Commercial Immunoassay Research Articles

Photoinduced Electrochemiluminescence Immunoassays.

Optimization of electrochemiluminescence (ECL) immunoassays is highly beneficial for enhancing clinical diagnostics. A major challenge is the improvement of the operation conditions required for the bead-based immunoassays using the typical [Ru(bpy)3]2+/tri-n-propylamine (TPrA) system. In this study, we report a heterogeneous immunoassay based on near-infrared photoinduced ECL, which facilitates the imaging and quantitative analysis of [Ru(bpy)3]2+-modified immunobeads at low anodic potential. The photovoltage generated by the photoanode under near-infrared light promotes oxidation processes at the electrode/electrolyte interface, thus considerably lowering the onset potential for both TPrA oxidation and ECL emission. The anti-Stokes shift between the excitation light (invisible to the human eyes) and the visible emitted light results in a clear and stable signal from the immunobeads. In addition, it offers the possibility of site-selective photoexcitation of the ECL process. This approach not only meets the performance of traditional ECL immunoassays in accuracy but also offers the additional benefits of lower potential requirements and enhanced stability, providing a new perspective for the optimization of commercial immunoassays.

Ultrasensitive Biosensors Detecting m6A in Blood: Achieving Early Screening and Typing of Tumors.

N6-methyladenosine (m6A) modification is one of the most widespread RNA modifications in eukaryotes and is involved in cancer development and progression by regulating oncogene expression. Herein, a reticulated rolling circle amplification (RCA) cascade reaction was used to construct a novel electrochemical biosensor for ultrasensitive detection of m6A, employing ferrocene-tyramine (Fc-Tyr) molecules as electroactive probes. In this strategy, the RCA cascade reaction not only amplifies specific circular DNA in the designed template to reduce the binding with similar nucleic acid sequences but also generates a long ssDNA through multiple repetitions to capture a large number of electrochemical signal probes and achieve the amplification of electrochemical biosensing signals. The developed biosensor demonstrated high selectivity and sensitivity toward m6A in the range of 0.5 pM-150 nM, with a detection limit of 14.07 fM. Meanwhile, total RNA extracted from cell samples was analyzed for m6A expression levels using the developed biosensor and a commercial colorimetric immunoassay, the biosensor and immunoassay showed consistent results. In addition, m6A levels in clinical serum samples were assessed using the developed electrochemical biosensor, which showed that m6A expression was much lower in healthy individuals than in cancer patients, therefore the biosensor is promising for cancer typing. This study provides a new method for rapid and convenient tumor marker detection in clinical practice, as well as a new idea for sensitive detection of other biomolecules.

Prognostic value of serum 25-hydroxyvitamin D levels and malnutrition status on postoperative complications in patients following laryngectomy with neck dissection.

Postoperative complications (PCs) following total laryngectomy remain a significant challenge, with recent investigations directed toward the impact of nutrition status and vitamin D deficiency. To elucidate the association between preoperative vitamin D level status, malnutrition risk score, and surgical and survival outcomes in patients with advanced laryngeal cancer following total laryngectomy. Prospective cohort study. Sixty-four patients with advanced laryngeal carcinoma treated with total laryngectomy were included in the study. Serum levels of 25(OH) D3 were measured employing a commercial chemiluminescent immunoassay kit, while nutrition status was evaluated using the nutrition risk index (NRI) and Malnutrition universal screening tool (MUST). The mean serum 25(OH) D level was 37.1 ± 19.4 nmol/L (range 11.0-100.6 nmol/L), with 47% of patients exhibiting vitamin D deficiency and 31% displaying insufficiency. Medium/high MUST score had 53% of patients, and moderate/severe NRI was verified in 48% of patients. Univariate logistic regression analysis identified MUST score, GPS score, neutrophil-to-lymphocyte ratio, and circulating 25(OH) D levels as predictive for the occurrence of PCs. In multivariate analysis, MUST score and circulating 25(OH) D levels remained significantly associated with PCs. Patients with high nutrition risk had significantly lower two-year OS rates compared to the medium and low nutrition risk groups, respectively (30% vs. 62% and 83%, p = 0.010). Early identification of malnourished or patients with vitamin D deficiency and those who would benefit from specific nutritional support could be beneficial for minimizing the risk of development of surgical complications and help improve our clinical outcomes.

Factors Associated with IgG/IgM Levels after SARS-CoV-2 Vaccination in Patients with Head and Neck Cancer.

This study evaluated the factors influencing IgG/IgM antibody levels in 120 patients with head and neck cancer (HNC) following vaccination with inactivated SARS-CoV-2 vaccines. Each patient's demographic and clinical data were documented, and serum IgG and IgM antibodies were detected using a commercial magnetic chemiluminescence enzyme immunoassay kit. The results indicated that while all patients had received at least one vaccine dose, 95 tested positive for IgG and 25 were negative. A higher proportion of IgG-positive patients had received three vaccine doses. Comparatively, gamma-glutamyl transferase levels were elevated in IgM-negative patients. The study further differentiated patients based on their treatment status: 46 were treatment-naive and 74 had received chemotherapy combined with immune checkpoint inhibitors (ICT) at enrollment. Despite similar baseline characteristics and time from vaccination to antibody detection, IgM positivity was significantly lower in the ICT group, with no significant difference in IgG positivity between the treatment-naive and ICT groups. A multivariable analysis identified the number of vaccine doses as an independent factor of IgG positivity, while ICT emerged as an independent risk factor for IgM positivity. Additionally, IgG titers generally declined over time, although patients with higher baseline IgG levels maintained higher titers longer. In conclusion, ICT in patients with HNC does not significantly affect IgG levels post-vaccination. However, booster vaccinations have been shown to be associated with higher IgG positivity, although these levels gradually decrease over time.

8408 Comparison of Free Thyroxine (fT4) Reference Ranges in Pregnancy: Evaluanting a Novel Equilibrium Dialysis Coupled with Liquid Cromatography Tandem Mass Spectrometry Analytical Method Versus a Standard Routine Immunoassay

Abstract Disclosure: R.M. Biscolla: Employee; Self; Fleury Medicina e Saude. A. Lebkuchen: Employee; Self; Fleury Medicina e Saude. K.H. Cardozo: Employee; Self; Fleury Medicina e Saude. V.M. Carvalho: Employee; Self; Fleury Medicina e Saude. M.T. Justa: Employee; Self; Fleury Medicina e Saude. J.G. Vieira: Owner/Co-Owner; Self; Fleury Medicina e Saude. M.I. Chiamolera: Employee; Self; Fleury Medicina e Saude. There is a difficulty in assessing thyroid function during the gestational period and one of the most important reasons is the changes in TBG during pregnancy, resulting from the increase in estrogen, lead to interference in the dosages of free thyroxine (fT4) measured by commercial competitive immunoassays, posing limitations on the accuracy and sensitivity of existing assessment methods. Therefore, it is necessary to establish specific reference values for each trimester and for the different methods, so the analysis of fT4 results could be more confidently used in clinical practice.Objective: This study aimed to describe a new direct method for measuring free thyroxine (fT4) utilizing equilibrium dialysis (ED) coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS). Additionally, the performance of this method was compared to a commercial immunoassay, and trimester-specific normal reference ranges for fT4 in pregnancy were determined. Methods: We developed a novel ED LC–MS/MS analytical method employing 96-well dialysis plates for measuring fT4 that demonstrated an adequate sensitivity, precision, and linearity. A total of 291 samples from pregnant women were analyzed, the gestational trimester was determined using ultrasound. Additionally, serum fT4 was assessed using an immunoassay with Beckman Coulter kits to compare the two methods. Results: The lower limit of detection (LLOQ), and upper limit of detection (ULOQ) of the new method were respectively calculated to be 0.4 ng/dL and 20.8 ng/dL. The comparison between the two methods showed median FT4 concentrations of 0.7 ng/dL (range, 0.5–1.1 ng/dL) for the immunoassay and 1.1 ng/dL (0.6–2.1 ng/dL) for the ED LC–MS/MS assay (paired t test, p < 0.0001). A significant correlation between methods was observed (Spearman’s r= 0.4898, p < 0.0001). fT4 reference values for ED LC–MS/MS (2.5–97.5 percentiles) were 0.9–1.9 ng/dL, 0.7–1.4 ng/dL, and 0.7–1.5 ng/dL for the first, second, and third trimesters, respectively, whereas median TSH levels were 1.7 mIU/L (0.05–4.0 mIU/L), 1.7 mIU/L (0.44–3.7 mIU/L), and 1.9 mIU/L (0.28–3.5 mIU/L) for the first, second, and third trimesters, respectively.Conclusions: The study with a recent developed LC-MS/MS fT4 method pointed to the differences with an immunoassay method and underscores the importance of establishing trimester- and method- specific normal reference ranges in pregnancy. Presentation: 6/3/2024

Smartphone-based dual-mode aptasensor with bifunctional metal-organic frameworks as signal probes for ochratoxin A detection

Ochratoxin A (OTA) poses significant risks to human health, being potentially nephrotoxic, carcinogenic, genotoxic, and immuno-toxic. In this work, we developed a dual-mode aptasensor for OTA analysis, integrating colorimetric, electrochemical, and smartphone-based detection. The bifunctional Fe-MIL-88 metal-organic framework, acting as both a nanozyme capable of catalyzing the 3,3′,5,5′-tetramethylbenzidine substrate and an electrochemical signal amplifier, enabled OTA quantification through current response or changes in color and absorbance intensity. Besides, the spatial confinement effect enhances the local concentration of Fe-MIL-88 signal probes through rolling circle amplification reaction, thereby contributing to a substantial enhancement in sensitivity. The proposed technique is simple, disposable, highly sensitive and selective, enabling OTA detection in the range of 1 fg/mL to 250 ng/mL, with a limit of detection of 0.22 fg/mL (3σ rule). Furthermore, we successfully detected OTA in corn, wheat, and red wine samples, with results good concordance with those obtained using commercial enzyme-linked immunoassay kits.

6848 Improvement Of Free Thyroxine Measurement Quality Through The CDC Clinical Standardization Programs

Abstract Disclosure: A. Ribera: None. N. Vazquez: None. S. Knoblock: None. O. Sugahara: None. T. Buchannan: None. L. Collins: None. D. Spector: None. L. Zhang: None. U. Danilenko: None. H.W. Vesper: None. Thyroid function tests are among the most requested tests in patient care, and reliability of these measurements are crucial for appropriate diagnosis and management of thyroid disorders. In response to concerns from the clinical and research community about the quality of thyroid function tests, the CDC Clinical Standardization Programs (CSP) has created the hormone standardization (HoSt) program for free thyroxine (FT4) in partnership with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC). As a first step, CDC CSP has completed an interlaboratory comparison study of commercial immunoassays (IA) and lab-developed tests (LDT) to determine the current agreement among assays. Participants received 40 blinded, high quality individual donor serum samples with FT4 reference values assigned by the CDC FT4 reference measurement procedure (RMP). FT4 concentrations ranged between 11-32 pmol/L. Samples were analyzed in duplicate over 2 days and results were compared to the CDC FT4 RMP. To assess the potential impact of standardizing measurements to the CDC HoSt FT4 program, reported results were normalized to the CDC RMP results (recalibrated in silico using linear regression). Mean percent bias to the reference value was evaluated pre- and post-recalibration. In addition, inter-assay agreement of classification (assignment of hypo-, eu- or hyperthyroid status according to FT4 reference intervals) were assessed pre- and post-recalibration using the assay-specific and CDC RMP reference intervals, respectively. A total of 19 methods participated, consisting of 15 IA and 4 LDT. All participants showed a negative mean bias to the RMP, with IA showing a higher magnitude of bias (average: -20.7 ± 0.9%) than LDT (average: -4.7 ± 1.1%). Of the 40 samples measured, only 53% were classified uniformly by all 19 participants. Post-recalibration, mean percent bias improved profoundly (IA average: 0.0 ± 0.8%, LDT average: 0.1 ± 1.1%), and classification agreement improved by 33% to 86% of samples with consistent classification. The CDC CSP interlaboratory comparison among FT4 assays highlights the critical need for measurement standardization to improve accuracy of FT4 measurements and increase consistency in result interpretation. This study demonstrates that it is possible to develop common FT4 reference intervals via measurement standardization, which would allow for universal evidence-based clinical guidelines to be used in patient care. Presentation: 6/3/2024

8551 Comparability of Free Testosterone Measurements

Abstract Disclosure: H. Zhou: None. O. Sugahara: None. U. Danilenko: None. H.W. Vesper: None. Assessment of free testosterone (FT) has been recommended as a biomarker for diagnosis and management of hypogonadism and female hyperandrogenism. Reliable laboratory measurements are essential for properly diagnosing and treating these diseases. FT is currently measured using direct commercial immunoassays, assays using equilibrium dialysis (ED) or other separation techniques, or estimated by calculating free testosterone (cFT) using different formulas based on the levels of total testosterone and sex hormone binding globulin (SHBG). There are concerns about the accuracy and reliability of current methods that may negatively impact correct detection, treatment, and prevention in patient care. CDC’s Clinical Standardization Programs (CDC CSP) has developed an accurate, high throughput method using ED coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ED-UHPLC-MS/MS). The ED step uses an internationally recognized procedure and is performed in a custom designed dialyzer system that allows maintaining equal ratio of sample-to-buffer volume. The method uses certified pure primary reference material (National Measurement Institute-M914) as calibrator. The method has a limit of detection of 0.6 pg/mL, with an accuracy of ±5%, and precision less than 10% CV. The measurement range is 2 – 2000 pg/mL. The analytical performance of this method as well as other FT methods used in patient care was assessed using single-donor serum samples. The evaluated methods included two commercially available platforms utilizing Chemiluminescence Immunoassay or radioimmunoassay techniques, as well as six laboratory-developed assays using ED-LC-MS/MS. The results of the interlaboratory comparison study will help assessing the current level of agreement among FT assays, to identify potential factors causing differences among assays, and to assist with developing materials to improve the agreement among assays. Presentation: 6/1/2024

Establishing the TSH reference intervals for healthy adults aged over 70 years: the Australian ASPREE cohort study.

As thyroid disorders are common amongst the elderly, this study aims to evaluate the reference interval (RI) for thyroid stimulating hormone (TSH) in healthy adults aged 70 years and over. A proposed RI was determined from the Australian participants of the ASPirin in Reducing Events in the Elderly (ASPREE) randomised trial. Participants had nohistory of cardiovascular disease, thyroid cancer, dementia, or life-threatening illnesses. Participants prescribed with any thyroid-related medication at baseline were excluded. TSH levels were measured using a commercial chemiluminescence microparticle immunoassay. The RI was determined using the middle 95th percentile of the logarithmic transformed data of baseline TSH. Cox proportional hazard regression models were used to validate the RI by assessing disease incidence over time. A total of 10,995 participants had baseline TSH measures. Median (IQR) age was 73.9 (71.8-77.3) years. We propose a RI of 0.34-3.75 mU/L. TSH levels did not differ by age or sex. At baseline, there was no association between symptoms associated with thyroid disease and levels of TSH. Over the follow-up period of up to 11 years, no association was seen between baseline TSH levels and relevant disease outcomes for participants within the RI. From a group of initially healthy, community-dwelling adults aged >=70 years, we propose a RI of TSH to best represent euthyroidism. This concentration was not associated with an increased risk of thyroid related symptoms or outcomes, confirming its appropriateness for clinical use.

B-318 Serum PSA in Extracellular Vesicles: Immunoreactivity to Commercial Kits and Elimination Kinetics

Abstract Background Prostate Specific Antigen (PSA) also circulates in blood bound to extracellular vesicles (EVs), and observed higher ratio of PSA in EVs respect total serum PSA when serum concentrations are <4 μg/L, and can represent up to 59% of total circulating PSA. Moreover, this molecular form of PSA (ev-PSA) is recognized and can be quantified with commercial methodologies designed for serum quantification. We aimed to characterize ev-PSA studying its immunoreactivity to different commercial assays and analyzing its presence in international standards used for assays standardization. Serum half-life and elimination kinetics of ev-PSA were also studied. Methods Serum EVs from 32 prostate cancer (PCa) patients were isolated by ultracentrifugation (100,000xg, 90 minutes). PSA was assessed in serum (srm-), serum supernatant free of EVs (sn-) and EVs (ev-) with total PSA (T-PSA) commercial immunoassays (Elecsys® from Roche Diagnostics and Immulite® 2000 from Siemens Healthineers), and Immulite/Elecsys ratio was calculated in all samples. The analysis of the differences between methods was performed with Bland-Altman test, representing the percentage of the difference respect the mean. WHO 96/670 PSA Standard was ultracentrifuged and analyzed by Nanoparticle Tracking Analysis (NTA) in a NanoSight LM20.For half-life estimation, serum samples from 10 localized PCa patients were collected basal and 2 days after radical prostatectomy, and EVs were isolated by Size Exclusion Chromatography using Exo-spinTM kit (Cell Guidance System). The Research Ethic Committee approved the study. Results Significant differences were observed between Elecsys and Immulite immunoassays in ev-T-PSA (p<0.001) concentrations. ev-T-PSA was quantified in all samples with Elecsys (median: 0.134 μg/L; Q1-Q3: 0.033-0.391 μg/L), but only in 22 patients (68.75%) with Immulite (median: 0.206 μg/L; Q1-Q3: 0.074-0.340 μg/L). In those 22 quantifiable patients, the median Immulite/Elecsys ratio was similar (p=0.057) between sn-T-PSA and srm-T-PSA (0.96 and 0.92, respectively), but much lower for ev-T-PSA (0.77; p<0.001). Bland-Altman graphical analysis showed that Elecsys overestimates the concentrations of T-PSA in all samples compared to Immulite, being this overestimation more marked in the case of PSA contained in EVs, and amplified at lower PSA concentrations. Besides, NTA analysis of WHO 96/670 PSA Standard showed that it does not contain EVs, and therefore, ev-PSA. The percentage of remnant PSA calculated in samples obtained post-surgery respect to baseline concentration in prostatectomized patients, was significantly higher for ev-T-PSA compared to soluble srm-T-PSA (p=0.014) and srm-F-PSA (p=0.002). The elimination half-life of ev-T-PSA was 3.99 days, much higher than those estimated for serum T-PSA and F-PSA, that were 1.76 days and 0.64 days, respectively. Conclusions The quantification of ev-PSA is affected by the different immunoreactivity of the diverse commercial assays. Thus, Elecsys T-PSA immunoassay shows higher immunoreactivity compared to Immulite technique, being this different reactivity more marked at lower PSA concentrations. ev-PSA is not considered in the International WHO 96/670 PSA Standard used for methods standardization. PSA bound to EVs remains longer in circulation than soluble serum PSA.

B-178 A Sensitive LC-MS/MS Assay for Total Testosterone Quantification

Abstract Background Total testosterone immunoassay is a commonly ordered test for the diagnosis of gonadal disorders. However, immunoassays suffer from low specificity and sensitivity, which leads to inaccuracy especially in the low concentration range (< 100 ng/dL). As a result, commercial testosterone immunoassays have difficulties to accurately measure total testosterone levels for women, children, and hypogonadal men. LC-MS/MS has emerged as the method of choice for the accurate quantitation of steroid hormones. We presented here a sensitive laboratory-developed total testosterone assay, which was validated on two LC-MS/MS instruments. Methods An internal standard containing [2,3,4-13C3]-testosterone was added to each sample. The spiked serum sample was loaded on a supported liquid extraction (SLE) cartridge and eluted using methylene chloride. The elute was dried down under nitrogen and reconstituted in sample reconstitution buffer. Chromatographic separation was achieved using a Restek Ultra C18, 50 x 2.1 mm, 3µm analytical column and a total run time of each sample was 5 minutes. The assay was validated on two triple quadrupole mass spectrometers, API 6500 and API 4500. Results were compared with those of a reference laboratory. Results The assay was linear in the range of 1 to 1200 ng/dL for total testosterone and the AMR was set at 2-1200 ng/dL. The interday and intraday imprecision were less than 8%. Split sample comparison to a reference laboratory demonstrated acceptable accuracy with the slope of 1.053 and correlation coefficient (R2) of 0.991. The limit of detection (LOD) was 0.2 ng/dL and the limit of quantitation (LOQ) was 0.5 ng/dL. Results obtained from two instruments agreed well with the slope of 0.996 and the R2 of 0.999. No carryover or interferences was observed for this assay. Conclusions A LC-MS/MS based method was developed and validated for measuring total testosterone levels for female and children. This sensitive and specific assay will improve the diagnosis of hypogonadism and monitoring of testosterone-suppressing treatment.

A-075 Comparability of Free Testosterone Measurements- Results of the Interlaboratory Study

Abstract Background Assessment of free testosterone (FT) has been recommended as a biomarker for diagnosis and management of hypogonadism and female hyperandrogenism. Reliable laboratory measurements are essential for correctly diagnosing and treating these diseases. FT is currently measured using direct commercial immunoassays, assays employing equilibrium dialysis (ED) or other separation techniques, or it is estimated by calculating free testosterone (cFT). There are concerns about the accuracy and reliability of current methods that may impact patient care. To obtain more information about variability of FT measurements used in patient care, CDC’s Clinical Standardization Programs (CDC CSP) conducted an interlaboratory comparison study. Methods CDC CSP developed an accurate, automated method using ED coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ED-UHPLC-MS/MS). The ED step follows an internationally recognized procedure. The method is calibrated with primary reference material (National Measurement Institute-M914) and was used to assign target values to sera used in the study. The FT concentrations ranged from 3 to 339 pg/mL. In the interlaboratory comparison study single-donor serum samples were provided to participants operating FT methods used in patient care. The participating methods included commercially available platforms utilizing Chemiluminescence Immunoassay or radioimmunoassay techniques, as well as laboratory-developed assays using ED-LC-MS/MS. Results Preliminary findings of the CSP interlaboratory comparison study found large variability among participating assays. The agreement among IA and among ED-LC-MS/MS assays was close. However, FT concentrations obtained by IAs were in average 6 times lower compared to the ED-LC-MS/MS methods. Results estimated using the Vermeulen equation overestimated FT in average by 15% compared to the ED-LC-MS/MS methods and by over 6 times compared to IAs. Conclusions Large variability is observed between FT laboratory developed tests (LDTs) and IAs. As both LDTs and IAs are used in patient care, standardization of FT measurements to assure better patient care is needed. CDC CSP is addressing the need by developing highly accurate and precise FT method and conducting an interlaboratory comparison studies that can aid with improvement of the agreement among assays and identifying potential factors causing differences among assays.

Anti-SARS-CoV-2 Nucleocapsid Antibody Positivity Three Years after COVID-19.

The accurate identification of individuals without prior infection with severe acute respiratory syndrome coronavirus 2 is pivotal for seroepidemiological studies and vaccine trials. Owing to widespread vaccination against coronavirus disease 2019 (COVID-19), the anti-nucleocapsid antibody continues to serve as a valuable marker for individuals without a history of COVID-19. This study aimed to comprehensively assess anti-nucleocapsid antibody positivity using diverse commercial and in-house immunoassays among individuals who contracted COVID-19 more than three years earlier. We enrolled 44 participants with laboratory-confirmed COVID-19 between January and May 2020 from the Seoul National University Hospital and its community treatment centers. The results showed anti-nucleocapsid antibody positivity ranging from 45.5% to 87.9%, depending on the immunoassay. This study highlights the importance of considering the limited anti-nucleocapsid antibody positivity in individuals, with a history of distant COVID-19, in seroepidemiological or vaccine research.

Dynamics of inflammatory markers in patients with normal and impaired glucose metabolism during lung exacerbation treatment

Cystic fibrosis (CF) is one of the most common autosomal-recessive inherited diseases. The primary genetic defect in CF is aligned CFTR gene mutation which encodes a membrane protein functioning as cAMP-depended chloride channel. Classic phenotypical manifestations of CF include chronic obstructive pulmonary disease with bronchiectasis, persisting infection (St. aureus, Ps. aeruginosa, B. cepacia) and aberrant inflammatory response, as well as exocrine pancreatic insufficiency with malabsorption, hypotrophy and growth retardation. CFTR deficiency is also accompanied by β-cell pancreatic dysfunction, causing glucose metabolism disturbances and CF-related diabetes. The aim of the study was the comparison of inflammatory markers dynamics in patients with normal and disturbed glucose metabolism during pulmonary exacerbation treatment. The study included 10 patients with impaired glucose tolerance (Group 1) and 24 patients with normal carbohydrate metabolism (Group 2). Patients of the two groups did not significantly differ in demographic characteristics, pulmonary function test and body mass index parameters, as well as in the number of F508del mutation carriers and in the number of those who were infected with Ps. aeruginosa and B. cepacia complex. Blood sampling was performed twice: before and after a routine course of antibiotic therapy. Plasma levels of biomarkers including the antibodies to single- and double-stranded DNA (ss-DNA-IgG, ds-DNA-IgG, respectively), the hormones (dehydroepiandrosterone (DHEA) and DHEA sulfate), C-reactive protein (CRP), Mn-dependent superoxide dismutase (Mn-SOD), and the cytokines (tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), IFNα, tissue growth factor-β1 (TGF-β1), interleukin-4 (IL-4), IL-6, IL-10, IL-17A) were assessed using commercial immunoassay kits. Our study shows that antibiotic treatment did not have a sufficient influence on levels of inflammatory markers in patients with disturbances of glucose metabolism while patients with normal glucose tolerance demonstrated a significant reduction in inflammatory marker values after the therapy. The data may suggest both impaired effectivity of antibiotic treatment and aberrant inflammatory response in patients with glucose intolerance.

Comparative Analysis of Commercial Immunoassays for the Determination of Total, Intact, and Nonintact Luteinizing Hormone in Urine.

In our recent publications, we reported the identification of three different molecular forms of total luteinizing hormone (LH) in urine, the intact LH, the free beta-subunit (LHβ), and its core fragment of LHβ (LHβcf), the latter two establishing the nonintact portion of LH. Following the discontinuation of the Delfia immunofluorometric assay (IFMA) (Wallac, PerkinElmer Finland, Finland), a leading method for detecting urinary LH for 30 years, this study seeks to assess the efficacy of three alternative commercial immunoassays in identifying various forms of U-LH. Diluted urine samples underwent gel filtration to separate them into fractions, each containing different forms of LH. These were then assayed using Delfia IFMA, Architect LH (Abbott, USA), Elecsys LH Cobas (Roche, Switzerland), and Immulite 2000 LH (Siemens, Germany) immunoassays. Both Delfia and Immulite assays detected total U-LH, that is, all three forms of U-LH, including intact LH, LHβ, and LHβcf. Cobas detected only intact LH and LHβ, whereas Architect detected solely the intact LH. Immulite assay can be an alternative tool to detect all forms of urinary LH, a feature likely to be instrumental in developing noninvasive, practical, and scalable solutions for evaluating total U-LH changes during minipuberty in neonates, during the onset of central puberty in peripubertal children, puberty-associated disorders in adolescents, and the fertility window in women, with a special focus on postpeak changes.

Comparison of vancomycin assays in patients undergoing hemodialysis

Vancomycin is a glycopeptide antibiotic mainly excreted by glomerular filtration. Therefore, patients undergoing hemodialysis tend to accumulate its crystalline degradation product, which has been associated with cross-reaction in commercial immunoassays. The aim of this study was to assess the performance of two commercial immunoassays for measuring vancomycin levels in patients undergoing hemodialysis. This method-comparison study enrolled patients undergoing hemodialysis at two hospitals in Porto Alegre, Brazil. Vancomycin serum concentrations measured by Chemiluminescent Microparticle Assay (CMIA) and measured by Kinetic Interaction of Microparticles in Solution (KIMS) were compared with Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). A total of 64 samples from 42 patients and 54 samples from 23 patients were included in CMIA and KIMS groups. Both measurements were highly correlated with LC-MS/MS, with Spearman rank correlation coefficient r = 0.840 (p < 0.001) and r = 0.926 (p < 0.001), respectively. No deviation of linearity was observed (p = 0.81 and p = 0.49, respectively). The mean difference between CMIA and LC-MS/MS was -1.19 μg/mL and between KIMS and LC-MS/MS was -2.28 μg/mL. LC-MS/MS measured levels were, on average, 2.64 % higher than CMIA and 8.81 % higher than KIMS. CMIA and KIMS revealed accurate commercial methods to measure vancomycin serum concentrations in patients undergoing hemodialysis.

Serum amyloid A increases following routine vaccination of healthy adult horses.

To measure the effect of routine vaccination on serum amyloid A (SAA) concentration in apparently healthy horses. We hypothesized that routine vaccination would increase SAA in healthy horses. 21 apparently healthy client-owned horses and 15 Kansas State University College of Veterinary Medicine-owned horses. In experiment 1 (n = 8 horses), a blinded, randomized, prospective, crossover study was performed. Horses were either vaccinated (rabies, tetanus, West Nile, Eastern and Western equine encephalomyelitis, equine herpesvirus-1/-4, influenza) or administered saline, and SAA was measured at 6, 12, and 24 hours and daily until day 10 with a commercial lateral-flow immunoassay. In experiment 2 (n = 28 horses), a prospective, observational study measured SAA after vaccination at 12 and 24 hours and daily until day 10. A linear mixed-effect model with repeated measures over time blocked by horse tested the effect of treatment on SAA. A repeated-measures correlation tested the correlation between SAA and temperature. Over time, vaccinated horses had increased model-adjusted SAA compared to unvaccinated horses without clinical evidence of adverse reaction (P < .01). In experiment 1, the model-adjusted SAA after vaccination peaked on day 2 (median, 1,872 µg/mL; IQR, 1,220.8 to 2,402.5 µg/mL) and returned to normal (< 20 µg/mL) by day 9 (median, 6 µg/mL; IQR, 0.8 to 23.5 µg/mL) after vaccination. In experiment 2, vaccinated horses had increased SAA over time; temperature and SAA were not correlated (P = .78). Results of this study indicated that routine vaccination results in increased SAA concentration and provided evidence for a period of convalescence following vaccination. Measuring SAA for 10 days following vaccination cannot be used as an indicator of illness.

A self-driven microfluidic immunosensor for rapid separation, enrichment, and detection of biomarkers in serum.

Biomarkers and their concentration levels are critical indicators of metabolomics for clinical applications. Rapid and sensitive analysis methods are essential for realizing timely and efficient quantitation of those significant biomarkers. In this work, a self-driven microfluidic immunosensor was developed for rapid all-in-one separation, enrichment, and detection of biomarkers. This immunosensor was constructed from a cyclic olefin copolymer (COC) channel layer and a polydimethylsiloxane (PDMS) sensing layer. The COC channel layer was modified through protein adsorption, immobilization, and remaining active site blocking. The obtained hydrophilic microchannels not only reduce the nonspecific adsorption, but also provide stable capillary-driven flow generation with linear velocities up to 20mm/s for aqueous solution auto-injection. The PDMS sensing layer was modified using capture antibodies to accomplish affinity recognition of target biomarkers. Procalcitonin (PCT) and serum amyloid A (SAA) were selected as model biomarkers in the feasibility study on applying the self-driven microfluidic immunosensor to bioassay. The limits of detection of PCT and SAA were 7.9ng/L and 7.6μg/L, respectively. Moreover, the whole process can be accomplished within 60min with excellent selectivity and reproducibility. In clinical serum sample analysis, satisfactory recoveries were achieved for PCT and SAA in the ranges of 85.0-103.0% and 95.5-106.0%, respectively, with relative standard deviations less than 5.3%. The method accuracy was further confirmed by the results of commercial immunoassay kits. This simple and easily operated immunosensor provides a rapid and sensitive biomarker analysis tool, and promotes the further development of automated and easy-to-use microfluidic immunoassays.

Generation of High-Affinity Aptamers for Indazole Synthetic Cannabinoids.

Synthetic cannabinoids are a widely abused class of dangerous psychoactive substances, especially among youths and young adults. Dozens of such drugs have been identified to date, and new ones continue to emerge. The ability to detect these drugs is important for interdiction efforts and the diagnosis of drug overdose, but existing analytical methods lack broad cross-reactivity to diverse members of this drug family. Here, we have utilized library-immobilized SELEX to generate DNA aptamers that can broadly recognize various members of the indazole-3-carboxamide synthetic cannabinoid family. Using two representatives of this family, AB-FUBINACA and 5F-AMB, we identify two aptamers FUB4 and AMB2F with respective dissociation constants (KDs) of 138 ± 15 and 411 ± 20 nM for their targets. These aptamers can recognize many indazole-based synthetic cannabinoids with high affinity and excellent specificity against natural cannabinoids as well as other structurally similar interferents like serotonin and tryptophan. We use these two aptamers to develop fluorescence strand-displacement sensors that successfully detect these synthetic cannabinoids at concentrations as low as 50 nM in human serum. The sensors can also detect up to 14 different drugs from this family─a major improvement over the six recognized by an existing commercial immunoassay.

Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 μL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.