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Hybridoma technology remains a cornerstone of monoclonal antibody (mAb) discovery. Classical screening practices such as ELISA, western blot, and dot blot require laborious, time-consuming procedures, rendering them inefficient in time-restricted decision-making. Additionally, due to these assays' practical and technical limitations, specificity testing of the mAbs is usually omitted during the primary screening of hybridoma libraries. Herein, we present a rapid, dipstick immunoassay (DIA) designed for mAbs screening in cell culture supernatant. The integrated proprietary setup is based on antibody capture and comprises accessible materials such as conjugate pad, nitrocellulose membrane, and absorbent pad. The critical element of this technology is the design of the assay. During the assay run, dipsticks are inserted into supernatant-containing microtiter wells, initiating capillary flow of mAbs towards the conjugate pad where a pre-dried HRP-labeled anti-host species antibody is embedded. This interaction forms an immunocomplex, which migrates to the nitrocellulose membrane and binds to the pre-immobilized target antigen while simultaneously encountering immobilized putative cross-reacting proteins in a multiplex-line fashion. Upon addition of HRP-oxidizable substrate, signal acquisition is performed using a simple camera (colorimetry) or a CCD sensor (chemiluminescence). The system was quantitative up to 2500 ng mL -1 and showed a minimum detectable concentration of 0.61 ng mL -1 . Compared to the gold-standard ELISA, the sensitivity was 8-fold higher, while the dynamic range was similar. Critically, the assay allows for the concurrent assessment of antibody specificity in one run. This immunoassay's unique configuration, simplicity, and rapidity can facilitate antibody discovery., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robert S. Marks & Frans Kokojka have a patent pending to Ben-Gurion University of the Negev-BGN Technologies Ltd., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The assessment of stress-related hormone levels using non-invasive methods has gained popularity in mammal and bird welfare, yet its application in reptiles remains limited. Particularly, the exploration of physiological measures such as faecal corticosterone metabolites (FCMs) for reptilian welfare has scarcely been explored. This study aims to validate two enzyme immunoassays (5α-pregnane-3ß,11ß,21-triol-20-one and 11-oxoaetiocholanolone EIA) for monitoring FCM levels in the European common wall lizard (Podarcis muralis). We collected daily faecal samples before (baseline) and after (post-treatment phase) inducing elevated corticosterone levels using transdermal administration of corticosterone (pharmacological treatment) and handling/confinement (biological treatment). We also conducted daily behavioural observations to explore the relationship between stress-related corticosterone changes and behaviour. Although treatments induced significant increases in FCM levels, the effect was much larger in the pharmacological one. Transdermal corticosterone induced a cumulative increase in FCMs over the treatment period, with a higher response observed in females. In contrast, the biological treatment yielded smaller FCM peaks, with no significant sex differences. Overall, 5α-pregnane-3ß,11ß,21-triol-20-one EIA appeared to be more sensitive in detecting these effects. Regarding lizard behaviour, both treatments led to increased hiding and decreased basking compared to baseline. The effects were more pronounced in animals subjected to handling/confinement, despite smaller FCM increases. Our results confirm the suitability of an EIA for monitoring FCMs in both male and female common wall lizards and provide insights into the complexities of using integrated approaches to assess stress, highlighting the need for further research on direct measures to evaluate reptile welfare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: We evaluated the application of a newly improved enzyme immunoassay (EIA) kit, Mumps IgG Seiken®, by comparing antibody responses to different EIA kits, and neutralization tests (NTs), using clinical samples., Methods: Serum samples were collected before and 4-6 weeks after vaccination from 128 children who had no history of mumps or mumps vaccination. Using three different EIA kits, Mumps IgG Seiken®, a commercial kit from Enzygnost®, and an in-house kit, mumps-specific IgG antibodies were measured. Anti-mumps antibody responses between each of the EIA kits and NTs were evaluated., Results: Before vaccination, all samples were seronegative, except for three serum samples measured by Mumps IgG Seiken®, which exhibited equivocal. The antibody-positivity rates and equivocal rates of Mumps IgG Seiken® post-vaccination (71.1 % and 23.4 %, respectively) were comparable to those of Enzygnost® (74.2 % and 19.5 %, respectively). However, antibody-positivity and equivocal rates were significantly higher and lower than those of in-house kit (51.6 % and 40.6 %, respectively) (p < 0.05). The concordance rates of the kit (68.7 %) were comparable to those of Enzygnost® (71.1 %) and higher than those of in-house-developed kit (58.6 %) (p < 0.05). The area under curve of the three EIA kits were 0.801, 0.804, and 0.859; however, the differences did not reach significance. Correlations of the values obtained using three EIA kits with NTs were 0.71, 0.61, and 0.78., Conclusion: The newly approved Mumps IgG Seiken® showed good correlation with NTs and had lower equivocal rates compared to in-house kit, comparable to Enzygnost®. This kit may be clinically acceptable for the evaluation of vaccine immunogenicity., Competing Interests: Declaration of competing interest Tetsuo Nakayama received consultant fees from Daiichi Sankyo Co., Ltd.; scholarship research funding from Eiken Chemical Co., Ltd.; lecture fees from Takeda Pharmaceutical Co., Ltd., MSD K.K., and Meiji Seika Pharma Co., Ltd. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

To determine the potency of the inactivated rotavirus vaccine (IRV), we developed an enzyme immunoassay (EIA) using a biotin-conjugated RV VP7-specific monoclonal antibody. RV VP7, a pivotal structural protein in the outer capsid layer, governs RV G genotypes and prompts host immune responses, including neutralizing antibodies. This EIA showed high specificity, good linearity, high precision, and high accuracy, with a low limit of detection (LOD) and a limit of quantitation (LOQ) of 0.037 µg/ml RV antigen. The EIA was evaluated and proved suitable for establishing the long-term stability of IRV drug substance (DS) and aluminum-formulated drug product (DP) when stored at -70±10°C and 5±3 °C, respectively. Our results support the use of this EIA to examine the stability and determine the potency, antigen dose, lot-to-lot consistency, and lot release of IRV products. This RV potency assay may serve as an alternative to in vivo potency tests, making it suitable for quality control tests of cGMP IRV lots in clinical trials., Competing Interests: Declaration of Competing Interest The Centers for Disease Control and Prevention (CDC) hold patents for rotavirus strains developed by Baoming Jiang and Yuhuan Wang. No other conflict to report. The findings and conclusions in this report are those of the authors and do not necessarily represent the official positions of the Centers for Disease Control and Prevention., (Copyright © 2024. Published by Elsevier B.V.)

Background: Obtaining non-directed samples from the upper bronchial tree is easier to perform and poses fewer risks for critically ill patients than deep bronchoalveolar lavage (BAL). Since invasive pulmonary aspergillosis is associated with a high mortality in critically ill patients, timely diagnosis and rapid initiation of treatment are of utmost importance., Objectives: The objective of this study was to compare Galactomannan (GM) testing by Enzyme Immunoassay (EIA), GM Lateral Flow Assay (LFA) and the detection of Aspergillus DNA by Polymerase Chain Reaction (PCR) between directed BAL and non-directed bronchial lavage (BL) in critically ill patients., Methods: In this prospective, exploratory pilot study, we analysed 120 samples from 40 patients admitted to 12 mixed intensive care units. Inclusion criteria required either risk factors for IPA or positive Aspergillus assessments and met the criteria published by the European Society of Clinical Microbiology and Infectious Diseases guidelines for IPA diagnosis. Both respiratory secretions and blood were collected. In each patient, LFA and PCR were performed on BAL, BL and blood serum, respectively. The EIA test was applied to the BL and BAL of each patient, and the serum of 24 patients. The study was registered on clinicaltrials.gov (NCT04848831)., Results: In a total of 80 respiratory samples, Aspergillus GM EIA yielded optical density indices (ODI) ranging from 0.04 to ≥ 3.5. We observed a high correlation between BAL and BL samples for Aspergillus GM EIA (Pearson's r = 0.78 [95% CI 0.62, 0.88]; intraclass correlation coefficient 0.78). At an ODI cutoff of 0.8 for BAL and 1.2 for BL, the sensitivity of Aspergillus GM EIA was 0.94, while the specificity was 0.67. Increasing the BAL cutoff to 1.0 ODI improved the specificity to 0.86. Aspergillus PCR examination showed good agreement between the two compartments, with a Cohen's kappa coefficient of 0.75 (95% CI 0.48, 1.00). The correlation of Aspergillus GM LFA between BAL and BL was weak., Conclusions: Our findings demonstrate that the detection of Aspergillus GM using EIA or Aspergillus PCR in BL is comparable to that in BAL. Thus, BL samples can be reliably used for diagnosing invasive pulmonary aspergillosis., (© 2025 The Author(s). Mycoses published by Wiley‐VCH GmbH.)

The laboratory diagnosis of Clostridioides difficile infection (CDI) is controversial. Nucleic acid amplification tests (NAAT) and toxin enzyme immunoassays (EIA) are most widely used, often in combination. However, the interpretation of a positive NAAT and negative toxin immunoassay (NAAT+/EIA-) is uncertain. PubMed and EMBASE were searched for studies reporting clinical outcomes in NAAT+/EIA- versus NAAT+/EIA+ patients. Forty-six studies comprising 33,959 patients were included in this meta-analysis. All-cause mortality (RR 0.96, 95% CI 0.80-1.15), attributable mortality (RR 0.61, 95% CI 0.20-1.91), fulminant CDI (RR 0.83, 95% CI 0.57-1.20), radiographic evidence of CDI (RR 0.87, 95% CI 0.65-1.16), total CDI complications (RR 0.95, 95% CI 0.59-1.53), colectomies (RR 0.78, 95% CI 0.34-1.79), and ICU admission (RR 1.04, 95% CI 0.84-1.30) did not significantly differ between NAAT+/EIA- and NAAT+/EIA+ patients. However, rates of recurrent (RR 0.62, 95% CI 0.50-0.77) or severe (RR 0.74, 95% CI 0.63-0.88) CDI were significantly lower in NAAT+/EIA- patients than in NAAT+/EIA+ patients. The pooled prevalence of NAAT+/EIA- patients who were treated with antibiotics for CDI was 73.4% (pooled proportion 0.72, 95% CI 0.52-0.88). NAAT+/EIA- patients have lower rates of recurrence and are at reduced risk for severe CDI compared with NAAT+/EIA+ patients but have a risk of CDI-related complications and mortality comparable to that of NAAT+/EIA+ patients. Toxin results cannot rule in or rule out CDI, and the decision whether to treat symptomatic NAAT+/EIA- patients for CDI should be based on clinical presentation and not on the toxin result.IMPORTANCE Clostridioides difficile infection (CDI) is a common cause of healthcare-associated infections and the leading cause of antibiotic-associated diarrhea. However, the laboratory diagnosis of CDI, primarily done by nucleic acid amplification test (NAAT) and enzyme immunoassay (EIA), is controversial, especially in patients who test positive by NAAT but negative by EIA. In this systematic review, we compared the clinical outcomes of NAAT+/EIA- versus NAAT+/EIA+ patients and found that the two groups have similar risk of mortality and CDI-related complications. However, NAAT+/EIA- patients had significantly lower rates of recurrence and severe CDI than NAAT+/EIA+ patients, and most NAAT+/EIA- patients received CDI therapy. Toxin testing can help to predict the likelihood of CDI recurrence or severe infection, but the toxin result should not be a determining factor in the administration of CDI therapy. The decision on whether to treat NAAT+/EIA- patients should be based on clinical assessment., Competing Interests: The authors declare no conflict of interest.

Immunoglobulin A (IgA) has been investigated as a stress biomarker with the potential to complement glucocorticoid measurements in welfare assessments. This study aimed to develop the methodology and validate an enzyme immunoassay (EIA) for quantifying IgA in feces (FIgA) of lions (Panthera leo), investigate excretion patterns of FIgA under baseline conditions in captive lions, and explore its relationship with fecal glucocorticoid metabolites (FGM). Feces were collected from 11 lions housed in stable social groups at four Spanish zoos over a period of two to six weeks. FIgA was reliably quantified using a commercial EIA, with concentrations ranging from 0.28 to 794.17 μg IgA/g feces, showing substantial intra- and inter-individual variability. Females had significantly higher FIgA concentrations than males (113.10 vs 54.96 μg IgA/g feces; p < 0.01). Additionally, FIgA concentrations varied across zoos (p < 0.001). Positive correlations were found between FIgA and FGM for all samples combined (rho = 0.43, p < 0.001) and across individual means (rho = 0.70, p < 0.05), but not consistently when examining each lion separately. This study demonstrates for the first time that IgA can be reliably quantified in lion feces, paving the way for its application in welfare studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The continuous spread and evolution of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) necessitate the development of convenient and rapid detection methods. In this study, we developed a fluorescence enzyme immunoassay (FEIA) based on a nanobody (Nb)-alkaline phosphatase (ALP) fusion protein for detection of SARS-CoV-2 spike protein. The genetically modified anti-SARS-CoV-2 S-RBD Nb, Nb61, gene was fused with the ALP gene sequences via a flexible linker. Recombinant cloning was used to yield a recombinant prokaryotic expression plasmid, Nb61-ALP-His. The Nb61-ALP-His construct was transformed into E. coli BL21(DE3) and expressed in bacteria. Both Nb61 properties and ALP enzymatic activity were validated by colorimetric and fluorometric analysis. FEIA was optimized and established on the basis of the Nb61-ALP fusion protein. The detection limit of the FEIA was 3.18 ng/mL, with a linear range of 1.9-62.5 ng/mL. Comparison with a commercial kit indicated the reliability of the Nb61-ALP fusion-protein-based FEIA for monitoring the SARS-CoV-2 spike protein. This study highlights the potential of Nb-based enzyme immunoassays as a valuable tool for the rapid and accurate detection of SARS-CoV-2.

Histoplasmosis poses a significant risk to HIV patients, particularly in regions with limited access to antiretroviral therapy. Antigen detection assays are crucial in these settings for timely diagnosis and treatment, which can reduce mortality. While commercial antigen detection kits have performed well in diagnosing histoplasmosis, their effectiveness in monitoring treatment remains unclear. This study aimed to evaluate the correlation between urine antigen levels and clinical response using the clarus Histoplasma Galactomannan (GM) enzyme immunoassays (EIA) kit. The study followed 27 HIV patients diagnosed with histoplasmosis over 24 weeks, measuring urinary Histoplasma antigen (Ag) levels and clinical outcomes. Patients received amphotericin B as induction therapy, followed by maintenance with itraconazole. Results showed a significant decrease in Ag levels over time, with clinical scores improving in correlation with the decline in Ag levels. Four patients exhibited atypical Ag patterns due to immune reconstitution inflammatory syndrome or issues with itraconazole bioavailability. Despite these challenges, all patients showed improvement by week 24. The findings suggest that the clarus Histoplasma GM EIA kit could be a valuable tool for monitoring and evaluating the response to antifungal therapy in histoplasmosis patients., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Porcine circovirus 3 (PCV3) is a small non-enveloped circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS). It has occurred worldwide and poses a serious threat to the pig industry. However, there is no commercially available vaccine. PCV3 capsid protein (Cap) is an ideal antigen candidate for serodiagnosis. Here, a novel fully automated chemiluminescence enzyme immunoassay (CLEIA) was developed to detect antibodies (Abs) to Cap in porcine serum. Recombinant PCV3 Cap, self-assembled into virus-like particles (VLPs), was produced using baculovirus and coupled to magnetic particles (Cap-MPs) as carriers. Combined with an alkaline phosphatase (AP)-adamantane (AMPPD) system, Cap-Abs can be rapidly measured on a fully automated chemiluminescence analyzer. Under optimal conditions, a cut-off value of 31,508 was determined, with a diagnostic sensitivity of 96.8% and specificity of 97.3%. No cross-reactivity was observed with PCV1 and PCV2 and other common porcine pathogens, and both intra-assay and inter-assay coefficients were less than 5% and 10%, respectively. Prepared Cap-MPs can be stored at 4 °C for more than 6 months. Importantly, this CLEIA had a good agreement of 95.19% with the commercially available kit, demonstrating excellent analytical sensitivity and significantly reduced operating time and labor. A serological survey was then conducted, and showed that PCV3 continues to spread widely in South China. In conclusion, our CLEIA provides time and labor-saving, and a reliable tool for PCV3 epidemiological surveillance.

Objective A dedicated stool container is required for Helicobacter pylori stool antigen testing. If H. pylori fecal antigen can be measured from a fecal occult blood test container (S fecal collection container or S container), which is widely used for colorectal cancer screening, screening of the upper and lower gastrointestinal tract can be performed with a single stool sample. We investigated the clinical usefulness of an H. pylori stool antigen assay using an S container. Materials A total of 347 patients who underwent esophagogastroduodenoscopy (EGD) were included. After the procedure, H. pylori stool antigen was measured using the S container and collection container recommended for H. pylori stool antigen (BL-stool collection container or BL container), and the qualitative outcomes of each were compared. A bioluminescent enzyme immunoassay (BLEIA) was used to measure H. pylori stool antigen. Results The overall agreement between S containers and BL containers was 100% (347/347), indicating that the qualitative outcomes were equivalent. As a secondary analysis, the results of the S container samples were evaluated according to the diagnosis made by physicians, and the overall agreement rate was 99.7% (345/346), indicating a high correlation. Conclusion The detection of H. pylori stool antigen using the S container is clinically useful because the results are equivalent to those obtained by the usual method. Screening of the upper and lower gastrointestinal tract is expected to be possible with a single stool sample in the future.

Background: Diagnosis of Lyme borreliosis (LB) relies on clinical symptoms and detection of Borrelia-specific antibodies. Guidelines recommend a two-tier testing (TTT) strategy for disseminated LB: serological screening with a sensitive enzyme immunoassay (EIA) and confirmation with a specific immunoblot. Searching for the most sensitive and specific approach, this retrospective study evaluated standard (STTT) and modified (MTTT) strategies using a well-defined study population., Methods: Cases included patients with active Lyme neuroborreliosis (LNB; n = 29) or Lyme arthritis (LA; n = 17). Controls comprised patients treated for LNB (n = 36) or LA (n = 8), healthy individuals who were either untreated (n = 75) or treated for LB (n = 15) in the past, and patients with potentially cross-reactive diseases (n = 16). Sera were subjected to three EIAs and two immunoblots. Reactive screening results were confirmed by immunoblot (STTT) or EIA (MTTT). Solitary IgM results in the screening assay and effects of antibiotic treatment on isotype-specific seropositivity rates were also assessed., Results: Sensitivities of STTT strategies ranged from 90%-97% for LNB and were 100% for LA. MTTT strategies were 100% sensitive. Specificities ranged from 89%-95% for STTT and from 88%-93% for MTTT strategies. Differences between STTT and MTTT strategies were not statistically significant. Solitary IgM reactivity was common among controls. Antibiotic treatment significantly reduced IgM/IgG positivity for LNB patients; for LA patients, a decline was only observed for IgM., Conclusion: In conclusion, MTTT strategies showed a slightly higher sensitivity and similar specificity compared to STTT strategies. Since EIAs are more time- and cost-efficient, MTTT strategies seem more favorable for clinical use. IgG testing enhances specificity with minimal sensitivity loss., Competing Interests: Declarations. Ethics approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study “T-cell response in Lyme” (TRIL) was approved by the regional Medical Research Ethics Committees United (MEC-U), Nieuwegein, The Netherlands and registered with the Central Committee on Research Involving Human Subjects (CCMO) of the Netherlands under number NL36407.100.11 at 09–07-2020. Consent to participate: Informed consent was obtained from all individual participants included in the TRIL study. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The measurement of biomarkers in blood and excreta can enable immune status assessment and provide prognostic information on individual health outcomes. In this respect, the fecal measurement of secretory immunoglobulin A (sIgA), the primary mammalian antibody for mucosal defense, has recently received increased interest in a few anthropoid primates, but a fecal sIgA assay for use in strepsirrhine primates has not yet been reported. Here, we develop and analytically validate a cost-effective in-house sandwich enzyme immunoassay for the extraction and measurement of sIgA in feces of redfronted lemurs (Eulemur rufifrons). We also tested a simple method for sIgA extraction that can be used under remote field conditions and undertook experiments to assess the robustness of sIgA concentrations to variation in processing and storage conditions of fecal extracts. Our analytical validation revealed that the assay recognizes immunoreactive sIgA in redfronted lemur feces, that sIgA can be measured accurately with no potential interference from the fecal matrix, and that assay reagents and performance are highly stable over time. The field-friendly extraction procedure produced sIgA results strongly correlated with those generated by a standard laboratory extraction method. Short-term storage at room temperature resulted in a slight decline in sIgA concentrations, whereas freezing extracts at -20°C kept sIgA levels stable for at least 3 months. Longer-term storage of >5 months, however, led to a significant decline of sIgA concentrations. Multiple freeze-thaw cycles did not affect sIgA levels. This study, therefore, provides the basis for measuring fecal sIgA in lemurs and possibly other strepsirrhines. When samples are processed properly and stored frozen, and when sIgA analysis can be performed within 3 months upon sample collection, fecal sIgA measurements can become a valuable tool for monitoring aspects of immunity and health in both zoo-housed and wild-living lemurs., (© 2024 The Author(s). American Journal of Primatology published by Wiley Periodicals LLC.)

Soluble CLEC-2 is anticipated to have various clinical applications as a novel biomarker for in vivo platelet activation, assessable using plasma obtained through routine sampling procedures. While sCLEC-2 has been measured using ELISA, we have developed a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) reagent that can yield results in approximately 19 minutes. This study aims to assess its fundamental performance and explore the molecular forms of sCLEC-2 measured in patient samples. We examined the sensitivity, precision, linearity, influence of endogenous substances, residual platelets, as well as the correlation with the ELISA method, for the sCLEC-2 CLEIA reagent. The CLEIA method demonstrated sufficient sensitivity for levels observed in healthy donors, and its basic performance was satisfactory. It exhibited a strong correlation with the previously described ELISA method, with reference ranges that did not significantly differ from the ELISA data. The sCLEC-2 reference range for males was significantly higher than that for females. Since it is known that sCLEC-2 exists in shed form and microparticle form, we investigated molecular forms of sCLEC-2 measured by the CLEIA in in vitro-activated platelets and in patients' plasma using gel filtration. It is considered that the CLEIA method shows significantly stronger reactivity with the shed form compared to the microparticle form. Studies using gel filtration of patient samples also suggest that the shed form is being primarily measured. The sCLEC-2 CLEIA reagent exhibits robust performance and is promising for clinical applications.

African swine fever (ASF) is a high-consequence transboundary animal disease caused by African swine fever virus (ASFV). Given that vaccines are not widely available, ASFV detection, including by molecular and serologic assays, is paramount to efficacious control and mitigation of ASF. ASFV-specific antibodies can be detected as early as 7-10 d postinfection in infected animals and may persist for several months or longer. Accurate detection of ASFV-specific antibody is critical for the identification of chronically infected, subclinically infected, or recovered animals. ELISAs are commonly used for the rapid screening of large numbers of animals for ASFV antibodies. The World Organisation for Animal Health recommends that ELISA-positive results should be confirmed with a second serologic method, such as an indirect immunofluorescent assay, indirect immunoperoxidase test (IPT), or immunoblot test. Commercial kits are not available for those tests. We developed and validated an in-house IPT by using a currently circulating genotype II ASFV strain as antigen. The sensitivity and specificity of the in-house IPT are comparable to the reference IPT developed by an international ASFV reference laboratory and superior to a commercial blocking ELISA., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Accurate and timely diagnosis of Alzheimer's disease (AD) is necessary to maximize the effectiveness of treatment and using biomarkers for diagnosis is attracting attention as a minimally invasive method with few side effects. Electrochemical immunosensor (EI) is a method that is in the spotlight in the medical and bioanalytical fields due to its portability and field usability. Here, we quantified four AD specific biomarkers using EIs based on enzyme immunoassay. We selected and developed quantitative methods for the biomarkers using screen-printed gold electrodes. For three biomarkers, quantification was performed using competition immunoassays in which antigen-antibody premix mixtures were applied to antigen-immobilized electrodes and the limit of detection (LOD) values were secured, 1.20 ng/ml, 1.30 ng/ml, and 1.74 ng/ml, respectively. For the other, a sandwich immunoassay using antibody pair was selected for quantification and LOD was also achieved as 0.077 ng/ml. All four biomarkers in buffer samples were successfully quantified and reliable R 2 values were obtained, and reliable calibration curves were secured for three biomarkers in spiked human serum samples. The immunosensors developed and will be optimized are expected to be used in various fields, including detection of biomarkers for not only AD but also related diseases., (© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Background: Interstitial lung diseases (ILD) is a group of lung disorders characterized by interstitial lung thickening due to inflammatory and fibrotic processes. Krebs von den Lungen-6 (KL-6) is a molecule secreted by damaged type II alveolar pneumocytes in the alveolar space. The goal of the present study was to compare two detection methods of KL-6 in both bronchoalveolar lavage (BAL) and serum from ILD patients at the moment of diagnosis., Methods: Patients with suspicious of ILD and followed at two Italian referral centres for rare lung diseases were included in the study. BAL fluid and serum were collected and analysed by chemiluminescent enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) methods provided by Tosoh Biosciences., Results: A total of 158 (mean age ± standard deviation, 61.5 ± 13.7, 65 females) patients were enrolled. A total of, 36 had diagnosis of idiopathic pulmonary fibrosis (IPF), 74 sarcoidosis, 15 connective tissue disease-ILD (CTD-ILD) and 33 other ILD. Diagnostic agreement between two methods was demonstrated for both BAL (r = 0.707, p < 0.0001) and serum (r = 0.816, p < 0.0001). BAL KL-6 values were lower than serum (p < 0.0001). IPF patients had higher serum KL-6 concentration than other ILDs (p = 0.0294), while BAL KL-6 values were lower in IPF than in non-IPF (p = 0.0023)., Conclusion: This study explored KL-6 concentrations through the CLEIA method in serum and BAL of patients with various ILDs, showing significant differences of biomarkers concentrations between IPF and other non-IPF ILDs. Our findings are promising as they provided further knowledge concerning KL-6 expression across different ILDs and may suggest its utility in differential diagnosis., (© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background: We developed a fully automated quantitative immunoassay for the detection of prostaglandin E-major urinary metabolite (PGE-MUM). In this study, we evaluated the analytical performance of this assay., Methods: Sensitivity, within-run reproducibility, correlation with radioimmunoassay (RIA), cross-reactivity, dilution linearity, spike recovery performance, analyte stability, and effects of coexisting substances were evaluated. The assay was also used to measure PGE-MUM in 211 healthy people., Results: The limit of detection and quantification were 1.0 and 1.3 ng/mL, respectively. When the assay was performed six times in a single run, the coefficient of variation ranged from 1.4% to 2.2%. The coefficient of correlation with a preceding RIA method was 0.970 with a correlation slope of 0.88. There was no cross-reactivity with PGE-MUM analogs. Linearity of dilution was confirmed at up to 16-fold dilution with assay results within 100 ± 20% of the theoretical values calculated based on the undiluted sample. Spike recovery was good and ranged from 94% to 101%. Analyte stability was tested by storing samples at 25°C for 6 days, 10°C for 1 month, and by performing up to five freeze-thaw cycles. Assay results were all within 100 ± 10%, the values measured before storage and before the freeze-thaw process. Assay results in healthy people ranged from 3.1 to 162.7 ng/mL (mean: 35.8 ng/mL). After correction for creatinine, the 95% confidence interval was 8.68-42.25 μg/g creatinine., Conclusion: The assay precisely detects PGE-MUM., (© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H 2 O 2 ), and then HRP catalyzes H 2 O 2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

The PATHFAST TB LAM Ag assay is based on a chemiluminescent enzyme immunoassay to quantify lipoarabinomannan (LAM) in sputum within 1 h, and was developed as an alternative to conventional culture methods for monitoring tuberculosis (TB) treatment. This study aimed to evaluate the analytical performance and initial clinical feasibility of using five Mycobacterium tuberculosis variants, 178 non-tuberculous mycobacteria (NTM), 34 upper respiratory and oral cavity microorganisms, 100 sputum specimens from untreated patients, and potential interfering substances, including 27 drugs. The results reveled a single-site repeatability coefficient of variation (CV) of 5.2%-7.0%, and a multi-site reproducibility CV of 7.1%-8.4%. The limit of blank, limit of detection, and limit of quantification were 3.03 pg/mL, 6.67 pg/mL, and 7.44 pg/mL, respectively. Linearity was observed over the analytical measurement range (10.0 pg/mL-50,000 pg/mL), and no hook effect was observed. The assay tended to cross-react with slow-growing NTMs, but not with common upper respiratory and oral cavity microorganisms, except Nocardia asteroides , Nocardia farcinica , and Tsukamurella paurometabola . No interference was observed in the presence of mucin, blood, or major anti-TB, anti-HIV, and anti-pneumonia drugs. Regarding clinical performance, the assay had a sensitivity of 88.8% (95% CI: 80.0%-94.0%) and specificity of 100.0% (95% CI: 83.9%-100.0%) using mycobacterial culture as the reference standard, and a correlation (Spearman's r = -0.770) was observed between LAM concentration and time to detection of culture. These findings show, for the first time, that the PATHFAST TB LAM Ag assay has potential value for monitoring TB treatment., Competing Interests: A.A., M.T., T.Y., S.Matsushita, and Y.H. are employees of PHC Corporation (formerly LSI Medience Corporation). K.C. and S.Mitarai received joint research fees from PHC Corporation (formerly LSI Medience Corporation). T.I. and T.S. are employees of LSI Medience Corporation.

Background: Galactomannan (GM) testing using Platelia Aspergillus enzyme immunoassay (Platelia AGM) from bronchoalveolar lavage fluid (BALF) aids in early diagnosis of invasive pulmonary aspergillosis (IPA). Globally, only a minority of laboratories have the capability to perform on-site GM testing, necessitating accessible and affordable alternatives. Hence, we conducted a comparative evaluation of the new clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype) with Platelia AGM using BALF samples., Methods: This is a single-center, prospective, cross-sectional study, where Platelia AGM testing was routinely performed followed by clarus AGM prototype testing in those with true positive or true negative AGM test results according to the 2020 EORTC/MSG and the 2024 FUNDICU consensus definitions. Descriptive statistics, ROC curve analysis, and Spearman's correlation analysis were used to evaluate analytical performance of the clarus AGM prototype assay., Results: This study enrolled 259 adult patients, of which 53 (20%) were classified as probable IPA, while 206 did not fulfill IPA-criteria. Spearman's correlation analysis revealed a strong correlation between the two assays (rho = 0.727, p < 0.001). The clarus AGM prototype had a sensitivity of 96% (51/53) and a specificity of 74% (153/206) for differentiating probable versus no IPA when using the manufacturer recommended cut-off. ROC curve analysis showed an AUC of 0.936 (95% CI 0.901-0.971) for the clarus AGM prototype, while the Platelia AGM yielded an AUC of 0.918 (95% CI 0.876-0.959)., Conclusions: Clarus AGM prototype demonstrated a strong correlation and promising test performance, comparable to Platelia AGM, rendering it a viable alternative in patients at risk of IPA., (© 2024. The Author(s).)

In the present study, we validate and compare the second-generation Miravista Coccidioides IgG and IgM enzyme immunoassays (EIA) (MiraVista Diagnostics [MVD] Ab EIA) to Meridian Diagnostics Coccidioides IgG and IgM EIA (Meridian Ab EIA), immunodiffusion (ID) and complement fixation (CF). We also evaluated whether the addition of Coccidioides antigen testing to anti-Coccidioides antibody testing increased the sensitivity for the diagnosis of currently active coccidioidomycosis. We retrospectively studied 555 patients evaluated at Valleywise Health Medical Center between January 2013 and May 2017 for whom coccidioidomycosis was suspected and samples were submitted to MVD for testing. Specimens were tested for antigen in the MVD antigen enzyme immunoassay (MVD Ag EIA) and for IgG and IgM antibodies with MVD and Meridian Diagnostics EIAs. ID and CF were obtained from medical records. Sensitivity and specificity were 83.0% and 91.1% or MVD Ab EIA, 69.3% and 99.7% for Meridian Ab EIA, 85.4% and 100% for ID and 65.5% and 100% for CF. Combined MVD antigen and antibody detection by EIA and ID resulted in increased sensitivity in disseminated and pulmonary disease (MVD Ag/MVD Ab: 100%, 88.3%; MVD Ag/Meridian Ab: 98.2%, 78.6%; and MVD Ag/ID: 100%, 91.7%). The detection of antibodies by MVD EIA was more sensitive than Meridian EIA or CF but similar to ID. This study supports the use of antigen testing in immunocompromised patients and those with suspected disseminated disease. Furthermore, the addition of antigen detection by EIA to antibody detection resulted in higher sensitivity of all serological tests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Aspergillus species can colonize and infect immunocompetent and immunocompromised hosts. Conventional fungal identification depends on microscopic analysis and microorganism medium growth. Other diagnostic methods, non-growth dependent, to invasive fungal infections, are the biomarkers that detect circulating polysaccharides, for example, 1-3-β-d-Glucan and galactomannan. Both are polysaccharides present on the external layer of fungi cell wall and can be detected in clinical samples during the growth of the fungus in the patient. This study aimed to compare the galactomannan detection of Lateral Flow Assay and Enzyme Immunoassay methods in Bronchoalveolar Lavage Fluid. The galactomannan antigen in Bronchoalveolar Lavage Fluid was measured using Enzyme Immunoassay according to the manufacturer's instructions (PLATELIA ASPERGILLUS™ BioRad) and, using a Lateral Flow Assay according to the manufacturer's instructions (Galactomannan LFA IMMY©). The 71 samples were Bronchoalveolar Lavage Fluid of patients hospitalized at Unicamp Clinical Hospital between 2019 and 2021; of these samples 12/71 (16.9 %) resulted in positive Galactomannan-Lateral Flow Assay. In contrast, Galactomannan-Enzyme Immunoassay resulted as positive in 9/71 (12.6 %) samples, a difference that showed not significant statistically (p-value = 0.36) Comparing both assays' results identified 8 divergences between them, about 11 % of the total sample. The Sensitivity (73.3 %), Specificity (92.35 %), Positive Predictive Value (62.85 %) and Negative Predictive Value (95.15 %) of Lateral Flow Assay were calculated using the Galactomannan Enzyme Immunoassay as standard. The Lateral Flow Assay demonstrated good results when compared with the Enzyme Immunoassay., Competing Interests: Conflicts of interest The authors declare no have conflicts of interest., (Copyright © 2024 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Double labeling to identify different markers in the same tissue section represents a useful tool either for in situ diagnosis or characterization of molecular associations. Here, we present a protocol to detect senescence-associated β-galactosidase (SA-βGal) and immunoperoxidase (IPO) activity in fresh-frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-βGal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-βGal activity. For complete details on the use and execution of this protocol, please refer to Pacheco-Rivera et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Serum galactomannan (GM) testing is essential for diagnosing invasive aspergillosis (IA), particularly in immunocompromised individuals. The global lack of on-site GM testing capacities necessitates cost-effective alternatives, such as .the clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype)., Methods: This single-centre, cross-sectional study compared the diagnostic performance of the clarus AGM prototype (IMMY, Norman, Oklahoma) with the serological gold standard (=Platelia AGM assay; Bio-Rad, Marnes-la-Cocquette, France). IA was classified according to modified 2020 EORTC/MSG consensus and 2024 FUNDICU criteria. In total, 300 prospectively (May-Dec 2023) and retrospectively (2012-2015) collected samples were included., Results: Among 300 samples from 232 patients, 49 (16%) were classified as proven (n = 1) or probable IA (n = 48). In non-IA cases (n = 250), one patient was classified as possible IA. With the manufacturer recommended cut-off of ≥0.2, sensitivity and specificity of the clarus AGM prototype were 27% (13/49; 95% confidence interval [CI]: 15%-41%) and 99% (248/250; 95% CI: 97%-100%), respectively, while sensitivity and specificity were 78% and 79% when using the optimised Youden's cut-off of 0.0045 ODI. ROC curve analysis demonstrated an area under the curve (AUC) of 0.829 (95% CI: 0.760-0.898) for the clarus AGM prototype in distinguishing between proven/probable IA and non-IA. The AUC for the Platelia AGM was 0.951 (95% CI: 0.909-994). Spearman's correlation analysis showed a weak correlation between the two assays (0.382; p < .001)., Conclusions: The weak correlation between the clarus AGM prototype and Platelia AGM highlights the need for further investigation into the clinical performance of the clarus AGM prototype, giving the different antigen epitopes addressed., (© 2024 The Author(s). Mycoses published by Wiley‐VCH GmbH.)

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Objective: The serum squamous cell carcinoma antigen (SCCA) level is a well-known tumor marker for squamous cell carcinoma. In this study, we examined the impact of immunoglobulin (Ig)-bound macromolecular SCCA on serum SCCA levels measured by 2 different methods., Methods: Seventy-five serum samples with an SCCA level >5.0 ng/mL as determined by a chemiluminescent immunoassay (CLIA) were also analyzed using a chemiluminescent enzyme immunoassay (CLEIA). The levels of IgG- and IgA-type anti-SCCA antibodies, which form immunoglobulins and macromolecules, respectively, were determined using an enzyme-linked immunosorbent assay. An absorption test was performed to confirm the presence of anti-SCCA antibodies., Results: The correlation coefficient between the values measured by CLEIA and CLIA was 0.768. The ratio of SCCA levels measured by CLEIA to those measured by CLIA in 14 samples with IgG-type anti-SCCA antibodies was significantly lower than that in samples without these antibodies (P < .031). Absorption tests showed that SCCA levels measured by CLIA might be falsely high in samples with IgG-type anti-SCCA antibodies, probably due to reactions with SCCA1., Conclusion: The level of SCCA as measured by CLIA and CLEIA methods correlate well, but the presence of SCCA antibodies can affect the results of the CLIA method., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism., (© 2024. The Author(s).)

Aspergillus-specific antibodies are diagnostic indicators of allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA). Tests for detecting Aspergillus-specific antibodies were not used clinically in Japan, and the production of the Aspergillus precipitin test was discontinued. Thus, alternative tests for diagnosing aspergillosis are urgently needed. We retrospectively evaluated 64 patients with suspected ABPA and CPA who underwent precipitin antibody testing. Serum Aspergillus IgG levels were measured and compared using the Bordier Aspergillus fumigatus ELISA and the Platelia Aspergillus IgG (Bio-Rad) kits. Of the participants, 18 were diagnosed with CPA, and 8 were diagnosed with ABPA. Both the Bordier and Bio-Rad kits showed high sensitivity and specificity for CPA and ABPA. The area under the receiver operating characteristic curves for the Bordier and Bio-Rad kits were 0.97 and 0.95, respectively, for CPA, and 0.89 and 0.91, respectively, for ABPA. In contrast to the Bordier kit, the Bio-Rad kit showed relatively low anti-Aspergillus IgG levels and lower sensitivity to non-fumigatus Aspergillus infections. The Aspergillus-specific IgG ELISA tests showed sufficient diagnostic accuracy. Therefore, these assays are recommended as alternatives to the precipitin kit for diagnosing aspergillosis in clinical settings in Japan.

Introduction: The World Health Organization 2021 lung cancer classification highlights the central role of immunohistochemistry (IHC) in diagnostic pathology. Despite traditional IHC being essential, its limitation to one marker per tissue section brings challenges, particularly when facing cytological limitedly sized samples. To overcome these challenges, multiplex immunocytochemistry (mICC) techniques offer the simultaneous detection of multiple markers from a single section. These advances complement the highly complex imaging techniques that enable additional analyses of cellular interactions., Methods: The present study outlines a comprehensive mICC methodology of an automated multiplex immunoperoxidase staining method and multiple tissue hybrid controls for ICC/mICC. Protocols are presented in detail and demonstrate a careful approach to optimizing various markers for diagnostic workup including immunotherapy., Conclusion: Multiplex IHC/ICC emerges as a transformative force in biomedical diagnostics and research. Beyond simultaneous marker detection, it unravels complexities within tissues - unveiling co-localization nuances, deciphering expression patterns, and enhancing understanding of cellular populations. As personalized treatments gain prominence, the study emphasizes the heightened importance of diagnostic tools and sample adequacy. The present methodological study, encapsulating an automated multiplex immunoperoxidase staining method, symbolizes a stride towards precision in pulmonary carcinoma diagnosis. Multi-tissue controls represent a key element in quality assurance in pathology laboratories., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

The formation of toxic Amyloid β-peptide (Aβ) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aβ oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aβ oligomers produce strong SOBA signals, while monomeric and β-sheet protofibrillar Aβ do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aβ oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

The SARS-CoV-2 virus, which is driving the current COVID-19 epidemic, has been detected in wastewater and is being utilized as a surveillance tool to establish an early warning system to aid in the management and prevention of future pandemics. qPCR is the method usually used to detect SARS-CoV-2 in wastewater. There has been no study using an immunoassay that is less laboratory-intensive than qPCR with a shorter turnaround time. Therefore, we aimed to evaluate the performance of an automated chemiluminescence enzyme immunoassay (CLEIA) for SARS-CoV-2 antigen in wastewater. The CLEIA assay achieved 100% sensitivity and 66.7% specificity in a field-captured wastewater sample compared to the gold standard RT-qPCR. Our early findings suggest that the SARS-CoV-2 antigen can be identified in wastewater samples using an automated CLEIA, reducing the turnaround time and improving the performance of SARS-CoV-2 wastewater monitoring during the pandemic.

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less., (© 2021 Wiley Periodicals LLC.)

Background: Studies have demonstrated the diagnostic efficiency of antibody testing in COVID-19 infection. There is limited data on the IgM/IgG changes in asymptomatic and discharged patients with reoccurring positive nucleic acid test (RPNAT). This study aims to investigate these IgM/IgG changes., Methods: There were 111 patients with positive nucleic acid test (NAT) and 40 suspected patients enrolled in the study. The serum SARS-CoV-2 specific IgM/IgG antibody levels were retrospectively analysed with the disease progress in asymptomatic and RPNAT patients., Results: The best overall performance was found by combining the IgM, IgG, and CT; 95.1% sensitivity and 75% specificity. This was tested in 111 RT-PCR positive cases. The median IgM and IgG levels were lower in the asymptomatic group compared to the symptomatic group ( p  < .01). Among 15 RPNAT cases, the IgM levels of the RPNAT group at the time of discharge (IgM2.79, IQR: 0.95-5.37) and retest (IgM 2.35, IQR: 0.88-8.65) were significantly higher than those of the non-reoccurring positive nucleic acid test group (Non-RPNAT) (IgM on discharge: 0.59, IQR: 0.33-1.22, IgG on retest: 0.92, IQR: 0.51-1.58)., Conclusion: Serum SARS-CoV-2 specific IgM/IgG antibody levels remained at a low level during hospitalisation for asymptomatic patients. Elevated IgM levels may have implications in the identification of RPNAT patients before discharge. Key messages This study determined the IgM/IgG changes in asymptomatic and RPNAT patients. The rate of serum SARS-CoV-2 specific IgM/IgG antibody levels increase in the asymptomatic group was lower than in the symptomatic group during hospitalisation. The IgM level did not decrease significantly at discharge in the RPNAT patients, and was higher than that of the Non-RPNAT group on discharge. These results highlight the importance of timely monitoring of IgM levels to identify RPNAT patients before discharge.

Background: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests., Aims: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays., Methods: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned., Results: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r 2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed., Conclusions: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser., (© 2021 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Background: Parvovirus B19 is a pathogenic virus often diagnosed by serology, yet little is known about analytical performance of commercial enzyme immunoassays (EIAs)., Objective: To investigate performance of 4 EIAs for parvovirus B19 IgM and IgG: Liaison, Euroimmun, Mikrogen and Virion/Serion., Study Design: To compare 4 EIAs to Biotrin's ELISA on 168 samples and determine consensus score for discordant samples using Mikrogen's confirmatory line assay., Results: Two thirds of results for IgM/IgG were identical for all 4 EIAs and Biotrin. Liaison shows the highest IgM sensitivity, but has low specificity. Euroimmun lacks IgM sensitivity. Mikrogen had a good overall performance, but had the lowest IgG specificity. Virion/Serion had variable performance with a low IgM specificity and the most borderline and cross-reactive results., Conclusions: Liaison and Mikrogen have similar performance to Biotrin's ELISA. Euroimmun lacks sensitivity and Virion/Serion produced many borderline and cross-reactive results., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The importance of the detection of relevant toxins or toxin genes to diagnose Clostridioides difficile infection (CDI) or the prediction of clinical outcomes of CDI has been emphasized in recent years. Although stool culture of C. difficile is not routinely recommended in the era of nonculture methods as the preferred tools for CDI diagnosis, the clinical significance of toxigenic C. difficile growth (tCdG) in stool cultures was analyzed. A clinical study was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, in southern Taiwan. Diarrheal adults with fecal glutamate dehydrogenase and C. difficile toxin between January 2013 and April 2020 were included. Of the 209 patients with CDI, 158 (75.6%) had tCdG found in stool cultures, and the rest (51, 24.4%) had no tCdG in stool. Only prior ceftazidime or ceftriaxone therapy was independently associated with no tCdG in stool (odds ratio [OR] 2.17, P =  0.02). Compared to the patients with tCDG in stool, those without tCdG in stool experienced treatment success more often (97.1% versus 67.0%, P <  0.001) if treated with metronidazole or vancomycin but had a similar in-hospital mortality or recurrence rate. In the multivariate analysis among 114 patients with CDI treated with metronidazole or vancomycin, treatment success was independently associated with no tCdG in stool (OR 12.7, P =  0.02). Despite the limited utility of stool cultures in CDI diagnoses, no tCdG in stool culture heralds a favorable therapeutic outcome among adults with CDI treated with metronidazole or vancomycin. IMPORTANCE The importance of detecting toxins or toxin genes when diagnosing Clostridioides difficile infections (CDIs) or predicting the severity and outcomes of CDI has been emphasized in recent years. Although the yielding of C. difficile from stool cultures might implicate higher bacterial loads in fecal samples, in an era of nonculture methods for the standard diagnosis of CDIs, clinical significance of positive stool cultures of toxigenic C. difficile was analyzed in this study. Despite the limited ability of stool cultures in CDI diagnoses, no yielding of C. difficile growth might predict the successful CDI therapy.

Blood based β-amyloid (Aβ) assays that can predict amyloid positivity in the brain are in high demand. Current studies that utilize immunoprecipitation mass spectrometry assay (IP-MS), which has high specificity for measuring analytes, have revealed that precise plasma Aβ assays have the potential to detect amyloid positivity in the brain. In this study, we developed plasma Aβ40 and Aβ42 immunoassays using a fully automated immunoassay platform that is used in routine clinical practice. Our assays showed high sensitivity (limit of quantification: 2.46 pg/mL [Aβ40] and 0.16 pg/mL [Aβ42]) and high reproducibility within-run (coefficients of variation [CVs]: <3.7% [Aβ40] and <2.0% [Aβ42]) and within-laboratory (CVs: <4.6% [Aβ40] and <5.3% [Aβ42]). The interference from plasma components was less than 10%, and the cross-reactivity with various lengths of Aβ peptides was less than 0.5%. In addition, we found a significant correlation between the IP-MS method and our immunoassay (correlation coefficients of Pearson's r: 0.91 [Aβ40] and 0.82 [Aβ42]). Our new method to quantify plasma Aβ40 and Aβ42 provides clinicians and patients with a way to continuously monitor disease progression., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KY, SW, KI, MM, YI, JM, KH, and SI are employees of Sysmex Corporation. TK is an employee of Sysmex R&D Center Americas, Inc. TY is a board member of Sysmex Corporation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ochratoxins are mycotoxins that have been extensively studied lately due to the multiple toxic effects such as nephrotoxicity, hepatotoxicity, and carcinogenicity. These toxins contaminate plant and animal foods and after ingestion they reach into body fluids. The method of competitive direct enzyme immunoassay, in the solid phase, was validated through the determination of specific parameters (performance, linearity, recovery percentage, limit of detection, limit of quantification). The validated method was used to determine ochratoxin A in colostrum and cow's milk. The method applied for the determination of ochratoxin A was linear for the concentration range of 0.0-0.5 ng/mL, the value for the regression coefficient (r) was 0.9838. Ochratoxin A was present in 91.67% of the colostrum and in 93.33% of cow's milk samples. The linearity of the method, demonstrated for very low concentrations of analyte, the detection limit as well as the limit of quantification recommend the method for the determinations of micro-pollutants from foods, including biological fluids.

Fragmented cytokeratin 18 (fCK18) released from epithelial cells undergoing apoptosis is widely studied in various diseases. However, fCK18 measurement is not utilized in clinical practice due to imprecise disease-state cutoff values. Therefore, we set out to generate new monoclonal antibodies (mAbs) and a recombinant fCK18 (rfCK18) calibrator in an effort to develop a highly sensitive chemiluminescent enzyme immunoassay (CLEIA). New capture mAb (K18-624) had a high binding ability compared to the current commercial antibody. New detection mAb (K18-328) recognized 323S-340G of CK18. A rfCK18 was expressed in the soluble fraction of E. coli when the N-terminal region (260 amino acid residues) of CK18 was truncated. Analysis of performance and measurement of human fCK18 were evaluated using K18-624 and K18-328 in a highly sensitive CLEIA. The coefficients of variation (CV) for within-run and between-day repeatability were below 10% and the recoveries were in the range of 15%. The detection sensitivity was 0.056 ng/mL. Serum fCK18 levels were significantly increased in non-alcoholic steatohepatitis (NASH) patients when compared to healthy individuals. Our new fCK18 mAbs showed high affinity and sensitivity. CLEIA using our new antibodies will be useful in measuring fCK18 in human blood thereby generating accurate clinical diagnoses of human liver diseases., (© 2021. The Author(s).)

Background: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid-phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis., Methods: A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA)., Results: The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60., Conclusions: EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first-line screening technique allows the detection of antibodies, which could not be detected by IIF alone., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Background: Virological failure during antiretroviral treatment (ART) may indicate the presence of drug resistance, but may also originate from nonadherence. Qualitative detection of ART components using drug level testing may be used to differentiate between these scenarios. We aimed to validate and implement qualitative point-of-care drug level tests for efavirenz (EFV), lopinavir (LPV), and dolutegravir (DTG) in rural South Africa., Methods: Qualitative performance of immunoassays for EFV, LPV, and DTG was assessed by calculating limit of detection (LoD), region of uncertainty, and qualitative agreement with a reference test. Minimum duration of nonadherence resulting in a negative drug level test was assessed by simulation of treatment cessation using validated population pharmacokinetic models., Results: LoD was 0.05 mg/L for EFV, 0.06 mg/L for LPV, and 0.02 mg/L for DTG. Region of uncertainty was 0.01-0.06 mg/L for EFV, 0.01-0.07 mg/L for LPV, and 0.01-0.02 mg/L for DTG. Qualitative agreement with reference testing at the LoD in patient samples was 95.2% (79/83) for EFV, 99.3% (140/141) for LPV, and 100% (118/118) for DTG. After simulated treatment cessation, median time to undetectability below LoD was 7 days [interquartile range (IQR) 4-13] for EFV, 30 hours (IQR 24-36) for LPV, and 6 days (IQR 4-7) for DTG., Conclusions: We demonstrate that qualitative ART drug level testing using immunoassays is feasible in a rural resource-limited setting. Implementation of this technology enables reliable detection of recent nonadherence and may allow for rapid and cost-effective differentiation between patients in need for adherence counseling and patients who require drug resistance testing or alternative treatment., Competing Interests: T.H. is an employee of ARK Diagnostics, Inc. The remaining authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Human parvovirus B19 (B19) is a pathogen that threatens the quality of plasma products. Therefore, health authorities have mandated measures against B19 contamination of plasma pools. The US FDA has recommended a B19 genome level of 10 4  IU/ml or lower in pooled plasma lots. Therefore, the B19 nucleic acid amplification test (B19-NAT) has been introduced in many plasma fractionators. However, in the Japanese Red Cross, which is the only approved blood collector in Japan, the B19 antigen test has been introduced for screening donated blood in Japan. Therefore, to clarify whether the antigen test is robust enough to screen blood samples according to the FDA recommendation, we evaluated B19 genome levels in each pooled plasma lot from 2003 to 2020., Study Design and Methods: Data of 5576 pooled plasma lots from factories A and B, which were derived from plasma bags and passed the B19 antigen-based tests, receptor-mediated hemagglutination assay (B19-RHA), or chemiluminescent enzyme immunoassay (B19-CLEIA), during 2003 to 2020, were evaluated. The amount of B19 genome in each lot was determined using quantitative or semiquantitative B19-NAT., Results: The B19 genome levels in pooled plasma lots screened using B19-RHA did not meet the FDA recommendation, whereas the lots derived from B19-CLEIA fulfilled the FDA recommendation, even during the B19 epidemic in Japan., Discussion: The results suggest that the B19-CLEIA donor screening for plasma pools is also useful in light of the US FDA recommendation., (© 2021 AABB.)