlated with Tie-2 receptor expression levels. Stepwise linear regression analysis demonstrated that IL-1β levels correlated positively with PANSS positive subscores and BPRS total scores. PANSS negative subscores, general psychopathology subscores, and PANSS total scores had positive effects on the Tie-2 receptor. Receiver operating characteristic (ROC) curve analysis showed that IL-1β and Tie-2 were highly sensitive and specific for predicting first-episode SCZ symptoms and achieving an area under the ROC curve of 0.8361 and 0.6462, respectively.ConclusionOur results showed that patients with first-episode SCZ have low-grade inflammation. IL-1β and Tie-2 receptors may be important mediators between inflammation and vascular dysfunction in patients with SCZ and may underlie the increased cardiovascular disease in this population.Trial registrationThe clinical trial registration date was 06/11/2018, registration number was chiCTR1800019343.

Evaluation of Plasma Calcitonin Gene-Related Peptide as a Biomarker for Painful Temporomandibular Disorder and Migraine.

To assess associations of plasma calcitonin gene-related peptide (CGRP) with chronic temporomandibular disorder (TMD) myalgia/arthralgia or frequent/chronic migraine, alone and in combination, and to evaluate relations between the CGRP concentration and clinical, psychological, and somatosensory characteristics of participants. The cross-sectional study selected four groups of adult volunteers: healthy controls (HCs), TMD without migraine, migraine without TMD, and TMD with migraine. Each group comprised 20 participants, providing 94% power to detect statistically significant associations with CGRP concentration for either TMD or migraine. TMD and headache were classified according to the Diagnostic Criteria for TMD and the International Classification for Headache Disorders, 3rd edition, respectively. Plasma CGRP was quantified with a validated high-sensitivity electrochemiluminescent Meso Scale Discovery assay. Questionnaires and clinical examinations were used to evaluate characteristics of TMD, headache, psychological distress, and pressure pain sensitivity. Univariate regression models quantified associations of the CGRP concentration with TMD, migraine, and their interaction. Univariate associations of the CGRP concentration with clinical, psychological, and pressure pain characteristics were also assessed. Among 80 participants enrolled, neither TMD nor migraine was associated with plasma CGRP concentration (P = 0.761 and P = 0.972, respectively). The CGRP concentration (mean ± SD) was similar in all 4 groups: HCs 2.0 ± 0.7 pg/mL, TMD 2.1 ± 0.8 pg/mL, migraine 2.1 ± 0.9 pg/mL, and TMD with migraine 2.2 ± 0.7 pg/mL. CGRP concentration was positively associated with age (P = 0.034) and marginally with body mass index (P = 0.080) but was unrelated to other participant characteristics. In this well-powered study, interictal plasma concentration of CGRP was a poor biomarker for TMD and migraine.

Development of an electrochemiluminescent oligonucleotide-based assay for the quantification of prostate cancer associated miR-141-3p in human serum

MicroRNAs (miRNAs) are small oligonucleotides (18–25 bases), biologically relevant for epigenetic regulation of key processes, particularly in association with cancer. Research effort has therefore been directed towards the monitoring and detection of miRNAs to progress (early) cancer diagnoses. Traditional detection strategies for miRNAs are expensive, with a lengthy time-to-result. In this study we develop an oligonucleotide-based assay using electrochemistry for the specific, selective and sensitive detection of a circulating miRNA (miR-141) associated with prostate cancer. In the assay, the excitation and readout of the signal are independent: an electrochemical stimulation followed by an optical readout. A ‘sandwich’ approach is incorporated, consisting of a biotinylated capture probe immobilised on streptavidin-functionalised surfaces and a detection probe labelled with digoxigenin. We show that the assay allows the detection of miR-141 in human serum, even in the presence of other miRNAs, with a LOD of 0.25 pM. The developed electrochemiluminescent assay has, therefore, the potential for efficient universal oligonucleotide target detection via the redesign of capture and detection probes.

1294-P: Prevalence of Islet Autoantibodies in Adults from the General Population

Objective: Over half of cases of type 1 diabetes are diagnosed in adults, yet the prevalence of islet autoantibodies (IA) in general population adults is unknown. Methods: The Autoimmunity Screening for Kids (ASK) Study screened 1,087 adults (age 41±7 years, 78% female, 63% non-Hispanic white, 10% first-degree relatives) and 29,441 youth (age 1-17 years, 52%, 36%, and 5%, respectively) for IA to GAD, IA-2, insulin, and ZnT8 using electrochemiluminescence (ECL) or radiobinding assays (RBA). Results: In adults, 3.9% screened positive; 0.6% were positive for multiple IA, 1.7% were positive for a single high-affinity IA, and 1.6% were positive for a single low-affinity IA. GADA was most prevalent, found in 2.7%. Unexpectedly, the prevalence of IA in adults and youth screened by ASK was not statistically different (Figure). Conclusion: IA positivity is similar in youth and adults in the general population. Expanded access to type 1 diabetes screening and monitoring will aid in determining if early detection is similarly beneficial in adults as seen in youth, such as reducing rates of diabetic ketoacidosis at diagnosis of clinical type 1 diabetes. Disclosure M.Pauley: None. C.Geno rasmussen: None. F.Dong: None. K.M.Simmons: Advisory Panel; Provention Bio, Inc., Consultant; Dexcom, Inc., Provention Bio, Inc., Research Support; Novartis. M.Rewers: Research Support; Provention Bio, Juvenile Diabetes Research Foundation (JDRF), Hemsley Charitable Trust, Dexcom, Inc., Janssen Research &amp; Development, LLC. Funding National Institute of Diabetes and Digestive and Kidney Diseases (T325T32DK063687); JDRF (2-SRA-2022-1270-S-B)

Coreactant-free electrochemiluminescence of polyfluorene nanoparticle coupling double quencher for β-amyloid1-42 detection.

β-amyloid1-42 (Aβ1-42) is a humoral biomarker for early diagnosis of Alzheimer's disease (AD), and exists at a low level in human body. Its sensitive detection is very valuable. The electrochemiluminescence (ECL) assay of Aβ1-42 has attracted special attention owing to high sensitivity and simple operation. However, currently reported ECL assays for Aβ1-42 usually required the introduction of exogenous coreactants to improve the detection sensitivity. Introducing exogenous coreactants will lead to non-negligible repeatability and stability problems. This work exploited poly [(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadazole)] nanoparticles (PFBT NPs) as coreactant-free ECL emitters for detecting Aβ1-42. The PFBT NPs, first antibody (Ab1) and antigen Aβ1-42 were successively assembled on glassy carbon electrode (GCE). Silica nanoparticles served as a carrier to grow polydopamine (PDA) in situ, and further assembled Au nanoparticles (Au NPs) and second antibody (Ab2), producing the secondary antibody complex (SiO2@PDA-Au NPs-Ab2). With its assembly on the biosensor, the ECL signal decreased since both PDA and Au NPs could quench ECL emission from PFBT NPs. The limit of detection (LOD) of 0.55fg/mL and limit of quantification (LOQ) of 37.45fg/mL for Aβ1-42 were obtained. PFBT NPs coupling dual-quencher PDA-Au NPs created an excellent ECL system for bioassays, and constructed a sensitive analytical method for Aβ1-42.

Antibody detection by agglutination-PCR (ADAP) assays for the analysis of tissue transglutaminase autoantibodies in celiac disease

Background & aimsTissue transglutaminase autoantibodies (tTGA) are used as diagnostic markers of celiac disease. Different methods have been developed for the detection of tTGA of which enzyme-linked immunosorbent assays (ELISA), radiobinding assays (RBA) and electrochemiluminescence (ECL) assays are the most commonly used. Here we aimed to evaluate a novel antibody detection by agglutination-PCR (ADAP) assay for the detection of tTGA. MethodsIncluded were 126 children with untreated celiac disease (UCD), 64 disease controls (DC), 21 children with potential celiac disease (PCD), and 1501 children from the general population. Tissue TGA were determined using an automated ADAP assay platform and compared with two RBAs for the detection of IgA-tTG and IgG-tTG, respectively. ResultsADAP detected tTGA in 123/126 (97.6%) UCD children compared with 122/126 (96.8%) using RBA-IgA-tTG and RBA-IgG-tTG (p > 0.9999), respectively. Among DC, ADAP detected 5/64 (7.8%) children with tTGA compared with 4/64 (6.3%) with RBA-IgA-tTG (p > 0.9999) and 8/64 (12.5%) with RBA-IgG-tTG (p = 0.5600), respectively. Tissue TGAs were equally detected in children with PCD in both assays. In the general population, 4/1501 (0.3%) were tTGA positive using ADAP compared with 3/1501 (0.2%) for RBA-IgA-tTG and RBA-IgG-tTG (p > 0.9999), respectively. The area under the curves (AUCs) were 0.998 for ADAP, 0.994 for RBA-IgA-tTG, and 0.999 for RBA-IgG-tTG, respectively. ConclusionsNo difference in specificity and sensitivity of tTGA for the diagnosis of celiac disease was reported between ADAP and RBA. ADAP could be recommended as the first-line screening method of larger populations for celiac disease.

IL-6 Levels Correlate with Prognosis and Immunosuppressive Stromal Cells in Patients with Colorectal Cancer.

The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels. Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases. Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3+ and CD4+ T cells as well as FOXP3+ cells. Mass cytometry analysis showed that IL-6+ cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3highCD45RA- effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10+ cells in MDSCs and that of IL-10+ or CTLA-4+ cells in eTregs correlated with IL-6 levels. Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME.

O049 Interleukin-16 in flush effluent fluid as a biomarker for kidney viability prior to transplantation

Abstract Background Kidney transplantation is limited by the number of transplantable organs. Biomarkers that can predict organ viability will reduce the number of organs currently discarded (13% UK, 2021-22, NHSBT). The kidney microcirculation can be flushed, prior to transplantation, with preservation solution as part of the back-table preparation. The effluent fluid is usually discarded, but we propose this as a source of potential biomarkers. Interleukin-16 (IL-16) is a pro-inflammatory cytokine previously shown to predict viability in liver grafts. Here we examine IL-16 in the context of kidney viability. Methods IL-16 concentration was measured, in the flush effluent, using an electrochemiluminescent multiplex assay. The total protein content of each sample was concurrently measured to normalise the concentration. IL-16 concentrations were then compared to donor characteristics and early transplant outcomes. Results Samples were collected from 30 transplanted kidneys (10 DCD, 10 DBD, 10 live donor) and 5 declined kidneys (4 DCD, 1 DBD). IL-16 concentration was significantly higher in DCD kidneys (mean 81.8+/-38 (SD)) compared to live donor kidneys (17.7+/-18 pg/ml) (p=0.0001). CIT did not affect IL-16 concentration (β=-0.014, p=0.4), nor was IL-16 able to predict delayed graft function. However, IL-16 was significantly higher in kidneys declined for transplantation (transplanted: 54.7+/-30 vs non-transplanted: 98+/-43, p=0.03). Conclusion Flush effluent is a rich source of potential biomarkers. IL-16 concentration in flush effluent fluid correlates with organ quality. With real-time assessment, it will be possible to identify kidneys currently discarded; that are either transplantable directly or would benefit from advanced therapy prior to transplantation.

8 Comparison of oxytocin and pro-inflammatory cytokines in saliva and plasma in the post-partum mare as potential biomarkers of well-being

Although evaluating cortisol concentrations is a common method to assess stress in many species including horses, interpretation of results in these studies has proven challenging. Therefore, researchers are investigating alternative analytes as biomarkers of well-being. Historically thought to serve primary roles in parturition and lactation, oxytocin (OT) has been shown to act as a neurotransmitter to promote prosocial behavior and buffer stress. Pro-inflammatory cytokines (PICs) have been shown in humans to influence production of neurotransmitters such as serotonin and dopamine, implicating their potential role in psychosocial stress. Analyte levels are often measured in blood, but this sampling procedure can result in acute stress responses that may influence experimental results and conclusions. Evaluating analytes in saliva can serve as a less-invasive method to assess biomarkers of well-being. The purpose of this study was to determine the feasibility of measuring OT and PICs in saliva as an alternative to plasma sampling in horses. As studies have demonstrated that OT and PICs are elevated during parturition and lactation, 4 postpartum Quarter Horse mares (15.0 ± 0.82 yr) were chosen for this study. Plasma (P) and saliva (S) samples were collected 30 min post-foaling (P1, S1) and 30 min post first suckling (P2, S2). Samples were analyzedfor OT, IL1-β, IL-8, IL-6, and TNF-α via electrochemiluminescent (ECL) assay (Salimetrics). Data were analyzed using the PROC GLM and PROC CORR in SAS v 9.4. Mean analyte concentrations, with the exception of IL-8, differed by sample type (S OT = 45.16 ± 36.65 pg/mL, P OT = 96.10 ± 41.0 pg/mL, P < 0.05; S IL1-β = 13.98 ± 2.03 pg/mL, P IL1- β = 1.01 ± 0.18 pg/mL, P < 0.01; S IL-6 = 0.35 ± 0.14 pg/mL, P IL-6 = 1.32 ± 0.46 pg/mL, P < 0.01, S TNF-α = 0.36 ± 0.16 pg/mL, P TNF- α = 3.40 ± 0.86 pg/mL, P < 0.01). There were no differences in analyte concentrations due to time point. Though not significant (P > 0.5), time point comparisons across sample type showed moderate to strong Pearson correlations among OT (S1-P1; r = −0.56; S2-P2; r = −0.47), IL-8 (S1-P1; r = −0.69), IL-6 (S1-P1; r = 0.82), and TNF-α (S1-P1; r = 0.48). The established roles of OT and PICs in prosocial behavior and psychosocial stress suggest that measurement of these analytes may provide a targeted approach to assess biopsychosocial responses. While this assay has not yet been validated for use in horses, this study illustrates that PICs can be quantified in equine saliva which supports this less-invasive approach for studying biomarkers of well-being in horses.