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Assays investigated were vitamin B12 (B12), erythropoietin (EPO), folate (FOL), bone specific alkaline phosphatase (Ostase), pregnancy associated plasma protein A (PAPP-A), and thyroid peroxidase antibody (TPO). Onboard dilutions may provide quality, labor, and turnaround time advantages by eliminating unnecessary manual processes. © American Society for Clinical Pathology Am J Clin Pathol 2015;144:

Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets. This tutorial describes how to design nanoparticle-based LFAs for detecting biomolecules. The authors provide guidance on how to select the appropriate lateral-flow strip components and bioreceptors as well as detection strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in 2020. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA. We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, 2014, and Oct 4, 2016) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least 20 days post symptom onset (collected between Feb 1, 2020, and May 31, 2020). Receiver operating characteristic (ROC) curves were used to assess assay thresholds. At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2–94·8) and specificity was 99·9% (99·4–100%); for the DiaSorin assay sensitivity was 96·2% (94·2–97·7) and specificity was 98·9% (98·0–99·4); for the Oxford immunoassay sensitivity was 99·1% (97·8–99·7) and specificity was 99·0% (98·1–99·5); for the Roche assay sensitivity was 97·2% (95·4–98·4) and specificity was 99·8% (99·3–100); and for the Siemens assay sensitivity was 98·1% (96·6–99·1) and specificity was 99·9% (99·4–100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset. Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health. Public Health England and UK National Institute for Health Research.

Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for “strengths, weaknesses, opportunities, threats”). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included “immunochromatography”, “sol particle immunoassay”, “lateral flow immunoassay” and “dipstick assay”.

Ultrabright green-emissive AIE nanoparticles (AIENPs) were used as signal-amplification probes to enhance the detectability of lateral flow immunoassay (LFIA). The detection performances of the green-emissive AIENP probes in both sandwich and competitive LFIA formats were systematically evaluated. Benefiting from its remarkable fluorescent brightness, the developed AIENP-LFIA showed versatile applicability for the detection of small molecules and macromolecules by using ochratoxin A (OTA) and procalcitonin (PCT) as model analytes, respectively. Under the optimum conditions, the detection limits (LODs) of the fabricated AIENP-LFIA for OTA and PCT were 0.043 ng mL −1 and 0.019 ng mL −1 , respectively. These LOD values are significantly lower than those of conventional LFIA methods using gold nanoparticles as signal reporters. In addition, we demonstrated the practical application potential of AIENP-LFIA for the detection of OTA in real maize samples and PCT in real serum samples. These results indicated that the ultrabright green-emissive AIENPs were promising as signal output materials for building high-performance LFIA platform and broadening the application scenarios of LFIA. Graphical Abstract

Abstract Disclosure: K. Partridge: None. C. Sturgeon: None. E. Atkinson: None. M. Moore: None. P. Rigsby: None. B. Cowper: None. Alpha-fetoprotein (AFP) is a glycoprotein in the serum albumin family, primarily produced in the liver of foetuses during gestation. While AFP levels are normally low in the blood and serum of non-pregnant adults, levels increase during the first and second trimester of pregnancy. Higher than normal levels in pregnancy can also be indicators of birth defects such as anencephaly, omphalocele and spina bifida. Elevated levels in non-pregnant adults can also be indicative of disease, including hepatocellular carcinoma, germ cell tumours, and chronic active hepatitis. Measurement of AFP by immunoassays has therefore become widely established in the diagnosis and monitoring of disease states, responsiveness of patients to therapy, and in Triple and Quad pregnancy screening. Of the many commercially available AFP immunoassays on the market, the majority are traceable to the 1st WHO International Standard for Alpha Fetoprotein (1st WHO IS for AFP, 72/225), where it serves as a tool aimed at harmonisation of AFP immunoassay measurements. Stocks of the 1st WHO IS are now exhausted. Therefore, a replacement WHO IS, coded 22/216, has recently been produced at the MHRA. In this study, we describe the process of developing and characterising the 2nd WHO IS. With support from UKNEQAS, via an International Collaborative Study, we (i) evaluated the suitability of a highly purified preparation of AFP from human cord serum to serve as the 2nd WHO IS, (ii) value assigned the candidate standard, in international units (IU), in terms of the 1st WHO IS, by immunoassay, (iii) assessed commutability of the candidate standard with clinical samples in major commercial assays and (iv) evaluated long term stability of 22/216 by accelerated thermal degradation analysis. 20 different immunoassay platforms were used, including both manual and automated ELISA methods. Data from the study demonstrated good parallelism for the candidate IS, relative to the 1st WHO IS, indicating the candidate IS behaves in a similar manner to the current IS, allowing for the overall geometric mean potency value to be assigned as 7756 IU/ampoule (inter-lab GCV 8.73%). Commutability of candidate IS preparations with clinical samples was mixed, but comparable to those shown for the 1st WHO IS, 72/225, using a “calibration effectiveness” approach. Finally, accelerated thermal degradation samples were tested by immunoassay. Data indicated that 22/216 showed good stability with an estimated loss in potency of 0.07% per year when stored at -20°C Taken together, the data supports the introduction of 22/216 as the replacement IS for AFP, to continue to aid harmonisation of AFP immunoassays. 22/216 was established as the 2nd WHO IS by the WHOs Expert Committee on Biological Standardisation (ECBS), and replaces the 1st WHO IS, 72/225. Presentation: Monday, July 14, 2025

Abstract Disclosure: A.J. Newman: None. S. Mahrokhian: None. J. Chan: None. I. Hanna: None. A. Ferrebus: None. J.M. Brown: None. R.J. Auchus: None. A. Vaidya: None. Background: Accurate and specific assays for serum cortisol are essential to the clinical endocrinologist. However, the availability of cortisol assays varies across laboratories. We evaluated the correlation of serum cortisol measurements by liquid chromatography tandem mass spectroscopy (LC-MS/MS) with a modern cortisol immunoassay (IA) across the physiologic range of cortisol production in healthy participants. Methods: Healthy adults (n=97), without known or suspected cortisol excess or insufficiency, and who were not taking glucocorticoid medications, were prospectively recruited to undergo measurement of morning serum cortisol, post-overnight dexamethasone (1 mg) suppression testing (DST), and a 250 mcg cosyntropin stimulation (CST). Serum cortisol was measured by LC-MS/MS and with the Beckman-Coulter Access cortisol polyclonal IA. Inter-assay cortisol concentrations were evaluated using correlation analyses and Bland-Altman plots. Results: Median random morning cortisol by IA was 8.5 mcg/dL (IQR 6.8-11.5) and by LC-MS/MS was 8.7 mcg/dL (7.0-11.7). After 1 mg DST, median cortisol by IA was 0.8 mcg/dL (0.6-1.1) and by LC-MS/MS was 0.8 mcg/dL (0.6-1.2). Following CST, median cortisol by IA was 19.8 mcg/dL (17.4 - 22.1) and by LC-MS/MS was 20.9 mcg/dL (17.8-24.0). The correlation between LC-MS/MS and IA serum cortisol concentrations across the entire range was strong (r = 0.95, P < 0.001). In the range relevant to the evaluation for adrenal insufficiency, where cortisol < 5 mcg/dL, measured values were highly correlated (r = 0.77, P < 0.001). Similarly, in the range of values typically used to adjudicate a normal hypothalamic-pituitary-adrenal axis (10 - 20 mcg/dL), values were again highly correlated (r = 0.76, P < 0.001). CONCLUSIONS: Cortisol measured by the widely used Access immunoassay was highly correlated with the gold standard of LC-MS/MS across the physiologic range of cortisol production, including morning values, post-dexamethasone suppressed values, and post-cosyntropin stimulated values. These findings provide reassurance that the use of this specific Access IA serves as a reliable surrogate for the gold standard LC-MS/MS when evaluating cortisol across the spectrum of adrenal function. Presentation: Monday, July 14, 2025

Abstract Disclosure: E. Ng: None. H.Q. Shen: None. X. Lim: None. P. Marcus: None. S.M. Gwini: None. M. Thuzar: None. M. Stowasser: None. P.J. Fuller: None. J. Hu: None. J. Yang: None. In primary aldosteronism (PA), the variability in plasma aldosterone concentration (PAC) at a single time point is well documented. Measurement of 24-hour urinary aldosterone excretion (24h-UAE) may overcome this variability. An elevated 24h-UAE > 12 ug (33 nmol)/day, combined with high urinary sodium excretion (24h-UNa) > 200 mmol/day, is a recognised method for confirming PA. However, the rationale for this threshold and its diagnostic accuracy are not well established. This study aims to assess the diagnostic accuracy of the historical cut-off in Australian and Chinese cohorts and evaluate the optimal 24h-UAE threshold for diagnosing PA, using the saline suppression test (SST) as the reference standard. Patients referred for PA workup from 2018 to 2023 at two tertiary centres in Melbourne, Australia (n=215), and Chongqing, China (n=894) were included in the study if they had a 24h-UAE and SST performed. Blood and urine collections for aldosterone measurements were performed after washout of interfering medications. 24h urine sample collection was performed in an ambulatory setting for the Australian cohort and in an inpatient setting for the Chinese cohort. PAC, DRC and 24h-UAE were measured using an automated chemiluminescence immunoassays (DiaSorin). The diagnostic accuracy of 24h-UAE was compared to the SST result. The optimal 24h-UAE was determined using the Liu optimal cut-point and Youden index. PA was diagnosed in 138/215 (64%) of the Australian and 688/894 (77%) of the Chinese cohorts based on SST. The 24h-UAE cut-off of > 33 nmol/day (regardless of 24h-UNa) demonstrated 53% sensitivity and 79% specificity in the Australian cohort, and 36% sensitivity and 93% specificity in the Chinese cohort. The addition of 24h-UNa > 200 mmol/day improved sensitivity to 57 and 41%, respectively, but reduced specificity in the Australian cohort (75%), with no change in the Chinese cohort. In the Australian cohort, where 24h-UNa >190 mmol/day, the optimal 24h-UAE threshold was > 31 nmol/day, yielding 64% sensitivity and 77% specificity for diagnosing PA. In those with a DRC < 10 mU/L and 24h-UNa >190 mmol/day, a 24h-UAE > 22 nmol/day achieved 76% sensitivity and 83% specificity. Comparable diagnostic accuracy was seen in the Chinese cohort with the same DRC and 24h-UNa cut-off at a lower 24h-UAE cut-off of >18 nmol/day. This study highlights the suboptimal diagnostic accuracy of 24h-UAE measured by immunoassay and challenges the validity of the historical 24h-UAE cutoff of 33 nmol/day. Utility of the 24h-UAE may be improved by using liquid chromatography-mass spectrometry to measure aldosterone, but this remains to be formally evaluated. Presentation: Saturday, July 12, 2025

Background/ObjectivesA minority of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) have autoantibodies against components of nodal and paranodal structures in peripheral nerves. These autoantibodies are associated with lower likelihood of response to intravenous immunoglobulin (IVIg) treatment and increased responsiveness to B cell depletion. An easily available, reliable diagnostic assay for the detection of nodo-paranodal antibodies is a major unmet need.MethodsWe tested serum samples from patients with CIDP and healthy controls for the presence of antibodies against neurofascin 155 (NF155), neurofascin 186 (NF186), contactin-1 (CNTN1) and the contactin1/contactin-associated protein 1 (CNTN1/CASPR1) complex using a commercial transfected HEK-293 cell-based indirect immunofluorescence immunoassay (EUROImmun). We modified the manufacturer’s staining protocol using an additional incubation step to increase analytical sensitivity of the assay.ResultsWe detected nodo-paranodal antibodies in four out of 22 CIDP patients, including three who had previously tested positive for nodo-paranodal antibodies using enzyme-linked immunosorbent assay (ELISA), with no positive tests in 52 healthy controls. The modified staining protocol successfully increased the analytical sensitivity of the assay. Our modified assay demonstrated robustness in the presence of common interfering substances and serum with high nonspecific background immunofluorescent staining.ConclusionWe successfully modified and validated a commercial indirect immunofluorescence immunoassay for the qualitative detection of antibodies against NF155, CNTN1 and CNTN1/CASPR1. This could lead to more rapid diagnosis of patients with these autoantibodies, avoiding costly and ineffective treatments. Evaluation of the assay in a larger CIDP cohort and disease control group is ongoing. Testing is now available in Australia.