Immunoassay System Research Articles

Serum vitamin B12 concentration is associated with improved memory in older individuals with higher adherence to the Mediterranean diet

Vitamin B12 plays a crucial role in cognition, but its effect might be regulated by the presence of other micronutrients, such as folate. The aim was to evaluate the effects of vitamin B12 on cognitive performance according to adherence to the Mediterranean diet, and whether the Mediterranean dietalso results in increased folate or vitamin B12 levels. This is a cohort study nested in a randomized controlled clinical trial performed in Hospital Clinic in Barcelona, Spain. A total of 170 participants of the PREDIMED trial (Barcelona - Hospital Clinic site) aged 55-80 years at high cardiovascular risk were included. Adherence to the Mediterranean diet was assessed using a validated 14-item questionnaire, memory function was evaluated with a battery of neuropsychological tests and serum vitamin B12 and folate were determined using an automated electrochemiluminiscence immunoassay system. In the multivariable adjusted linear regression model, serum vitamin B12 concentration presented a significant correlation with memory function (r2=0.57; P=0.028) in participants with high adherence to the Mediterranean diet whereas the correlation was weak and inverse for those who presented a low adherence to the Mediterranean diet (r2=0.37, P=0.731). Mediterranean diet adherence showed a positive association with serum folate, but not with serum vitamin B12. In an older Mediterranean population at high cardiovascular risk, changes in serum vitamin B12 correlate with better memory function only in the context of a high adherence to the Mediterranean pattern, suggesting that the effects of vitamin B12 goes further than a mere nutritional requirement. The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of the 11 participating centres. The study was registered with the International Standard Randomized Controlled Trial Number (ISRCTN) 35739639 (https://www.isrctn.com/ISRCTN35739639).

Broadly Reactive SARS-CoV-2-Specific T-Cell Response and Participation of Memory B and T Cells in Patients with Omicron COVID-19 Infection.

January 2022 onward, India witnessed a sudden increase in Omicron COVID-19 infections, having a mild course that prompted us to identify the key host factors/immune molecules modulating disease course/outcomes. The current study evaluated the percentages of lymphocyte subsets by flowcytometry, SARS-CoV-2 specific T-cell immune response by ELISPOT, estimation of plasma cytokine/chemokine levels on a Bio-plex Multiplex Immunoassay System and anti-SARS-CoV-2 IgG levels by enzyme-linked immunosorbent assay in 19 mild Omicron infected patients, 45 mild SARS-CoV-2 (2020) patients and 36 uninfected controls from India. Natural killer cells, B and memory B cells were high in vaccinated and total Omicron-infected patients groups compared to the mild SARS-CoV-2 (2020) patient group, while CD8+ T cells were high in total Omicron-infected patients group compared to the uninfected control group (p < 0.05 each). Omicron-infected patients had T-cell response against SARS-CoV-2 whole virus, S1 proteins (wild type and delta variant) in 10 out of 17 (59%), 10 out of 17 (59%), and 8 out of 17 (47%), respectively. The current study of Omicron-infected patients elucidates broadly reactive antibody, T-cell response, and participation of memory B and T cells induced by vaccination/natural infection. The limited effect of Omicron's mutations on T-cell response is suggestive of protection from severity. Pro-inflammatory IL-6, IFN-γ, chemokines CCL-2, CCL-3, CCL-4, CCL-5, and IL-8 as potential biomarkers of Omicron infection may have future diagnostic importance. The cellular immune response data in Omicron-infected patients with parental Omicron lineage could serve as a starting point to define the readouts of protective immunity against circulating Omicron subvariants.

Reagentless impedimetric immunosensor for monitoring of methotrexate in human blood serum using multiwalled carbon nanotube@polypyrrole/polytyramine film electrode

Ensuring effective monitoring of methotrexate (MTX) levels in the bloodstream of cancer patients undergoing high-dose methotrexate chemotherapy is crucial to prevent potentially harmful side effects. However, the absence of portable analytical devices suitable for point-of-care bedside monitoring has presented a significant obstacle to achieving real-time MTX monitoring. In this study, we developed an impedimetric immunosensor that doesn't require reagents for measuring MTX levels in undiluted human blood serum. This reagentless approach simplifies the assay process, enabling rapid and straightforward MTX quantification. The immunosensor transducer was fabricated by electrodepositing conductive network of porous multiwalled carbon nanotube@polypyrrole/polytyramine on screen-printed gold microchip electrode (SP–Au/MWCNT70@PPy-PTA). Polyclonal anti-MTX antibodies were immobilized on the film, acting as the immunorecognition element. Non-specific binding was prevented by blocking the transducer interface with denatured bovine serum albumin (dBSA) fibrils, resulting in SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA film electrode. When MTX binds to the SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA interface, the film conductance and electron transfer resistance changes. This conductivity attenuation allows for electrochemical impedimetric signal transduction without a redox-probe solution. The electrochemical impedance spectroscopy (EIS) results showed increased charge transfer resistance and phase angle as MTX concentrations increased. The SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA demonstrated high sensitivity, with a linear response from 0.02 to 20.0 μM and a detection limit of 1.93 nM. The detection limit was 50 times lower than the intended safe level of MTX in human serum. The immunosensor exhibited minimal cross-reactivity with endogenous MTX analogs and serum proteins. The SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA immunosensor presents a simple and rapid method for therapeutic drug monitoring compared to traditional immunoassay systems.

Serum Level of Tumor Marker In Breast Cancer Women In Different Age Groups

Breast cancer is the most common crucial cancer in women worldwide. The tumor marker Carcinoma Antigen 15-3 is frequently used for screening and monitoring breast cancer. Carcinoma antigen 15–3 (CA15-3) is used to monitor the prognosis for patients after treatment. This study aims to estimate the level of serum tumor marker CA15-3 in different age groups. Participants women diagnosed with breast cancer living in Iraq were admitted to the Middle East lab (private lab), Al-Harthia city. The study involves 176 females aged from 30 to 80 years. Data was collected from the period between March to November 2021. An automated electrochemistry luminescence immunoassay system (ROCHE E170, Germany) measured serum CA15-3 levels. When the CA15-3 marker level was above or below the cut-off value of 25 U/mL, the marker was counted positive or negative. The current study reveals that CA15-3 in the age group between (33-39) was the highest in 39 years (27.7 U/ml). Furthermore, 47 years exhibited the highest level of CA15-3 (150 U/ml) among (40-49 years) category. Meanwhile, 55 years participants showed the highest level of CA15-3 (101 U/ml) in (50-59 years) age group. 66 years old, on the other hand, showed an elevated level of CA15-3 (72 U/ml) compared to other participants in the (60-69) age group.

A-213 Assessing the Function of High-Density Lipoprotein and the Risk of Cardiovascular Disease Using Fully Automated Immunoassay Analyzer, HISCLTM

Abstract Background High-density lipoprotein (HDL) function has earned an attention as a way of assessing the risk of cardiovascular disease. Conventionally, cholesterol efflux capacity (CEC) has been used to measure the function of the HDL, but because of its time-consuming and complex procedure it has been withheld from being widely used in clinical fields. We have previously introduced an immunoassay-based technique that measures cholesterol uptake capacity (CUC) as a novel measure of HDL functionality. There we have demonstrated that CUC is capable of risk stratification in patients with coronary artery disease, in which coronary lesions was inversely associated with CUC (Fujimoto et al., Atherosclerosis 2022; Murakami et al., Scientific Reports 2023). However, since this method uses a specialized washing solution with mild surfactant to prevent delipidation from HDL rather than using the conventional washing solution, it had to be operated as a dedicated device. In other words, CUC could not be measured with automatic analyzer along with the existing immunoassay items. Here we propose an upgraded version that intends for IVD use. Method Automated immunoassay system, HISCL™ was used to measure CUC in serum. This rapid and sensitive immunoassay is based on anti-ApoA1 antibody coated magnetic beads that captures HDL particles from the sample, while HDL simultaneously takes in the PEGylated biotin-labeled cholesterol. After the cholesterol uptake reaction, magnetic beads are washed with the HISCL™ washing solution, a commercially available reagent used in conventional immunoassay. Then, using alkaline phosphatase labeled streptavidin and specific substrate, signals are detected. As for the samples, myeloperoxidase-mediated oxidized HDL, recombinant Lecithin-cholesterol acyltransferase (rLCAT) added HDL, and commercially available serum (as control) were measured using above mentioned method to evaluate functional change in HDL. Results Despite using the conventional washing solution, we were able to maintain specific signals corresponding to the dynamics of cholesterol probe by optimizing the reaction sequence and reducing the number of washing process. In this condition, the assay was linear between 0 and 50 nL of serum (R2 &amp;gt; 0.99), and CUC correlated with CEC (r = 0.897, P &amp;lt; 0.01). Furthermore, statistically significant difference (P &amp;lt; 0.05) in CUC level was seen with HDL in different functional states. In specific, CUC dropped with HDL oxidation while an opposite movement was found in HDL that’s been treated with rLCAT, an enzyme that esterifies cholesterol thereby improves cholesterol uptake of HDL. This result suggests that this assay is capable of accurately depicting the function of HDL. In our previous measuring system, CUC demonstrated its utility in coronary risk stratification. Here, we were able to see a significant correlation (r = 0.973, P &amp;lt; 0.01) between our previous data and our current data with high reproducibility (Coefficient of Variation &amp;lt; 10%). Conclusion Our results suggest that we are inching ever closer to expanding the research of HDL functionality to the clinical settings and diagnosing patients with high risk of cardiovascular disease.

A-006 Evaluation of the ADVIA Centaur NT-proBNPII Assay Vs Standard-of-care Natriuretic Peptide Testing in the Emergency Department

Abstract Background The Siemens Healthineers ADVIA Centaur® NT-proBNPII (PBNPII) assay* was developed for in vitro diagnostic use in the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in human serum and plasma (EDTA and lithium heparin) using the ADVIA Centaur XP Immunoassay System. Siemens Healthineers performed a clinical study to evaluate the ADVIA Centaur XP PBNPII assay in an Emergency Department (ED) patient population. The study also involved collection of standard-of-care (SOC) natriuretic peptide (NP) results, either NT-proBNP or BNP. Objective To compare results from the ADVIA Centaur PBNPII assay in ED populations with SOC NP results and adjudicated diagnosis of heart failure. Methods 3128 subjects were enrolled from 30 ED sites. Blood was collected at time of enrollment for subsequent ADVIA Centaur PBNPII measurement; results from SOC NP testing were obtained from chart review. Available SOC testing was performed within 24 h of enrollment draw for all except four patients. Adjudication of acute HF (de novo or worsening) or non-acute HF was determined by an independent central adjudication panel of expert clinicians. Results Of the 3128 patients in the ED study, 1646 (52.6%) had an SOC BNP test performed, 1020 (32.6%) had an SOC NT-proBNP test performed, and 462 (14.8%) had neither. Agreement between the SOC NP results and PBNPII results was assessed based on concordance using cutoffs described in heart failure guidelines that include an indeterminate zone (1). For those tested with SOC NT-proBNP, there was 94.7% concordance, 5% partial concordance (indeterminate by one assay and positive or negative by the other), and 0.3% discordance. For those tested with SOC BNP, there was 73.8% concordance, 25.5% partial concordance, and 0.7% discordance. Agreement with adjudication was virtually identical for both the PBNPII assay and SOC PBNP at 63% concordance, 17% partial concordance, and 20% discordance. For those tested with SOC BNP, there was 63% concordance, 29.5% partial concordance, and 7.5% discordance, while in this same population the PBNPII assay had 67.8% concordance, 15.7% partial concordance, and 16.5% discordance. Conclusion Upon comparison of ADVIA Centaur PBNPII assay results to SOC NP testing in an ED population, Siemens Healthineers concludes that the ADVIA Centaur PBNPII assay performs similarly to commercial natriuretic peptide assays in routine use. These results agree with publications comparing diagnostic testing of NT-proBNP and BNP for suspected heart failure (2). *Disclaimer The products/features mentioned herein are not commercially available in all countries. Their future availability cannot be guaranteed.

A-012 Novel Method for Determining the Concentration of Total N-terminal Pro-hormone BNP (NT-proBNP) in a Sample with Mass Spectrometric Detection

Abstract Background NT-proBNP, the N-terminal fragment of the hormone B-type natriuretic peptide (BNP), is a useful clinical biomarker for diagnostic and prognostic evaluation of heart failure (HF). Recent studies show that commercially available NT-proBNP assays underestimate concentrations of NT-proBNP due to the usage of antibodies targeting the glycosylated central region of the NT-proBNP molecule. With this, a clinical need arises to establish a robust and reliable assay method which can measure the total amount of NT-proBNP, independent of the glycosylation status of the molecule. Methods To determine the concentration of NT-proBNP in patient samples, a known quantity of labelled NT-pro-BNP was added to each sample as standard. The capture of the analyte (native and standard) was performed by Dynabeads™ M-270 Epoxy beads coated with polyclonal goat antibodies, targeting non-glycosylated sites of the NT-proBNP molecule. Magnetic beads were added to the sample and incubated overnight. The isolated analytes were trypsinated directly on the beads after treatment for denaturation by unfolding and oxidation/alkylation. The reaction was stopped by addition of formic acid and the tryptic peptides were cleaned on C18 SPE columns, dried by SpeedVac and resuspended in acetonitrile for separation and analysis on LC-MS/MS (Agilent 1200 Series LC/MS system coupled to an Agilent G6490 triple quadrupole mass spectrometer). Results The Gentian LC-MS/MS validation was conducted including five studies such as Linearity, LoQ, Trueness, Precision and Carry-Over. All studies passed our defined acceptance criteria. To elucidate how glycosylation influences measurements of NT-proBNP, 81 plasma and serum samples covering a concentration range from 100 ng/L to 25 000 ng/L were measured by the Gentian LC-MS/MS method and the Roche Elecsys® proBNP I assay. Within the clinically relevant range of 100–2000 ng/L, the results between both methods exhibit a linear correlation of R2 = 0.97 and show that the commercial assay consistently underestimates the concentration of NT-proBNP by an average factor of 4.3 (3.5–5.8). Furthermore, results reveal that the concentration measurements between the commercial assay and the LC-MS/MS method converge at higher NT-proBNP concentrations. Method comparison was repeated using the Siemens ADVIA Centaur® and the Siemens IMMULITE Immunoassay systems, confirming the results of underestimation of NT-proBNP in clinically relevant areas. For method robustness investigations, the LC-MS/MS method was set up and used at two geographically different sites by different operators, using two different mass spectrometry systems (Agilent 1200 with triple quadrupole and the nano-LC-ESI-Orbitrap from Thermo Fisher Scientific), resulting in CVs from 13% to 2% for NT-proBNP concentrations ranging from 220 ng/L to 9700 ng/L. Conclusion Gentian AS presents a robust and reliable mass spectrometry method, capable of detecting the total amount of NT-proBNP in a sample, independent of its degree of glycosylation. This method is suitable for serving either as a reference method for calibrating and ensuring the accuracy of existing methods, harmonization of existing methods, or as a novel method in clinical chemistry for accurately determining the concentration of total amount of NT-proBNP.

B-318 Development of Non-competitive Immunoassay System for Cyclosporine a Using Alpaca Naïve Library-derived VHH

Abstract Background Cyclosporine A is a potent immunosuppressive drug. It has a narrow therapeutic index, and therefore the measurement of cyclosporine’s blood concentration is essential to obtain optimal therapy. Cyclosporine A, a small cyclic polypeptide, is generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. In this study, we report a novel sandwich immunoassay for cyclosporine A using an alpaca naïve library-derived variable domain of heavy chain of heavy chain antibody (VHH) developed against an immunocomplex of cyclosporine A and anti-cyclosporine A mouse monoclonal antibody. Methods We generated a VHH against an anti-cyclosporine A mouse monoclonal antibody complexed with cyclosporine A by alpaca naïve VHH phage display library. A bivalent tandem VHH was generated using the established VHH, and a new sandwich immunoassay for measuring cyclosporine A was constructed using the tandem VHH conjugated with alkaline phosphatase. A prototype fully automated sandwich immunoassay was developed on a fully automated chemiluminescence analyzer using magnetic particles immobilized with the mouse monoclonal antibody and the tandem VHH conjugated with alkaline phosphatase. The prototype assay was further developed by implementing an automated whole blood pretreatment process with strong denaturing agents. The performances of the prototype immunoassays were investigated. Results The established VHH showed a highly specific reaction to the complex of cyclosporine A and the anti-cyclosporine A mouse monoclonal antibody, and a sandwich immunoassay for cyclosporine A was successfully developed using the VHH and the mouse monoclonal antibody. The tandem bivalent VHH exhibited higher reactivity to the complex than the monomeric VHH. The fully automated reagent prototype achieved a measurement range of 30 to 1500 ng/mL. Measurement of cyclosporine A in whole blood samples by the prototype reagent showed a correlation coefficient of 0.96 with mass spectrometry, and a correlation coefficient of 0.95 with a commercial IVD reagent. Conclusion We successfully developed a sandwich reagent prototype for cyclosporine A using VHH established by alpaca naïve VHH phage display library. This is the first report of a fully automated reagent for cyclosporine A measurement including an automated whole blood pretreatment process.

A-008 Comparison of Roche 5th Generation Troponin T Analytical Outlier Rates Between the e411 Analyzer Using a Biotin-sensitive Reagent and the e601 Analyzer Using Reformulated (Biotin-insensitive) Reagent

Abstract Background Analytical outliers (fliers) occur with most troponin methods and can adversely affect patient management. We compared the analytical outlier rates for the Roche 5th generation Troponin T STAT (cTnT) reformulated (biotin-insensitive) reagent using a Cobas e601 analyzer (Indianapolis, IN) to rates observed in a previous study using the biotin-sensitive reagent on a Cobas e411 analyzer. Methods Plasma separator tubes (PST) were collected from hospital patients with orders for clinical cTnT testing. PST samples were centrifuged for 3 min at 4000g and analyzed using the biotin-insensitive cTnT Gen 5 assay on a Roche Cobas e601 immunoassay system. If samples displayed measurable cTnT Gen 5 (≥6 ng/L), samples were stored at 2–8 °C within 2 h of initial measurement (N = 2555). Within 24 h of initial analysis, samples were warmed to room temperature, aliquoted, re-centrifuged at 4000g for 3 min, and cTNT was re-measured on the same analyzer. Outlier rates were compared to a previous study (N = 1997) using nearly identical study design but using biotin-sensitive Roche 5th Gen cTnT reagent on an e411 analyzer. We defined analytical outliers by three methods: Repeat value (after aliquoting and re-centrifugation) that differed from the initial value for all samples with measurable cTnT by a critical difference (CD): where CD = z × √2 × SDanalytical, with a z-value of 3.719 and using SD from best fit line of precision vs cTnT concentration. Precision experiments were performed using four plasma pools in 5 × 5 precision experiment on the e411 analyzer. Using the same CD but considering only initial cTnT values &amp;lt;100 ng/L, those are most likely to be clinically significant or lead to misinterpretation. Using CD for initial values &amp;lt;100 ng/L, and a fixed 10% difference between replicates for initial values ≥100 ng/L, accounting for our practice of reporting the cTnT “delta” in percent (rather than absolute) for values ≥100 ng/L. Comparisons of outlier rates between the biotin-sensitive and biotin-insensitive reagents were performed using Pearson’s chi-squared test. Results Using the CD the total number of outliers that were observed for the biotin-insensitive reagent on the e601 was 208/2555 (8.1%), compared to 68/1997 (3.4%) on the biotin-sensitive reagent measured on the e411 (P &amp;lt; 0.0001). Outliers were observed in 138 (6.4%) of samples with initial values &amp;lt; 100 ng/L using the biotin-insensitive reagent on the e601 compared to 45 (2.6%) of such samples on the e411 (P &amp;lt; 0.0001). Using the Z score method to define outliers for initial values &amp;lt;100 ng/L and a fixed 10% to define outliers for initial values ≥100 ng/L, the outlier rate was 151 (5.9%) for the biotin-insensitive reagent on the e601 compared to 53 (2.7%) on the e411 (P &amp;lt; 0.0001). Conclusions Analytical outliers occurred more frequently using the reformulated (biotin-insensitive) reagent on the Roche Cobas e601 analyzer, compared to outlier rates observed for biotin-sensitive reagent on the Cobas e411.

Boosting Electrochemiluminescence Immunoassay Sensitivity via Co-Pt Nanoparticles within a Ti3C2 MXene-Modified Single Electrode Electrochemical System on Raspberry Pi.

Point-of-care testing plays a crucial role in diagnostics within resource-poor areas, necessitating the utilization of portable and user-friendly devices. The adaptation of biosensors for point-of-care applications requires careful considerations, such as miniaturization, cost-effectiveness, and streamlined sample processing. In recent years, the electrochemiluminescence (ECL) immunoassay has gained significant attention due to its visual detection capabilities and ability to facilitate high-throughput analysis. However, the development of a practical and cost-effective ECL device remains a challenging task. This study presents the development of an integrated MXene-modified single-electrode electrochemical system (SEES) for visual and high-throughput ECL immunoassays incorporating a Raspberry Pi system. The SEES was designed by affixing a plastic sticker with multiple perforations onto a single carbon ink screen-printed electrode, which operates based on a resistance-induced potential difference. Leveraging the excellent adsorption and bioaffinity properties of the carbon ink screen-printed electrode, effective immobilization of antibodies was achieved. Furthermore, the incorporation of Co-Pt nanoparticles enhanced the ECL intensity and electron transfer kinetics, enabling the sensitive detection of SARS-CoV-2. The developed system comprised 18 individual reaction cells, allowing for simultaneous analysis while maintaining sample isolation. Impressively, the system achieved a remarkable minimum virus detection limit of 10-14 g mL-1, accompanied by a high R2 value of 0.9798. These findings highlight the promising potential of our developed system for efficient point-of-care testing in resource-limited settings.

Bioluminescent immunoassay for serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as a substrate

A bioluminescent immunoassay system was developed to determine serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as the substrate. A monoclonal antibody against the synthetic phosphorylated serine peptide (K9P peptide) of histone H3 (19 amino acid residues), referred to as the H3S10P antibody, was chemically conjugated to maleimide-activated aequorin to prepare aequorin-labeled H3S10P (AQ-S-H3S10P). For the serine/threonine kinase assay, a non-phosphorylated serine peptide (K9C peptide) coated on a microplate was incubated with serine/threonine protein kinase in the presence of ATP and Mg2+. The resulting phosphorylated K9C peptides (K9P peptide) were identified using AQ-S-H3S10P. Thus, after the removal of unbound AQ-S-H3S10P though washing, the serine/threonine kinase activity was determined by the luminescence activity of aequorin from AQ-S-H3S10P bound to the K9P peptide. This assay system, in combination with the K9C peptide and AQ-S-H3S10P, could be used to screen inhibitors of various serine/threonine protein kinases in general.

M13 Bacteriophage-Assisted Recognition and Signal Spatiotemporal Separation Enabling Ultrasensitive Light Scattering Immunoassay.

The demand for the ultrasensitive and rapid quantitative analysis of trace target analytes has become increasingly urgent. However, the sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. To address this dilemma, we describe versatile M13 phage-assisted immunorecognition and signal transduction spatiotemporal separation that enable ultrasensitive light-scattering immunoassay systems for the quantitative detection of low-abundance target analytes. The newly developed immunoassay strategy combines the M13 phage-assisted light scattering signal fluctuations of gold nanoparticles (AuNPs) with gold in situ growth (GISG) technology. Given the synergy of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation, the practical detection capability of this strategy can achieve the ultrasensitive and rapid quantification of ochratoxin A and alpha-fetoprotein in real samples at the subfemtomolar level within 50 min, displaying about 4 orders of magnitude enhancement in sensitivity compared with traditional phage-based ELISA. To further improve the sensitivity of our immunoassay, the biotin-streptavidin amplification scheme is implemented to detect severe acute respiratory syndrome coronavirus 2 spike protein down to the attomolar range. Overall, this study offers a direction for ultrasensitive quantitative detection of target analytes by the synergistic combination of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation.

Pediatric reference intervals for endocrine markers in healthy children and adolescents on the Liaison XL (DiaSorin) immunoassay system

ObjectivesProminent physiological changes occurring throughout childhood and adolescence necessitate the consideration of age and sex in biomarker interpretation. Critical gaps exist in pediatric reference intervals (RIs) for specialized endocrine markers, despite expected influence of growth and development. The current study aimed to establish and/or verify RIs for six specialized endocrine markers on a specialized immunoassay system. MethodsSamples were collected from healthy children and adolescents (5 to <19 years) and apparently healthy outpatients (0 to <5 years) as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER). Serum samples were analysed for aldosterone, renin (plasma), thyroglobulin, anti-thyroglobulin, growth hormone, and insulin-like growth factor-1 (IGF-1) on the Liaison XL (DiaSorin) immunoassay platform. RIs (2.5th and 97.5th percentiles) were established for aldosterone, renin, thyroglobulin, anti-thyroglobulin, and growth hormone. Manufacturer-recommended pediatric RIs for IGF-1 were verified. ResultsAge-specific RIs were established for aldosterone, renin, and thyroglobulin, while no age-specific differences were observed for anti-thyroglobulin or growth hormone. IGF-1 was the only endocrine marker studied that demonstrated significant sex-specific differences. Manufacturer-recommended IGF-1 RIs were verified for children aged 6 to <19 years, while those for children aged 0 to <6 years did not verify. ConclusionsThis study marks the first time that pediatric RIs for aldosterone and renin were established in the CALIPER cohort and highlights the dynamic changes that occur in water and sodium homeostasis during the first years of life. Overall, these data will assist pediatric clinical laboratories in test result interpretation and improve clinical decision-making for patients tested using Liaison immunoassays.

Plasma myoglobin indicates muscle damage associated with acceleration/deceleration during football.

Monitoring muscle damage in athletes assists not only coaches to adjust the training workload but also medical staff to prevent injury. Measuring blood myoglobin concentration can help evaluate muscle damage. The novel portable device utilized in this study allows for easy on-site measurement of myoglobin, providing real-time data on the player's muscle damage. This study investigated the relationship between external load (global positioning system parameters) and internal loads (myoglobin concentration and creatine kinase activity) in 15 male professional football players before and after a match. Whole blood samples from participants' fingertips were collected before the match (baseline) and at 2, 16, and 40 h after the match. Myoglobin concentrations were measured using the IA-100 compact immunoassay system. Creatine kinase concentrations were measured in a clinical laboratory, and match loads were monitored using a global positioning system device. The mean myoglobin concentration was significantly higher at 2 h than at the other time points (P<0.05), and decreased to baseline levels within 16 h post-match. The mean creatine kinase concentration increased after the match but did not reach a significant level. Muscle damage monitored by myoglobin after football match-play was strongly associated with acceleration/deceleration metrics rather than the sprint/high-speed running distance. Our findings indicate that myoglobin is a more sensitive marker of muscle damage than creatine kinase after football match-play. Monitoring myoglobin in athletes can aid in determining their recovery status from the previous training load and help practitioners manage the training load.

Ethno-demographic disparities in humoral responses to the COVID-19 vaccine among healthcare workers.

The COVID-19 pandemic had a profound impact on global health, but rapid vaccine administration resulted in a significant decline in morbidity and mortality rates worldwide. In this study, we sought to explore the temporal changes in the humoral immune response against SARS-CoV-2 healthcare workers (HCWs) in Augusta, GA, USA, and investigate any potential associations with ethno-demographic features. Specifically, we aimed to compare the naturally infectedindividuals with naïve individuals to understand the immune response dynamics after SARS-CoV-2 vaccination.A total of 290 HCWs were included and assessed prospectively in this study. COVID status was determined using a saliva-based COVID assay. Neutralizing antibody (NAb) levels were quantified using a chemiluminescent immunoassay system, and IgG levels were measured using an enzyme-linked immunosorbent assaymethod. We examined the changes in antibody levels among participants using different statistical tests including logistic regression and multiple correspondence analysis.Our findings revealed a significant decline in NAb and IgG levels at 8-12 months postvaccination. Furthermore, a multivariable analysis indicated that this decline was more pronounced in White HCWs (odds ratio [OR] = 2.1, 95% confidence interval [CI] = 1.07-4.08, p = 0.02) and IgG (OR = 2.07, 95% CI = 1.04-4.11, p = 0.03) among the whole cohort. Booster doses significantly increased IgG and NAb levels, while a decline in antibody levels was observed in participants without booster doses at 12 months postvaccination.Our results highlight the importance of understanding the dynamics of immune response and the potential influence of demographic factors on waning immunity to SARS-CoV-2. In addition, our findings emphasize the value of booster doses to ensure durable immunity.

Diagnostic Utility of Aldosterone/Direct Renin Concentration Ratio in Iraqi Patients with High Arterial Blood Pressure: A Pilot Study

&#x0D; &#x0D; &#x0D; Objectives: Primary aldosteronism is a prevalent contributor to secondary hypertension, posing an elevated risk of morbidity and mortality. The initial step in diagnosing this condition involves screening individuals suspected of having it.&#x0D; Methods: In our study, we enrolled 50 patients who were suspected of having hyperaldosteronism. We provided specific recommendations and instructions to these patients concerning drug therapy and implemented measures to enhance testing accuracy. The tests were conducted using the Chemiluminescence Immunoassay (CLIA) system procedure, relying on plasma direct renin concentration rather than activity. Our findings were meticulously validated, compared, and aligned with the references from ARUP laboratories.&#x0D; Results: Among our participants, 4 patients (8%) were unequivocally diagnosed with primary aldosteronism based on the Aldosterone Direct Renin Concentration (ADRR) criteria. These patients exhibited the trifecta of hypokalemia, elevated aldosterone levels, and diminished renin levels, obviating the need for further confirmatory testing. Furthermore, 13 patients (26%) were deemed likely candidates for primary aldosteronism, given their plasma aldosterone levels exceeding 15 ng/dl and renin levels below 2.5 pg/ml. Additionally, 7 patients (14%) displayed strong indications of primary aldosteronism, characterized by plasma aldosterone levels surpassing 21 ng/dl and plasma renin concentrations below 2.5 pg/ml, accompanied by high ADRR values. However, both the "likely" and "strong indication" groups necessitated confirmatory testing. Notably, our results revealed no significant disparities in age, gender, personal or family history of atherosclerotic cardiovascular disease (ASCVD), or the presence of adrenal adenomas between patients diagnosed with primary aldosteronism and those in the non-aldosteronism group within the study.&#x0D; Conclusion: Primary aldosteronism is a prevalent health concern, warranting the screening of highly suspicious patients. Utilizing direct renin concentration, instead of renin activity, offers a straightforward, cost-effective, rapid, and reproducible method for diagnosis.&#x0D; &#x0D; &#x0D;

Bioscreening specific peptide-expressing phage and its application in sensitive dual-mode immunoassay of SARS-CoV-2 spike antigen

Biorecognition components with high affinity and selectivity are vital in bioassay to diagnose and treat epidemic disease. Herein a phage display strategy of combining single-amplification-panning with non-amplification-panning was developed, by which a phage displaying cyclic heptapeptide ACLDWLFNSC (peptide J4) with good affinity and specificity to SARS-CoV-2 spike protein (SP) was identified. Molecular docking suggests that peptide J4 binds to S2 subunit by hydrogen bonding and hydrophobic interaction. Then the J4-phage was used as the capture antibody to establish phage-based chemiluminescence immunoassay (CLIA) and electrochemical impedance spectroscopy (EIS) analytical systems. The as-proposed dual-modal immunoassay platform exhibited good sensitivity and reliability in SARS-CoV-2 SP and pseudovirus assay. The limit of detection for SARS-CoV-2 SP by EIS immunoassay is 0.152 pg/mL, which is dramatically lower than that of 42 pg/mL for J4-phage based CLIA. Further, low to 40 transducing units (TU)/mL, 10 TU/mL SARS-CoV-2 pseudoviruses can be detected by the proposed J4-phage based CLIA and electrochemical immunosensor, respectively. Therefore, the as-developed dual mode immunoassays are potential methods to detect SARS-CoV-2. It is also expected to explore various phages with specific peptides to different targets for bioanalysis.

Nanozymatic Biofuel Cell-Enabled Self-Powered Sensing System for a Sensitive Immunoassay.

Self-powered sensing system (SPSS) integrating the enzymatic biofuel cell and biosensing platform has attracted tremendous interest. However, natural enzymes suffer from the intrinsic drawbacks of enzymes and enzymatic proteins. Nanozymes with enzyme-like activities are the ideal alternatives to enzymes, and it is greatly challenging to explore high-performance nanozymatic biofuel cell for SPSS. Herein, the advanced nanozymatic biofuel cell-enabled SPSS is developed for the sensitive detection of the prostate-specific antigen (PSA), where Ir single atoms supported by nitrogen-doped carbon and Au nanozymes serve as the cathode and anode, respectively. Based on the excellent electrochemical activity and stability, the resultant nanozymatic biofuel cell exhibits a higher power output and open-circuit potential than the Pt/C-based counterpart, which is beneficial for the application of SPSS. As a proof of concept, the nanozymatic biofuel cell-enabled SPSS shows a wide detection range of 0.2-500 ng mL-1 with a detection limit of 62 pg mL-1 for PSA, which provides new insight into broadening the application scenarios of nanozymes.

Aggregation of Magnetic Nanoparticles in Biological Solvents Evaluated by HTS-SQUID Magnetic Immunoassay System

Magnetic nanoparticles (MNPs) have been studied for various medical applications by taking advantage of their unique magnetic properties. Magnetic immunoassay (MIA) is a technique for the rapid detection of target biomarkers by antigen-antibody reaction between antibody-modified MNPs and the target biomarker. In this study, we aim to improve the accuracy of the MIA, we evaluated the AC magnetic properties of MNPs in the biological solvents. To measure the magnetic signal of MNPs, we used the developed HTS-SQUID magnetic immunoassay system. First, we evaluated the instability of the HTS-SQUID magnetic immunoassay system using pellets of manganese (II) fluoride. The results show that the device instability due to operating time does not affect the measurement of magnetic signal changes in MNPs due to biological solvents. Since the magnetic properties of MNPs depend on particle size and viscosity, we measured the time evolution of the magnetic signal of Resovist in glycerin, human serum, sheep whole blood, and NaCl. It was found that the magnetic signal of Resovist decreased exponentially with ions contained in the solvent. The results are fitted as the exponential double function, suggesting that the magnetic signal of MNPs in biological solvents is affected by the aggregation of MNPs in the blood and that there are at least two steps in the mechanism of the aggregation.

Development of matrix-based reference materials for 17 beta-estradiol by the recommended reference method of ID-LC-MS/MS.

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 β-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 β-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0pmol/L, and 2017012 was 761.9 ± 35.5pmol/L.