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Herein a split‐type photoelectrochemical (PEC) immunosensor based on in situ grown gold nanoparticles (Au NPs) on the surface of ZnO microrods (ZnO MRs) was devised for the detection of alpha‐fetoprotein (AFP) by employing dopamine (DA)‐loaded liposomes as the signal amplification strategy. Specifically, as the level of AFP rises, a proportional number of sandwich‐type immunocomplexes form in the well of the microplate, which can introduce a corresponding amount of DA‐encapsulated liposomes at the same time. Encapsulated DA molecules will be released with the stimulation of Triton X‐100, thereby resulting in the marked enhancement of the PEC signal. Under optimized conditions, the proposed PEC immunosensing platform displays specific photocurrent responses toward AFP in a broad dynamic working range of 0.05 −50 ng mL−1, achieving an impressively low limit of detection (LOD) of 18.7 pg mL−1. Furthermore, this methodology not only displays high specificity, and satisfactory storage stability but also ensures acceptable accuracy and practicality for the analytical applications of human serum samples. [ABSTRACT FROM AUTHOR]

HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including type 1 diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble a and b chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of interchain disulfide engineering by introducing paired cysteines to juxtaposed positions in the a and b chains of HLA-DQ7, encoded by HLA-DQA1*05:01 and HLADQB1*03:01 respectively. We identified three variant peptideHLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83CE5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, highaffinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents. [ABSTRACT FROM AUTHOR]

Introduction: Hypercholesterolemia is a risk factor for premature arteriosclerosis. Inflammation and oxidative stress are thought to contribute to endothelial dysfunction preceding vasculopathy. We investigated the association between inflammation, glycocalyx biomarkers, endothelial function, and vascular parameters in children with hypercholesterolemia. Methods: In 22 patients (LDL-cholesterol >130 mg/dL; median age [IQR]: 13 [2.3] years) and 22 controls (13 [2.5] years), tumor necrosis factor-alpha (TNF-α), oxidized cholesterol (oxLDL), and glycocalyx biomarkers (Syndecan-1, Hyaluronan) were measured using immunoassays. Endothelial function was assessed by peripheral arterial tonometry, sublingual glycocalyx and microcirculation by videomicroscopy and carotid intima-media thickness by ultrasound. Results: OxLDL was significantly higher in patients (78.9 [38.2] vs. 50.3 [16.6] U/L, p = 0.002), whereas all other experimental parameters were comparable between groups. Multivariate analysis revealed a significant association of Syndecan-1 with TNF-α (β = 0.75, p < 0.001) and with hypercholesterolemia (β = 0.31, p = 0.030). The interaction term combining TNF-α and hypercholesterolemia showed a significant effect (p = 0.034). Sex was an independent predictor of endothelial function. Conclusion: The combined effect of hypercholesterolemia and inflammation on glycocalyx perturbation and the impact of sex in the premature development of arteriosclerosis deserve further evaluation. Therapeutic approaches tackling low-grade systemic inflammation may offer potential to prevent or delay progression of cardiovascular disease and cardiovascular complications. [ABSTRACT FROM AUTHOR]

Objective: To develop multiplex cytokine detection reagents to analyze expression levels of cytokines, angiogene sis and bone remodeling proteins in relapse/refractory multiple myeloma (RRMM) . Methods: Multiplex bead-based immunoassay by flow cytometry was used to develop quantitative detection reagents of multiplex cytokines, which were applied to detect serum samples from 55 RRMM patients and 22 healthy controls. Expression levels of cytokines, angiogenesis, and bone remodeling proteins in pa tients, and their correlation with clinical characteristics were analyzed. Results: Detection reagents of 10-plex cytokine immunoassay were successfully developed in this study, with average sensitivity of 7.1 pg/ml, average recovery rate of 97.4%, average intra-assay CV of 4.8%, and average inter-assay CV of 9.0%. In addition, results of RRMM samples found that levels of IL-2, IL-17, DKK1, RANKL and OPG were positively correlated with the level of IgG monoclonal protein, and TIMP1 was positively correlated with levels of IgG and IgA monoclonal protein. Conclusion: In this study, ten kinds of cytokine detection reagents with high sensitivity and speci ficity are developed, and we found that IL-2, IL-17, DKK1, RANKL, OPG and TIMP1 have potential value in tracking disease pro gression in RRMM. The established development process of multiplex cytokine reagents has important reference significance for expanding the development and application of multiplex detection reagents for protein markers in the future. [ABSTRACT FROM AUTHOR]

(1) Background: In vitro diagnostic (IVD) tests are the main means of obtaining diagnostic information for clinical purposes. The electrochemiluminescence immunoassay (ECLIA) has become an important in vitro diagnostic technique. It has unique advantages and broad market prospects due to its sensitivity, detection limit, detection range and reagent stability. At present, there is a need to develop and optimize electrochemiluminescence immunoassay subsystems to achieve high-throughput outputs and accurate detection of instruments to promote their clinical application. (2) Methods: On the basis of the demand for clinical testing instruments with high detection accuracy and speed, this study constructed an electrochemiluminescence immunoassay system by designing magnetic separation modules, introducing PMT and optimizing the system timing regulation capability. (3) Results: The magnetic separation modules can increase the detection accuracy, the PMT system increases the detection sensitivity and optimized system timing can achieve maximum test output. Furthermore, this system performs well in terms of its linearity, detection limit, signal-to-noise ratio, precision and accuracy. (4) Conclusions: The electrochemiluminescence immunoassay system is capable of high throughput with good sensitivity and accuracy, meeting the basic requirements of clinical applications for detection capacity and output throughput. [ABSTRACT FROM AUTHOR]

Produced by the mitochondria and endoplasmic reticulum, neurosteroids such as allopregnanolone are neuroprotective molecules that influence various neuronal functions and regulate neuroinflammation. They are reduced in neurodegenerative diseases, while in the Wobbler mouse model, allopregnanolone and its precursor progesterone showed protective effects on motor neuron degeneration. This single-center case-control study included 37 patients with amyotrophic lateral sclerosis (ALS) and 28 healthy controls. Cerebrospinal fluid (CSF) neurosteroid levels were quantified using liquid chromatography–electrospray tandem mass spectrometry and compared between the two cohorts. Neurosteroid concentrations have been correlated with neuroinflammation and neurodegeneration biomarkers detected through an automated immunoassay, along with disease features and progression. Pregnenolone, progesterone, allopregnanolone, pregnanolone, and testosterone levels were significantly lower in ALS patients' CSF compared to healthy controls. A significant inverse correlation was found between neurofilament and neurosteroid levels. Neurosteroid concentrations did not correlate with disease progression, phenotype, genotype, or survival prediction. Our study suggests the independence of the disease features and its progression, from the dysregulation of neurosteroids in ALS patients' CSF. This neurosteroid reduction may relate to disease pathogenesis or be a consequence of disease-related processes, warranting further research. The inverse correlation between neurosteroids and neurofilament levels may indicate a failure of compensatory neuroprotective mechanisms against neurodegeneration. [ABSTRACT FROM AUTHOR]

Background Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective, and antimicrobial factors to the neonate. Objective To evaluate method reproducibility of AOAC First Action Official Method   2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. Methods Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct-assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a four-parameter calibration regression. Results After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable Horwitz Ratio (HorRatR) values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin cleanup LC–UV), showed negligible difference between results. Conclusion The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing (MLT) study in July 2023 and recommended Official Method   2021.07 for adoption as a Final Action Official Method SM. Highlights A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma is the deadliest of all skin cancers. Therapy paradigms in cutaneous melanoma have changed significantly over the past 20 years, following both changes in the initial staging work-up of melanoma as well as in the available therapies. Regional recurrences after initial surgery are normally treated with nodal basin resection. However, some regional recurrences may present as bulky masses that can pose significant challenges in the treatment and control of the disease. It is well established that radiation therapy (RT) plays a role in locoregional control of malignancies in general. This review will attempt to discuss evidence for radiation therapy's role in reducing regional recurrence in the adjuvant setting in melanoma, identify adjuvant systemic therapy options, discuss systemic therapy–radiation therapy combinations, and identify paradigms and emerging evidence for the use of radiation therapy after neoadjuvant systemic therapy strategies are employed. In the landscape of Stage III locoregionally advanced cutaneous melanoma treatment, the post-immunotherapy era has sparked a number of questions on the management of the nodal basin. However, much of the available literature is not focused on radiation therapy as an adjuvant therapy. This literature review aims to illuminate the evidence surrounding radiation therapy's potential to mitigate regional recurrences in the adjuvant setting for melanoma. Additionally, it seeks to identify adjunct systemic therapy options and explore the synergy between systemic therapy and radiation. Despite strides in surgical techniques and systemic therapies, controlling regional Stage III melanoma remains a formidable clinical hurdle. While historical data strongly suggest the efficacy of adjuvant radiation therapy in reducing regional recurrence risk, its evaluation predates the advent of MAPK pathway inhibitors and robust immunotherapy options. Notably, clinical trials have yet to definitively demonstrate a survival advantage with adjuvant radiation therapy. Additional research should focus on refining the definition of high risk for regional recurrence through gene expression profiling or tumor immune profiling scores and elucidate the optimal role of adjuvant radiation therapy in patients treated with neoadjuvant systemic therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Total thyroidectomy and dissection of cervical lymph node compartments, depending on serum calcitonin levels and ultrasound findings, is standard treatment for patients with medullary thyroid cancer. The aim of this study was to evaluate whether pre-operative calcitonin and CEA levels can be useful as biomarkers of the extent of lymph node metastases at diagnosis. Results indicate that pre-operative serum calcitonin and CEA levels can predict presence, number, and site of lymph node metastases and, more specifically, values of 90 pg/mL for calcitonin and 17 ng/mL for CEA accurately indicate the N1b status. Since surgery is the only curative treatment for medullary thyroid cancer and there is not a strong indication regarding the extent of lymphadenectomy, these findings may help in the choice of the extent of neck dissection. Background: In medullary thyroid cancer (MTC), lymph node metastases are often present at diagnosis and the extent of surgery is usually based upon pre-operative calcitonin and CEA levels as well as ultrasound findings. The aim of this study was to evaluate the role of pre-operative calcitonin and CEA levels as predictive markers of the burden of lymph node metastases at diagnosis. Methods: we conducted a retrospective study analyzing 87 MTC patients. Results: The median levels of calcitonin and CEA were 88.4 pg/mL and 7.0 ng/mL, respectively, in patients with no lymph nodes metastases; 108.0 pg/mL and 9.6 ng/mL, respectively, in patients with metastases to 1–5 lymph nodes; 520.5 pg/mL and 43.2 ng/mL, respectively, in patients with metastases to >5 lymph nodes. There were no significant differences in pre-operative calcitonin and CEA values between N0 and N1a patients, whereas they were significantly higher in N1b patients. Pre-operative cut-off levels distinguishing N0/N1a from N1b patients were 90 pg/mL for calcitonin (sensitivity 100%, specificity 59.3%, AUC = 0.82) and 17 ng/mL for CEA (sensitivity 100%, specificity 75%, AUC = 0.89). Conclusions: in patients with MTC, pre-operative serum calcitonin and CEA levels may drive the decision-making process to better define the extent of surgery. [ABSTRACT FROM AUTHOR]

Objective: Vitamin D deficiency is a common public health issue worldwide. The purpose of this study was to investigate the vitamin D status and its potential determinants in children residing in Sardinia (40°N), Italy. Methods: Children were enrolled over a 12-month period. Serum 25(OH)D was measured by an immunochemiluminescence assay. A questionnaire was used to gather information on other variables, including passive smoke exposure. Results: A total of 182 children (males: 51.7%; median age: 9 years) were included. Mean±standard deviation serum 25(OH)D was 25.2±8.3 ng/mL for the whole group. The majority (n=123, 67.6%) had vitamin D sufficient values >20 ng/mL, while 32.4% (n=59) had vitamin D insufficient/deficient values (≤20 ng/mL). Among the variables investigated, passive smoke exposure was significantly associated with insufficient 25(OH)D levels (p<0.0001). Conclusion: Our results confirm that hypovitaminosis D is common in Italian children. Furthermore, passive smoke exposure was identified as a significant risk factor for hypovitaminosis D. [ABSTRACT FROM AUTHOR]

Background: Pauci‐immune glomerulonephritis (GN) due to antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) is a common cause of crescentic GN. Despite advances in treatment, rates of mortality and progression to end‐stage kidney disease remain high. Renal involvement is diagnosed by histological examination of kidney tissue. Serum ANCAs play a significant role in AAV; however, the value of serum ANCA quantification to predict renal involvement is not well‐established. Aim: We aimed to evaluate the diagnostic accuracy of serum ANCA titres in diagnosing AAV with renal involvement. Methods: We conducted a retrospective study of consecutive native kidney biopsies reported at our centre from 2016 to 2021. We included all adults who had both a kidney biopsy and ANCA serology. ANCA serology was tested using indirect immunofluorescence with reporting of titres. Antibodies to proteinase 3 and myeloperoxidase were measured using a chemiluminescent immunoassay. Results: Eight hundred and forty‐eight native kidney biopsies were reported during the study period. Five hundred and seven cases were included. The biopsy prevalence of pauci‐immune GN in paired samples was 41/507 (8.1%). Most of the cohort had haematuria (66.6%), proteinuria (93.4%) and/or acute kidney injury (65.0%). A positive ANCA at any titre demonstrated a sensitivity of 97.6% and a specificity of 71.2% for a diagnosis of pauci‐immune GN. The area under the curve for the receiver operator characteristic was 0.93 (95% confidence interval [CI]: 0.89–0.97). A cutoff ANCA titre of 1:160 provided the optimum balance between a sensitivity of 75.6% (95% CI: 59.7%–87.6%) and a specificity of 94.0% (95% CI: 91.6%–96.0%). Conclusions: ANCA titres are highly predictive of pauci‐immune GN in the appropriate context. While serum ANCA quantitation may not replace renal biopsy, reporting will assist in the decision to start treatment early for patients with organ or life‐threatening disease. [ABSTRACT FROM AUTHOR]

Objectives Myositis-specific and associated autoantibodies are important biomarkers in routine clinical use. We assessed local testing performance for myositis autoantibodies by comparing line immunoassay (LIA) to protein radio-immunoprecipitation and identifying clinical characteristics associated with each myositis autoantibody in the MyoCite cohort. Methods Serum samples from patients within the MyoCite cohort, a well-characterized retro-prospective dataset of adult and juvenile idiopathic inflammatory myopathy (IIM) patients in Lucknow, India (2017–2020), underwent LIA at Sanjay Gandhi Postgraduate Institute of Medical Science (SGPGIMS), Lucknow. Immunoprecipitation of 147 IIM patients' serum samples (125 adult-onset, 22 juvenile-onset) was conducted at the University of Bath, with researchers blind to LIA results. LIA performance was assessed against immunoprecipitation as the reference standard, measuring sensitivity, specificity and inter-rater agreement. Univariate and multivariate logistic regression determined clinical associations for specific myositis-specific autoantibodies. Results Immunoprecipitation identified myositis autoantibodies in 56.5% (n  = 83) of patient samples, with anti-Jo1 (n  = 16; 10.9%) as the most common, followed by anti-MDA5 (n  = 14, 9.5%). While LIA showed good agreement for anti-Jo1, anti-PL7 and anti-PL12 (Cohen's κ 0.79, 0.83 and 1, respectively), poor agreement was observed in other subgroups, notably anti-TIF1γ (Cohen's κ 0.21). Strongly positive samples, especially in myositis-specific autoantibodies, correlated more with immunoprecipitation results. Overall, 59 (40.1%) samples exhibited non-congruence on LIA and immunoprecipitation, and κ values for LIAs for anti-TIF1γ, anti-Ku, anti-PmScl, anti-Mi2 and anti-SAE ranged between 0.21 and 0.60. Conclusion While LIA reliably detected anti-Jo1, anti-PL7, anti-PL12, anti-MDA5 and anti-NXP-2, it also displayed false positives and negatives. Its effectiveness in detecting other autoantibodies, such as anti-TIF1γ, was poor. [ABSTRACT FROM AUTHOR]

When faced with increasing drug‐related deaths and decline in practicing forensic pathologists, the need to quickly identify toxicology‐related deaths is evident in order to appropriately triage cases and expedite turnaround times. Lateral flow immunoassays conducted pre‐autopsy offer quick urine drug screen (UDS) results in minutes and are used to inform the need for autopsy. Over 1000 medicolegal cases were reviewed to compare UDS results to laboratory enzyme‐linked immunosorbent assay (ELISA) blood results to evaluate how well autopsy UDS predicted laboratory findings. Mass spectral analysis was performed on ELISA‐positive specimens and these data were used to investigate UDS false‐negative (FN) results when possible. Five different UDS devices (STAT One Step Drug of Abuse dip card and cassette, Premiere Biotech multi‐drug and fentanyl dip cards and ATTEST 6‐acetylmorphine (6‐AM) dip card) were tested encompassing 11 drug classes: 6‐AM, amphetamine/methamphetamine, benzodiazepines, benzoylecgonine, fentanyl, methadone, opioids, phencyclidine, and delta‐9‐tetrahydrocannabinol. Sensitivity, specificity, efficiency, and positive and negative predictive values >80% indicated that UDS was useful for predicting cases involving benzoylecgonine, methadone, methamphetamine, and phencyclidine. UDS was unreliable in predicting amphetamine, benzodiazepines, fentanyl, and opiates‐related cases due to a high percentage of FN (up to 11.2%, 8.0%, 12.4%, and 5.5%, respectively) when compared to ELISA blood results. For the later analytes, sensitivities were as low as 57.5%, 60.0%, 72.2%, and 66.7%, respectively. Overall results support that UDS cannot replace laboratory testing. Because UDS is subject to false‐positive and FN results users must understand the limitations of using UDS for triage or decision‐making purposes. [ABSTRACT FROM AUTHOR]

Objective: Our objective was to evaluate the association of serum biomarkers with baseline psoriatic arthritis (PsA) disease activity, pharmacodynamic effects of deucravacitinib on biomarker levels, and the relationship between biomarkers and clinical responses to deucravacitinib. Methods: The phase 2 trial (ClinicalTrials.gov identifier: NCT03881059) randomly assigned 203 patients with PsA 1:1:1 to placebo, deucravacitinib at 6 mg once daily (QD), or deucravacitinib at 12 mg QD. Serum biomarkers associated with the interleukin 23 (IL‐23) pathway (IL‐17A, β‐defensin [BD‐2], and IL‐19), type I interferon pathway, inflammation, and collagen matrix turnover were measured by immunoassay. Clinical responses (≥75% improvement from baseline in the Psoriasis Area and Severity Index [PASI75] and ≥20% improvement from baseline in American College of Rheumatology criteria [ACR20] responses) were measured at week 16. Hematologic variables were also assessed. Results: IL‐17A, BD‐2, and IL‐19 had a modest association with PASI scores (r = 0.4, r = 0.56, and r = 0.5, respectively) at baseline. In deucravacitinib groups, IL‐17A, BD‐2, IL‐19, C‐X‐C motif ligand 9 (CXCL9), CXCL10, C‐reactive protein, matrix metalloproteinase 3, and collagen type 4 degradation marker levels were significantly reduced at week 16 versus baseline (P < 0.01); higher levels of IL‐23 pathway–associated biomarkers predicted higher PASI75 and ACR20 response rates in deucravacitinib‐treated patients. Significantly higher PASI75 response rates were seen in patients with high baseline IL‐17A (odds ratio 15.76) and BD‐2 levels (odds ratio 15.41) versus low baseline IL‐17A and BD‐2 levels. Changes in hematologic variables that are characteristic of JAK inhibition were not observed with deucravacitinib. Conclusion: Deucravacitinib significantly impacted biomarkers associated with Tyk2 signaling pathways of key inflammatory cytokines, including IL‐23 and type I interferon, and those related to collagen matrix turnover. These biomarkers may predict treatment responses to deucravacitinib. [ABSTRACT FROM AUTHOR]

Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Nontreponemal tests are used to monitor disease activity. Toluidine red unheated serum test (TRUST), as one of nontreponemal tests, is generally applicable to hospitals at different levels. However, accurate judgment of TRUST results is inseparable from an experienced and accurate operator. To reduce current shortcomings of manual TRUST method, we attempted to convert the manual TRUST test into automatic TRUST test, that is, to determine the degree of aggregation of toluidine red particles by detecting the absorbance value of serum after reaction with toluidine red particles. 50 μL of serum sample and 80 μL toluidine red particles were added to 96-well plate. Then, the 96-well plate was placed on a microplate reader at medium grade for 8 min to mix. Then, plasma reagin reacted with toluidine red particles and promoted the aggregation of toluidine red particles to form a large clot, which eventually caused a decrease in the absorbance at 540 nm. The results showed that the specificity of the automatic TRUST test was 100%, the sensitivity was 87%. And this method showed 93.5% correlation with manual TRUST test. The developed method is simple and involves less subjectivity in reading results, opening new avenues for syphilis diagnostic testing. Turbidimetric immunoassay can avoid the shortcomings of subjective interpretation, time-consuming and manual operation of manual TRUST method, and is more suitable for large-scale screening in health examination. [ABSTRACT FROM AUTHOR]

Context.--Pemphigus is an autoimmune blister disease that causes blisters on the skin and mucosal surfaces. Direct immunofluorescence (DIF) testing is critical for the clinical diagnosis of pemphigus. However, it is limited to fresh tissue specimens and fluorescence microscopy. Objective.--To assess the value of C3d immunohistochemistry (IHC) on paraffin-embedded skin tissue for the diagnosis of pemphigus by comparing C3d-IHC results to DIF and enzyme-linked immunosorbent assay testing in pemphigus and other blister-related skin diseases. Design.--C3d-IHC assays were retrospectively performed on paraffin-embedded skin tissue sections from 115 patients (63 with pemphigus and 52 controls). Both the case group and the control group underwent the same protocol, and cases with C3d position in the peripheral spinous layer were considered as positive samples. Results.--C3d-IHC and DIF testing had similar performance for pemphigus diagnosis, with a sensitivity of 71.0% (95% CI, 51.8%-85.1 %) and 77.4% (95% CI, 58.5%-89.7%), specificity of 96.4% (95% CI, 79.8%-99.8%) and 100% (95% CI, 85.0%-100%), positive predictive value of 95.7% (95% CI, 76.0%-99.8%) and 100% (95% CI, 82.8%-100%), and a negative predictive value of 75.0% (95% CI, 57.5%-87.3%) and 80.0% (95% CI, 62.5%-90.9%), respectively. Conclusions.--Our study indicated that C3d-IHC results for paraffin-fixed tissues were not significantly different from DIF results for the diagnosis of pemphigus. The C3d-IHC assay has the potential for routine diagnosis of pemphigus, especially in the absence of fresh-frozen tissue. [ABSTRACT FROM AUTHOR]

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Bioluminescence is a natural and fascinating phenomenon in which living organisms produce light by enzymatic activity. Over many years, researchers have been studying the genetic sequence, biochemical, and physiology of bioluminescent organisms. In nature, forty different bioluminescent systems are found in that seven light-emitting reactions are known, and only two chemical pathways have been studied completely. Bioluminescence is used for many purposes but in the last decade, it has been used tremendously in several advanced fields. The ability to isolate and transfer the lux genes in both prokaryotic and eukaryotic organisms has potentially made the use of bioluminescent bacteria. It is applied to detect protein-protein interaction, to check the efficiency of drugs, and biosensors to validate environmental pollution, immunology, oncology, neuroscience, the food industry, and so on. This paper will review the discoveries and current applications of marine bioluminescence organisms in the field of biosensors tagged with bioluminescent gene and their applications in various purposes, bioluminescent assay in food industries, and molecular methods, bioluminescent tagged gene and their potential in anti-cancer immunotherapy, bioluminescent imaging to detect the infected cells in animal model, and immunoassay applications of RLux gene extracted from Renilla reniformis. [ABSTRACT FROM AUTHOR]

Half of patients with renal cell carcinoma (RCC) develop metastases. New and noninvasive biomarkers are needed for the diagnosis of RCC. The study aims to develop an EV‐based assay for the detection of RCC using a highly sensitive nanoparticle‐aided time‐resolved fluorescence immunoassay (NP‐TRFIA). To confirm the presence of tetraspanins on EVs, size exclusion chromatography is used to separate EV‐ and PE‐fractions from RCC4, 786‐O, and HEK293 cell lines. EV‐ and PE‐fractions are quantified using NP‐TRFIA assays established for CD9, CD63, CD81, and CD151. Tetraspanins are measured from RCC CCM and serum samples of RCC (n = 14), benign (n = 17), and healthy (n = 9) individuals. Among the tetraspanins, CD63 exhibits 3‐5‐fold higher expression on RCC4 and 786‐O CCM compared to HEK293. A sandwich CD63‐CD63 assay demonstrates significant discrimination of RCC patients from benign (p = 0.0003), and healthy (p = 0.005) individuals, respectively. Similarly, the CD81‐CD81 assay also enables significant separation of RCC patients compared to benign (p = 0.014), and healthy (p = 0.003) controls, respectively. This suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63‐ and CD81‐EVs compared to controls. Detection of these EVs using NP‐TRFIA approach may play a vital role in the detection of RCC. [ABSTRACT FROM AUTHOR]

The pSMILE project aims to ensure high-quality proficiency testing coverage for international laboratories participating in clinical trials. It evaluates and develops the capability of laboratories to participate in studies conducted outside the United States. The project facilitates appropriate proficiency testing coverage for every specified analyte according to sponsor requirements by identifying proficiency testing providers, assessing the suitability of their panels, and monitoring laboratory performance. Customization is done when standard panels are not available. The project also provides feedback and assistance to laboratories in troubleshooting and improving their performance. The article discusses the challenges faced by laboratories in selecting the correct proficiency testing panels for specific testing methods and emphasizes the importance of tailoring panels to meet the specific needs of each laboratory. Collaboration between proficiency testing providers and laboratories is highlighted as a way to improve panel offerings. [Extracted from the article]

Background: Given the opioid epidemic, fentanyl screening in urine has become increasingly important. Immunoassays remain the most common screening methodology due to the high throughput and ease of integration into automated chemistry systems. The fentanyl ARK II from Ark Diagnostics is a widely used immunoassay, while a novel fentanyl assay called FEN2 by Lin-Zhi has become available on the Roche platform. Here, we evaluate and compare their performance. Methods: Four hundred and thirty-four urine samples were analyzed for fentanyl across the Lin-Zhi FEN2 and ARK II assays on the Cobas c502 platform. Samples were analyzed immediately upon request for drug of abuse screening or frozen for subsequent analysis. For confirmation testing, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a limit of detection of 1 ng/mL for fentanyl/norfentanyl was used. Any sample with either fentanyl or norfentanyl above the LC-MS/MS cutoff was deemed positive. Results: The ARK II had 11 false negatives and 7 false positives, while the Lin-Zhi FEN2 had 12 false negatives and 2 false positives. This resulted in ARK II having a sensitivity and specificity of 90.4% and 97.8% respectively, while Lin-Zhi FEN2 had a sensitivity and specificity of 89.5% and 99.4%. Conclusions: Both the ARK II and Lin-Zhi FEN2 immunoassays detected fentanyl well. Overall, the Lin-Zhi assay had slightly better specificity than ARK II, in our data set. While some discrepant results were observed between the 2 immunoassay systems, most occurred near the immunoassay detection cutoffs. [ABSTRACT FROM AUTHOR]

Background: Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin treatment caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Accurate diagnosis of HIT is essential but remains challenging. The aim of our study was to explore the performance of our optimized diagnostic laboratory algorithm, based on Chemiluminescence (CliA) and ELISA immunoassays, on suspected HIT patients. The study compared the prevalence of HIT diagnosis in A.O. Mauriziano with the literature. Methods: 163 consecutive patients were investigated for suspected HIT with CliA HemosIL Acustar HIT-IgG, Werfen. HIT was ruled out in all patients with CliA <0.13 U/mL. All patients with CliA >0.13 U/mL were further investigated with Zymutest-HIA anti-PF4 IgG ELISA immunoassay. In these patients, HIT was ruled out on the combination of CliA between 0.13 and 1.0 U/mL followed by ELISA assay <0.300 OD. HIT was ruled in patients whose plasma tested positive or doubtful with CliA and positive with ELISA immunoassay and confirmed positive with a platelet aggregation test (PAT). Suspicion of HIT was revealed with clinical 4Ts score or recent suggestive anamnestic history. Results: Our diagnostic algorithm ruled out HIT diagnosis in 144/163 patients (88%) and predicted a positive PAT in 5/19 (26%) of CliA positive (4/5) or ELISA positive and CliA doubtful (1/5) patients. Conclusions: Our prevalence was 3.1%, comparable with the literature. The approach combining 2 quantitative immunoassays' (CliA and ELISA) results and 4Ts score probability was able to rule out the diagnosis within 1 h in 66% of patients with suspected HIT and within 24 h in 88% of patients. In the remaining 12% of cases, management decisions have to be based on individualized judgment while awaiting functional confirming results (48-72 h). [ABSTRACT FROM AUTHOR]

Background: Many fentanyl immunoassays are limited in their ability to detect norfentanyl. Urine specimens collected from individuals who have been exposed to fentanyl frequently have detectable concentrations of norfentanyl (≥2 ng/mL) but low concentrations of fentanyl (<2 ng/mL) by LC-MS/MS. The Lin-Zhi Fentanyl II Immunoassay (Lin-Zhi) claims 100% cross-reactivity with norfentanyl and therefore may detect exposure missed by other assays. Methods: In addition to verifying the manufacturer's analytical sensitivity claims, we selected 92 urine specimens with low-positive Lin-Zhi results (1-99 absorbance units, lowest 10%) for analysis by the Immunalysis Health Equity Impact Assessment and ARK II fentanyl methods. The accuracy of the 3 immunoassays was compared to LC-MS/MS as the reference method. Results: Spiking studies using purified fentanyl and norfentanyl and a set of 100 consecutive specimens confirmed the manufacturer's claims of limit of detection for fentanyl (3.8 ng/mL) and norfentanyl (5.0 ng/mL). However, the 92 low-positive patient specimens demonstrated concentrations of norfentanyl and fentanyl below 2.0 ng/mL by LC-MS/MS, with 47 (51%) having only norfentanyl detected. When comparing Lin-Zhi to the Immunalysis and ARK II immunoassays, only 27 (29%) of the 92 specimens were concordant. Fifty-two (57%) of the specimens were positive by LC-MS/MS and Lin-Zhi but false negative by one or both other immunoassays. Seven specimens (8%) were positive by Lin-Zhi but negative by the other immunoassays and had undetectable concentrations (<2 ng/mL) of fentanyl and norfentanyl by LC-MS/MS. Conclusions: The clinical sensitivity of the Lin-Zhi exceeds the manufacturer's claims, providing results comparable to LC-MS/MS methods. [ABSTRACT FROM AUTHOR]

Background: The objective of this study is to analyze the transplacental transmission of SARS-CoV-2 antibodies, their persistence in newborns, the factors that may influence this transmission, and the protection these antibodies confer over time. Methods: This prospective cohort was conducted in a tertiary pediatric hospital in the Barcelona Metropolitan Region, Spain. It included neonates born to mothers who had SARS-CoV-2 infection during pregnancy or delivery between August 2020 and January 2022. We followed the recruited children for at least six months, and blood tests were performed to determine the presence of SARS-CoV-2 antibodies. Results: A total of 101 children were recruited. Among the serologies performed on children under three months of age, 44/82 were positive (53.7%). Newborns whose mothers presented more severe disease exhibited higher seropositivity odds (coefficient 9.747; p = 0.002). There were increased preterm deliveries when maternal infection occurred closer to the time of delivery. No severe SARS-CoV-2 infections were detected in children during the follow-up. Conclusions: Slightly more than half of the SARS-CoV-2 serologies performed in the first three months were positive. This appears to confer protection during early childhood. The severity of maternal infection is the most significant factor influencing the transmission of antibodies in children born to unvaccinated mothers. [ABSTRACT FROM AUTHOR]

Background: 25-hydroxyvitamin D [25(OH)D] deficiency is a global health concern, particularly in pediatric populations, with implications for musculoskeletal health and overall well-being. This study aimed to evaluate serum 25(OH)D levels in a pediatric population and assess the prevalence of deficiency and insufficiency. Methods: A retrospective analysis was conducted on data from 741 pediatric patients (2–17 years old) who visited an urban hospital for children for acute respiratory and gastrointestinal conditions in the span of 2 years. Serum 25(OH)D levels were measured using chemiluminescent microparticle immunoassay. Statistical analyses were performed to assess the prevalence of deficiency and insufficiency, seasonal variations, and correlations with age and daylight exposure. Results: Of the 739 pediatric patients analyzed, a substantial proportion exhibited insufficient (31.80%) or deficient (36.54%) serum 25(OH)D levels. While younger age groups generally had higher mean 25(OH)D levels, a negative correlation was observed between 25(OH)D levels and age. Sunlight exposure variations did not significantly impact serum 25(OH)D levels. Despite diverse daylight exposure patterns, there were no significant differences between longer and shorter daylight periods. Conclusions: This study highlights the high prevalence of 25(OH)D deficiency and insufficiency in the pediatric population, emphasizing the need for public health monitoring and targeted supplementation strategies. Findings underscore the importance of regular consultations with healthcare providers to ensure optimal 25(OH)D levels in children, with potential implications for revising current sufficiency thresholds. Addressing 25(OH)D deficiency is crucial for promoting musculoskeletal health and overall well-being in children. [ABSTRACT FROM AUTHOR]

The specific detection of serum IgE antibodies specific to allergens (sIgE Abs) that can crosslink the plural high-affinity IgE receptor (FcεRIα) molecules on the surface of mast cells or basophils with a multivalent allergen can reduce the false-positive diagnoses observed in chemiluminescent and fluorescence enzyme immunoassays for type-I allergic patients. In this study, we detected sIgE Abs to the egg-allergen ovalbumin (OVA) and the wheat-allergen gluten in the sera of rats sensitized with each allergen using an amplified luminescence proximity homogeneous assay by crosslinking (AlphaCL). OVA and gluten were reacted with each sIgE Ab in the sera. Then, acceptor and donor beads labeled with the human FcεRIα were added to the reacted solution. The luminescence intensity for anti-OVA IgE Abs in the sera with the removal of IgG Abs was observed in five of seven (71.4%) of the sensitized rats, whereas no signals were observed in any of the unsensitized rats. The AlphaCL could also detect anti-gluten sIgE Abs in the sera of sensitized rats, but not of unsensitized rats. In conclusion, we successfully detected sIgE Abs in the sera of rats sensitized to two allergens using the AlphaCL. This detection method has the potential to be used as a new diagnostic tool for type-I allergic patients. [ABSTRACT FROM AUTHOR]

Diffusion layer dispersed electrodes were presented for application in electrochemical immunoassays. The diffusion layer refers to the region on the electrode surface where concentration gradients occur during the redox reactions of the target analytes. To control the diffusion layer, electrodes of the same area and size were fabricated, and the shape of the diffusion layer was controlled by adjusting each electrode distance. To estimate the properties of the electrodes, cyclic voltammetry (CV) analysis was performed using ferricyanide ([Fe(CN)6–3]) and 3,3′5,5′-tetramethylbenzidine (TMB) as a model redox couple. Changes in the diffusion layer shape according to electrode distance were compared through simulation. The redox currents for the target analytes increased as electrode distance increased. These results indicate that even if the electrode shape and area are the same, dispersing the diffusion layer by adjusting the distance between the electrodes can affect the microelectrode characteristics. Finally, the diffusion layer dispersed electrode was applied for the medical diagnosis of the human hepatitis B virus (hHBV) antigen, and the human hepatitis C virus (hHCV) antibody was compared with conventional assay methods, such as TMB-based chromogenic detection. [ABSTRACT FROM AUTHOR]

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Introduction: This study evaluated the pharmacokinetic (PK) equivalence between BP02 (a proposed trastuzumab biosimilar) and the reference trastuzumab approved in the EU (EU-trastuzumab) and the US (US-trastuzumab). Methods: In this phase 1, double-blind, parallel-group trial, 111 healthy male volunteers were randomized 1:1:1 to receive a single 6-mg/kg intravenous infusion of BP02, EU-trastuzumab, or US-trastuzumab and were evaluated for 78 days. Serum drug concentration–time data were analyzed by non-compartmental methods. The PK similarity of BP02 to the two reference products, and between EU-trastuzumab and US-trastuzumab, was determined using the standard 80–125% bioequivalence criteria. Results: Baseline demographics for the 111 subjects with evaluable pharmacokinetics were similar across all treatment groups. PK profiles were similar for the three products. The 90% confidence intervals (CIs) for the ratios of area under the serum concentration–time curve (AUC) from the time of dosing to infinity (AUC0-inf), AUC from the time of dosing until the time of the last quantifiable concentration (AUC0-t), and peak serum concentration of trastuzumab (Cmax) were within 80% to 125% for all three pairwise comparisons. Adverse events (AEs) were similar across all arms, with treatment-related AEs reported by 73.0%, 73.0%, and 89.2% of the subjects in the BP02, EU-trastuzumab, and US-trastuzumab groups, respectively. The most common AEs were headache, infusion-related reactions, and upper-respiratory-tract infections. Four subjects—three in the US-trastuzumab group and one in the BP02 group—discontinued the study due to AEs. All post-dose samples except for two tested negative for anti-drug antibodies. Conclusion: This study demonstrates the PK similarity among BP02, EU-trastuzumab, and US-trastuzumab. The safety and immunogenicity profiles observed for the three products in this study are consistent with previous reports for trastuzumab. Trial Registration: ANZCTR number: ACTRN12621000573853. [ABSTRACT FROM AUTHOR]

Abstract Background Plasma levels of d-dimer are elevated in patients with thromboembolisms. Here we investigated the existence of interfering antibodies as a potential cause for elevated d-dimer levels. Case presentation A 42-year-old white Caucasian woman with a prior history of pulmonary embolism during her first pregnancy (treated with heparin therapy for 6 weeks postnatally) and hypothyroidism had a persistent elevated d-dimer without any clinical or ultrasound-based signs of thromboembolic conditions during her second pregnancy. We obtained informed consent and plasma was obtained from the patient. d-dimer levels were measured using two different assays. We also tested for the presence of rheumatoid factor, performed dilution series, and finally used an antibody depletion strategy. The two d-dimer assays performed similarly. Using our antibody depletion technique, we observed that ~ 1/3 of the increased plasma levels of d-dimer may be attributed to interfering antibodies. Conclusions Our results identify interfering antibodies as a potential contributor to an increased d-dimer in this patient. Our case highlights the potential of heterophilic interference for increased d-dimer and provides a procedure to determine this analytically.

α-Amanitin (AMN) is one of the deadliest toxins from mushrooms, present in the deadly mushroom species Amanita phalloides. It is a bicyclic octapeptide and represents up to 40% of the amatoxins in mushrooms, damaging the liver and kidneys. Current methods of detecting amatoxins are time-consuming and require the use of expensive equipment. A novel label-free electrochemical immunosensor was successfully developed for rapid detection of α-amanitin, which was fabricated by immobilization of anti-α-amanitin antibodies onto a functionalized cellulose nanofibrous membrane-modified carbon screen-printed electrode. An oxidation peak of the captured amanitin on the tethered antibodies was observed at 0.45 V. The performance of the nanofibrous membrane on the electrode and necessary fabrication steps were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Due to its unique structural features and properties such as high specific surface area and microporous structure, the nanofibrous membrane as an immunosensor matrix for antibody tethering improved the electrochemical performance of the immunosensor by more than 3 times compared with cast membranes. Under the optimal conditions, the assembled immunosensor exhibited high sensitivity toward α-amanitin detection in the range of 0.009-2 ng mL-1 with a limit of detection of 8.3 pg mL-1. The results clearly indicate that the fabricated nanofiber-based-immunosensor is suitable for point-of-care detection of lethal α-amanitin in human urine without any pretreatment within 30 min.

Troponin is the American College of Cardiology and American Heart Association preferred biomarker for diagnosing acute myocardial infarction (MI). We provide a modeling framework for high sensitivity cardiac Troponin I (hs-cTnI) detection in chromatographic immunoassays (flow displacement mode) with an analytical limit of detection, i.e., LOD

A heavy-chain antibody (VHH) library against procymidone (PRM) was constructed via immunizing Bactrian camels. Through careful biopanning, seven nanobodies (Nbs) with different sequences were obtained. The variability in their performance was primarily attributed to the amino acid differences in complementarity-determining region 3 (CDR3), as analyzed by molecular docking. The Nb exhibiting the highest sensitivity, named NbFM5, was biotinylated and conjugated to streptavidin-labeled gold nanoparticles to preserve the epitope's activity and prevent a decrease in sensitivity due to traditional random electrostatic adsorption. Subsequently, a simple and sensitive immunochromatographic assay (ICA) was developed for rapid detection of PRM based on biotinylated Nb (btNb). The developed btNb-ICA showed a cut-off value of 200 ng/mL for visual judgment and a half-inhibitory concentration (IC50) of 6.04 ng/mL for quantitative detection. The limit of detection (LOD) was as low as 0.88 ng/mL. The recoveries in actual samples of crops ranged from 82.2 to 117.3%, aligning well with the results obtained from GC-MS/MS (R2 = 0.995). In summary, the developed btNb-ICA demonstrated high specificity and good accuracy for the rapid detection of PRM residues in vegetables. The total analysis time from preparing the sample to obtaining the result was less than 25 min.

Highly specific antibodies are the key reagents for developing immunoassays with a low false positive rate for environmental monitoring. Here, we provide evidence that nanobodies have the potential to achieve higher specificity than conventional antibodies and explain why from their structural features. Using sulfadimethoxine (SDM) as a model analyte, we constructed an immune phage display library and precisely isolated an ultra-specific nanobody (H1-17) by a crucial homologous antigen counter selection strategy. H1-17 showed no observable cross-reactivity (CR) with other structural analogs of 41 SDM tested, which has never been achieved by conventional antibodies. The structurally original specificity of H1-17 was illuminated and compared with that of one conventional antibody by homology modeling and site-directed mutagenesis validation. It was found that the noncanonical disulfide bond (C50-C104) of H1-17 helped CDR3 form a tailor-made binding pocket and divide it into two parts to accommodate the common structure of sulfonamides and the characteristic methoxyl group of SDM, respectively. Besides, the mutual-checking hydrogen bonds also played important roles in the specific recognition. Lastly, immunoassays with zero false positive rate were developed to screen SDM in water and milk samples, indicating that nanobodies could be reliable reagents for the accurate detection of chemical compounds.

To ensure proper dosage of a drug, analytical quantification of it in biofluid is necessary. Liquid chromatography mass spectrometry (LC-MS) is the conventional method of choice as it permits accurate identification and quantification. However, it requires expensive instrumentation and is not appropriate for bedside use. Using soluble epoxide hydrolase (sEH) inhibitors (EC5026 and TPPU) as examples, we report development of a nanobody-based enzyme-linked immunosorbent assay (ELISA) for such small molecules and its use to accurately quantify the drug chemicals in human samples. Under optimized conditions, two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL, respectively, and two order of magnitude linear ranges with high precision and accuracy. The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026. In addition, the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency. The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds. These methods could be easily implemented by the bedside, in the field in remote areas or in veterinary practice. This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy. Attributes of nanobody based pharmaceutical assays are discussed.

Introduction: Anaemia is a significant global health concern, particularly among pregnant women. This condition, primarily caused by Iron Deficiency (ID), poses risks to both maternal and foetal health. However, interpreting iron status through biochemical tests faces challenges. While serum ferritin concentration is a specific indicator of ID, its accuracy can be compromised by factors like inflammation. Serum Soluble Transferrin Receptor (sTfR) has shown promise in accurately diagnosing anaemia, especially in pregnant women, as it remains unaffected by pregnancy-related changes. Nonetheless, studies on its efficiency have yielded mixed results. Aim: To study the prevalence of Iron Deficiency Anaemia (IDA) among pregnant women and also to evaluate the accuracy of sTfR levels and the sTfR/Log Ferritin Index as diagnostic markers in predicting IDA among pregnant women. Materials and Methods: This cross-sectional study was conducted at NEIGRIHMS, Shillong, Meghalaya, India, between April 2021 and November 2022. A total of 92 pregnant women in their first trimester were included in the study. An automated clinical chemistry analyser measured serum iron and high-sensitivity C-Reactive Protein (hsCRP). Serum ferritin and sTfR levels were assessed via chemiluminescence assay. Sensitivity and specificity of sTfR levels and the sTfR/Log Ferritin Index in predicting IDA were determined utilising the Receiver Operating Characteristic (ROC) curve. Results: Most participants, 38 individuals (41.3%), were aged 20 to 25 years. The prevalence of anaemia was observed in eight individuals, comprising 8.7%, with sTfR proving highly sensitive (87.5%) and specific (75%) for IDA at 25.24 nmol/L. Both sTfR (r=-0.360) and sTfR-ferritin Index (r=-0.344) had significant negative correlations with Haemoglobin (Hb) (p-value

Aim. We present two cases with clearly discrepant results of clinical examination and cardiac troponin I (cTnI) and cardiac troponin T (cTnT) concentrations. In similar cases with discrepant results, the possibility of interference should be considered. Methods. Due to the suspicion of the presence of macrotroponin I in both of the presented cases, the patients were invited to our laboratory and both cTnI (Architect i1000, Abbott) and cTnT (Cobas 8000, Roche) concentrations were analysed. The samples were treated by preincubation in a heterophilic antibodies blocking tube (HBT) and analysed. Precipitation with polyethylene glycol solution (PEG) and molecular weight separation by gel filtration on Sephadex G100 was performed and concentrations of cTnI were analysed. Results. In the same blood sample, the cTnT and cTnI concentrations were 7 and 1782 ng/L, respectively, in Case 1, and 6 and 96 ng/L, respectively, in Case 2. Incubation of samples in HBT had no significant effect. CTnI concentrations after precipitation with PEG - presented as the percentage of initial concentrations - were 7.4% in Case 1 (and 26.8% in the control sample) and 1.4% in Case 2 (and 56.0% in the control sample). These results indicate a significant decrease in both cases, supporting presence of macrotroponin I. Finally, analyses of cTnI concentrations after gel filtration also supported the presence of macrotroponin I. Conclusion. The present cases show that the presence of macrotroponin can lead to unnecessary investigation of the patient. When the possibility of interference is suspected, cooperation with laboratory staff to help with interpretation or to perform more detailed analysis is crucial.

Anti‐immunocomplex (Anti‐IC) antibodies have been used in developing noncompetitive immunoassays for detecting small molecule analytics (haptens). These antibodies bind specifically to the primary antibody in complex with hapten. Although several anti‐IC antibody–based immunoassays have been developed, structural studies of these systems are very limited. In this study, we determined the crystal structures of anti‐testosterone Fab220 in complex with testosterone and the corresponding anti‐IC antibody FabB12. The structure of the ternary complex of testosterone, Fab220, and FabB12 was predicted using LightDock and AlphaFold. The ternary complex has a large (~ 1100 Å2) interface between antibodies. The A‐ring of the testosterone bound by Fab220 also participates in the binding of the anti‐IC antibody. The structural analysis was complemented by native mass spectrometry. The affinities for testosterone (TES) and three cross‐reactive steroids [dihydrotestosterone (DHT), androstenedione (A4), and dehydroepiandrosterone sulfate (DHEA‐S)] were measured, and ternary complex formation was studied. The results clearly show the ternary complex formation in the solution. Although DHT showed significant cross‐reactivity, A4 and DHEA‐S exhibited minor cross‐reactivity. [ABSTRACT FROM AUTHOR]

Traditional chemiluminescence (CL) imaging immunoassays usually rely on natural enzymes as catalytic probes, which has hampered their extensive application due to the susceptibility to inactivation of natural enzymes. In response, a gold brocade coated CoFe Prussian blue analogue (CoFe PBA@Au brocade) with enhanced peroxidase-like activity was synthesized and utilized as a powerful label probe for constructing a highly sensitive CL imaging immunosensor targeting disease biomarkers with excellent performance. This research offers a universal strategy for enhancing the sensitivity of CL imaging immunoassays and further expands the application of PBA nanozymes. [ABSTRACT FROM AUTHOR]

Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAbcoated beads that can detect other viruses may provide a useful diagnostic tool. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this work was to assess dynamic cytokine profiles associated with bloodstream infection (BSI) caused by Klebsiella pneumoniae (Kpn) and investigate the clinical features associated with mortality. Methods: A total of 114 patients with positive BSI-Kpn and 12 sepsis individuals without blood positive bacteria culture were followed up. Cytokine profiles were analyzed by multiplex immunoassay on the first, third, seventh and fourteenth day after diagnosis. The test cytokines included arginase, interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12 (p70), and IL-23. The minimum inhibitory concentration (MIC) of 24 antibiotics were tested for BSI-Kpn. Risk factors associated with the 30-day mortality and 120-day mortality were evaluated using logistic analyses and nomogram. Results: There were 55 out of 114 patients with BSI-Kpn were included. All isolates showed high susceptibility rate to novel avibactam combinations. The level of arginase was the highest in carbapenem-resistant Kpn (CRKP) patients. The AUCs of arginase, TNF-α and IL-4 reached 0.726, 0.495, and 0.549, respectively, whereas the AUC for the combination of these three cytokines was 0.805. Notably, 120-day mortality in patients with CRKP was higher than carbapenem-sensitive K. pneumoniae (CSKP). Furthermore, the long-term and high levels of IL-6 and IL-10 were associated with death. Conclusions: High expression of arginase is correlated with CRKP. In addition, BSI-CRKP could result in indolent clinic course but poor long-term prognosis. Continuous increase of IL-6 and IL-10 were associated with mortality. [ABSTRACT FROM AUTHOR]

Signal transducers are crucial in bioassay platforms for converting target detection into recordable signals. Commonly used color development for immunoassays involves enzymes and colorimetric substrates. However, due to cost and environmental issues, practical point‐of‐care testing requires alternative signal transducers. Growth‐induced extinction (absorption and scattering) of gold nanoclusters (AuNCs) is proposed as a novel approach for quantitative immunoassays. AuNCs devoid of localized surface plasmon resonance (LSPR) are used as seeds for growth reactions. Through reactions with a growth solution comprised of gold precursor and mild reductant, AuNCs of varying concentrations underwent controlled growth, resulting in nanoparticles of different sizes exhibiting distinct LSPR‐mediated extinction bands. Notably, the seed concentration exhibited a robust correlation with the resulting extinction of the grown particles on a small scale of 110 µL for a 96‐well microplate platform. To demonstrate this signal transduction mechanism, immunosorbent assays are performed using the conjugates of AuNC and detection antibody. The sandwich‐type assay successfully quantified a model antigen, human immunoglobulin G (hIgG), by monitoring LSPR wavelength and absorbance. This assay demonstrated a working range of 0.001–1 µg mL−1 and limit of detection of 1.19 ng mL−1. Signal transducers using the growth of AuNCs offer new alternative candidates for immunoassay platforms. [ABSTRACT FROM AUTHOR]

Here we report on the use of nonaqueous synthesis programming of gold nanoparticles (NPs) and the antibiotic metronidazole using reconfigurable paper comprising an asynchronous modular paperfluidic linear instrument-free (Ampli) chemical plug and play construction set. Liquid fractions and reactions are replaced with paperfluidic membranes on blocks that can be snapped together and disconnected for real time design of experiments. Reagents are embedded into the paper membranes of each block, and reactions are synthesized by snapping together blocks and adding solution, which runs through the blocks in a sequence. Reaction products were characterized by different spectroscopic techniques (Raman spectroscopy, optical absorption spectroscopy, and dynamic light scattering). The resulting products were extracted to form functional NPs used in diagnostic immunoassays, and metronizadole was functional in an antibacterial disk diffusion assay. We investigated how to map off-the-shelf reactions onto individual Ampli blocks and how to successfully generate and extract these products. The paper-based synthesis was faster, significantly more affordable, and lighter than traditional methods, with important implications for local and distributed manufacturing. [ABSTRACT FROM AUTHOR]

Background: Regional evidence on the role of vitamin D in the prevention of COVID-19 and its sequalae remains inconclusive and sparse. Objective: This study aimed to determine the association of COVID-19 status and severity with vitamin D among adults in Colombo District, Sri Lanka. Methods: A sex-matched case–control study was conducted among 104 RT-PCR-confirmed COVID-19 patients recruited from two major hospitals designated as COVID-19 treating centres and 104 RT-PCR negative adults recruited from the catchment area of the two hospitals. Non-fasting blood samples obtained from cases upon hospital admission and from controls upon release from 14-day quarantine period were analysed for serum 25(OH)D using chemiluminescence assay. Serum level < 50.0 nmol/L was identified as vitamin D deficiency (VDD). Results: Cases were older while the controls had fewer persons with co-morbidities. Cases (median = 30.0; IQR = 21.2 nmol/L) had significantly lower 25(OH)D compared to controls (median = 35.9; IQR = 28.3 nmol/L) (p = 0.02), which persisted after adjustments (p = 0.026), along with Sinhalese ethnicity (p < 0.001). VDD was significantly more prevalent in cases (83.7% vs. 71.2%; crude odds ratio (OR) = 2.1; 95% CI 1.1, 4.1); but was not an independent predictor of COVID-19 status (adjusted OR = 1.9; 95% CI 0.6, 5.7). A lower 25(OH)D level was observed in moderate/severe cases (median = 28.0; IQR = 20.4 nmol/L) vs. mild (median = 39.8; IQR = 19.0 nmol/L), however, neither low serum concentrations nor deficiency showed an independent relationship with severity (p > 0.05). Diabetes was the sole predictor of COVID-19 severity (p = 0.022). Conclusions: Vitamin D has potential as a primary preventive strategy in preventing COVID-19 like infections, but not as a secondary preventive strategy to reduce its severity. [ABSTRACT FROM AUTHOR]

Background/aims: Autoimmune hepatitis (AIH) is characterized by the presence of auto-antibodies and high blood immunoglobulin G (IgG) levels. In this study, the line immunoassay (LIA) was designed to assess various autoantibodies. Methods: In total, 1371 patients who underwent autoimmune liver disease antibody testing between July 2019 and November 2022 were enrolled. Autoantibodies including antinuclear antibody (ANA) and anti-mitochondrial antibody (AMA) were tested, and clinical data were collected. Statistical analyses were performed by categorizing the data based on diagnosis and IgG quantification separately. A scoring system was applied to identify individuals with AIH. Patients were also classified into the AIH and non-AIH groups. Results: The positivity rate for ANA was 80.2% in the AIH group. The IgG-high group had a high likelihood of the presence of detectable autoantibodies, with anti-Ro-52 being the most frequently detected antibody using LIA. The "Consider AIH" and "AMA" groups had 3–4 times more patients in the IgG-high group than in the "Not Considered" group. Conclusions: Among autoantibodies, the prevalence of ANA was the highest. As per LIA results, anti-Ro-52 was the most prevalent. AIH cannot be diagnosed based on IgG levels alone and must be distinguished via autoantibody testing. Therefore, extensive testing, including autoantibodies, IgG, ANA, and liver enzyme levels, will help accurately diagnose AIH. [ABSTRACT FROM AUTHOR]

The document is a contents list for the journal "Lab on a Chip," published by the Royal Society of Chemistry. It includes a range of papers on topics such as microfluidics, nanotechnology, and biomedical research. Some of the specific papers mentioned include a programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for single cell assay operations, an on-chip phytofluidic device for studying nutrient flow in plant roots, and optimized microfluidic formulation for improved lipid nanoparticle mediated genome editing. The journal covers a variety of scientific disciplines and offers valuable research for those interested in these fields. [Extracted from the article]

The extraction of targets from biological samples for immunoassays using organic solvents, such as methanol, is often necessary. However, high concentrations of organic solvents in extracts invariably lead to instability of the employed antibody, resulting in poor performance of the immunoassay. Evaluating the tolerance ability and exploring the molecular mechanisms of antibody tolerance in organic solvents are essential for the development of robust immunoassays. In this work, 25 monoclonal antibodies and methanol are utilized as models to address these questions. A novel protocol is initially established to precisely and rapidly determine antibody tolerance in methanol, identifying two distinct methanol effect patterns. Through a detailed investigation of the structural basis, a novel hypothesis regarding methanol effect patterns is proposed, termed "folding‐aggregation," which is subsequently validated through molecular dynamics simulations. Furthermore, the investigation of sequence basis reveals significant differences in residue types within the complementarity‐determining regions and ligand‐binding residues, distinguishing the two antibody methanol effect patterns. Moreover, the methanol effect patterns of the antibodies are defined by germline antibodies. This work represents the first exploration of antibody methanol effect patterns and associated molecular mechanisms, with potential implications for the discovery and engineering of tolerant antibodies for the development of robust immunoassays. [ABSTRACT FROM AUTHOR]

Parietal cell autoantibodies (PCAs), which recognize the enzyme H+/K+-ATPase as a target, are considered to be a diagnostic marker of autoimmune gastritis and pernicious anemia; these conditions are characterized by the presence of corpus atrophic gastritis. Circulating PCAs can be detected using several analytical methods that are commonly available in the clinical laboratory. Traditionally, indirect immunofluorescence (IIF) on rodent or primate stomach tissue is used as a screening test for the detection of PCAs. However, IIF suffers from a high inter-observer variability and lacks standardization. In addition, like immunoblotting, results are expressed only in a qualitative or semi-quantitative manner. Based on the few available studies that are reviewed herein, quantitative enzyme-linked immunosorbent assays (ELISAs) and fluorescence enzyme immunoassays (FEIAs) using purified H+/K+-ATPase perform better than IIF in the detection of PCAs, displaying higher sensitivity and utility in monitoring the disease. In light of their higher diagnostic accuracy, these solid-phase methods should be preferred to IIF in the screening of autoimmune atrophic gastritis. The use of methods to detect antibodies versus a specific subunit of H+/K+-ATPase (α or β) is currently confined to the world of research. Further investigation is required to define the clinical utility of H+/K+-ATPase subunit detection. [ABSTRACT FROM AUTHOR]

Marathon runners, subjected to intense training regimens and prolonged, exhaustive exercises, often experience a compromised immune response. Probiotic supplementation has emerged as a potential remedy to mitigate the impact of prolonged exercise on athletes. Consequently, this study sought to assess the influence of probiotic supplementation on monocyte functionality both before and after the official marathon race. Twenty-seven runners were randomly and double-blindly assigned to two groups: placebo (n 13) and probiotic (PRO) (n 14). Over 30 d, both groups received supplements – placebo sachets containing maltodextrin (5 g/d) and PRO sachets containing 1 × 1010 colony-forming unit Lactobacillus acidophilus and 1 × 1010 colony-forming unit Bifidobacterium bifidum subsp. lactis. Blood samples were collected, and immunological assays, including phagocytosis, hydrogen peroxide production, cytokine levels and monocyte immunophenotyping, were conducted at four different intervals: baseline (start of supplementation/30 d pre-marathon), 24 h-before (1 d pre-marathon), 1 h-after (1 h post-marathon) and 5 d-after (5 d post-marathon). Monocyte populations remained consistent throughout the study. A notable increase in phagocytosis was observed in the PRO group after 30 d of supplementation. Upon lipopolysaccharide stimulation, both PRO and placebo groups exhibited decreased IL-8 production. However, after the marathon race, IL-15 stimulation demonstrated increased levels of 5 d-after, while IL-1- β , IL-8, IL-10, IL-15 and TNF- α varied across different intervals, specifically within the PRO group. Probiotic supplementation notably enhanced the phagocytic capacity of monocytes. However, these effects were not sustained post-marathon. [ABSTRACT FROM AUTHOR]