Commercial Immunoassay Research Articles

Sporadic Hepatitis A Virus PCR False-Positive Results Observed during Reflex Testing of Serum Samples Previously Tested for Anti-HAV Antibodies and Caused by Contamination with HAV RNA Present in the Reagents of the Commercial Anti-HAV Immunoassay.

Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients' serological profiles and/or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay's reagents, thereby resulting in HAV RNA false positivity during the reflex testing. IMPORTANCE We present the first report of sporadic HAV PCR false-positive results that have been observed during the reflex testing of serum samples that have previously been tested for anti-HAV antibodies and have been caused by contamination with HAV RNA that is present in the reagents of the commercial anti-HAV immunoassay, with potentially serious clinical consequences. Although HAV RNA was consistently detected in the anti-HAV reagents of all three automated immunoassay analyzers that were in use in our laboratory, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results, and this was due to a peculiar pipetting mode of action in which the analyzer uses a single pipette tip to collect both anti-HAV reagents and a sample, which consequently causes the permanent contamination of the original sample with HAV RNA. Manufacturers should strongly consider the occasional need for reflex molecular testing from preprocessed samples and design their analyzers in a way that prevents contamination.

Analytical evaluation of a GAD65 antibodies chemiluminescence immunoassay for CSF in neurological syndromes.

Antibodies against glutamic acid decarboxylase isoform 65 (GAD-Ab) have been found in different severe neurological conditions associated with altered synthesis of γ-aminobutyric acid (GABA). Serum GAD-Ab can be found in up to 90 % of patients with type 1 diabetes mellitus (T1DM), mostly at relatively low concentrations, while high concentrations of GAD-ab are thought to be more frequently associate to a neurological condition, with levels 100-folds higher than those found in T1DM. Although CSF testing is recommended when suspecting a GAD-associated neurological syndrome, no commercial immunoassay is validated for this use and no cut-off is internationally recognized to support the diagnosis. In this study we validated CSF testing of GAD-Ab on an automated chemiluminescence (CLIA) immunoassay that had previously shown good agreement with ELISA on serum. We tested 43 CSF from patients with typical GAD-associated neurological disorders and patients with other neurological conditions, identifying a clinical cut-off of 18 kIU/L that discriminated GAD-disease with an area under the curve (AUC) of 0.921. CLIA showed good analytical performances on repeatability and recovery tests in CSF and confirmed an excellent agreement with ELISA. GAD-Ab associated neurological disorders are rare but CSF testing for GAD-Ab is a common request for neurologists when suspecting an insidious autoimmune central nervous system disease. CLIA platforms are expected to be increasingly adopted in clinical laboratories due to their flexibility and reliability, therefore studies on decisional levels should be implemented for improving the interpretation and utilization of laboratory data.

Cross sectional survey of Varicella-Zoster virus and measles seropositivity in people living with HIV in a Parisian suburb and a review of current immunization guidelines.

According to evidence-based guidelines, vaccines against measles and varicella are generally recommended to susceptible HIV-positive patients, as long as they are not severely immunocompromised. However, routine screening to determine serologic status is not recommended. We conducted a seroprevalence study of anti-measles and anti-Varicella-Zoster virus (VZV) antibodies in adults living with HIV (PLWHA) consulting at Avicenne University Hospital in a Parisian suburb. Sera were collected in years 2018-2020 and tested by commercial immunoassays in 268 patients. Most of the patients were born in Sub-Saharan Africa (55%) and only 23% in Europe. Measles and varicella seropositivity were present respectively in 91.4% and 96.2% of patients. One patient in ten was seronegative to at least one of tested diseases. In the univariate analysis, only younger age (p=0.027) was associated with a higher risk of measles seronegativity, while shorter time since arrival in France (p<0.001) and shorter time since HIV discovery (p=0.007) were associated with a higher risk of VZV seronegativity. In multivariate analysis no association was found. This study highlights the absence of specific risk factors for VZV and measles seronegativity in PLWHA and supports the importance of routine screening, in order to increase immunization rates and reduce risk of complications.

Validation of N Protein Antibodies to Diagnose Previous SARS-CoV-2 Infection in a Large Cohort of Healthcare Workers: Use of Roche Elecsys® Immunoassay in the S Protein Vaccination Era.

The aim of this study was to validate the detection of anti-nucleocapsid protein (N protein) antibodies for the diagnosis of SARS-CoV-2 infection in light of the fact that most COVID-19 vaccines use the spike (S) protein as the antigen. Here, 3550 healthcare workers (HCWs) were enrolled from May 2020 (when no S protein vaccines were available). We defined SARS-CoV-2 infection if HCWs were found to be positive by RT-PCR or found to be positive in at least two different serological immunoassays. Serum samples from Biobanc I3PT-CERCA were analyzed by Roche Elecsys® (N protein) and Vircell IgG (N and S proteins) immunoassays. Discordant samples were reanalyzed with other commercial immunoassays. Roche Elecsys® showed the positivity of 539 (15.2%) HCWs, 664 (18.7%) were found to be positive by Vircell IgG immunoassays, and 164 samples (4.6%) showed discrepant results. According to our SARS-CoV-2 infection criteria, 563 HCWs had SARS-CoV-2 infection. The Roche Elecsys® immunoassay has a sensitivity, specificity, accuracy, and concordance with the presence of infection of 94.7%, 99.8%, 99.3%, and 0.96, respectively. Similar results were observed in a validation cohort of vaccinated HCWs. We conclude that the Roche Elecsys® SARS-CoV-2 N protein immunoassay demonstrated good performance in diagnosing previous SARS-CoV-2 infection in a large cohort of HCWs.

Dual signal amplification detection of m6A in serum of hepatocellular carcinoma patient by hybridization chain reaction and biocatalytic silver deposition

N6-methyladenosine (m6A) modification has an impact on RNA metabolism, and it has been demonstrated that m6A regulatory mechanisms play an important role in tumorigenesis and may serve as a potential hepatocellular carcinoma (HCC) marker. We constructed a dual-signal amplification strategy electrochemical biosensor based on biocatalytic silver deposition and hybridization chain reaction (HCR). Enzymatic silver deposition was performed on the electrode and detected by linear sweep voltammetry (LSV), which was used for the quantitative detection of m6A. There was a nice linear relationship between the current response values and the logarithm of the m6A RNA concentration in the range of 0.5 pM-5 μM, with a detection limit as low as 25 fM. The concentration of m6A detected by the sensor constructed in this work is consistent with the results obtained using commercial colorimetric immunoassays. What's more, the analysis of m6A RNA in the serum of HCC patients using the developed sensor showed the high analytical efficiency of the designed strategy, which was superior to three other markers (AFP, CEA, CA199). The constructed electrochemical biosensor has good sensitivity, specificity and reproducibility for the detection of m6A RNA, and the sensing platform has promising clinical applications in the diagnosis and prognosis of HCC.

SARS-CoV-2-Neutralizing Antibody Response and Correlation of Two Serological Assays with Microneutralization

SARS-CoV-2 has caused a huge pandemic affecting millions of people and resulting innumerous deaths. A better understanding of the correlation between binding antibodies and neutralizing antibodies is necessary to address protective immunity post-infection or vaccination. Here, we investigate the humoral immune response and the seroprevalence of neutralizing antibodies following vaccination with adenovirus-based vector in 177 serum samples. A Microneutralization (MN) assay was used as a reference method to assess whether neutralizing antibody titers correlated with a positive signal in two commercially available serological tests:a rapid lateral flow immune-chromatographic assay (LFIA) and an enzyme-linked Fluorescence Assay (ELFA). Neutralizing antibodies were detected in most serum samples (84%). COVID-19 convalescent individuals showed high antibody titers and significant neutralizing activity. Spearman correlation coefficients between the serological and neutralization results ranged from 0.8 to 0.9, suggesting a moderate to strong correlation between commercial immunoassays test results (LFIA and ELFA) and virus neutralization.

Longer intervals between SARS-CoV-2 infection and mRNA-1273 doses improve the neutralization of different variants of concern.

The humoral immune response against SARS-CoV-2 variants of concern elicited by vaccination was evaluated in COVID-19 recovered individuals (Rec) separated 1-3 months (Rec2m) or 4-12 months (Rec9m) post infection and compared to the response in naïve participants. Antibody-mediated immune responses were assessed in 66 participants by three commercial immunoassays and a SARS-CoV-2 lentiviral-based pseudovirus neutralization assay. Immunoglobulin (Ig) levels against SARS-CoV-2 spike were lower in naïve participants after two doses than in Rec after a single dose (p<0.05). After two doses in Rec, levels of total Ig to receptor binding domain (RBD) were significantly increased in Rec9m compared to Rec2m (p<0.001). The neutralizing potency observed in Rec9m was consistently higher than in Rec2m against VOCs Alpha, Beta, Delta, and BA.1 sublineage of Omicron with 2.2-2.8-fold increases. Increasing the interval between SARS-CoV-2 infection and the vaccination with mRNA-based vaccines to more than 3 months generates a more efficient heterologous humoral immune response against VOCs by allowing enough time to mount a strong recall memory B cell response. This article is protected by copyright. All rights reserved.

Investigation of SARS-CoV-2 seroprevalence in relation to natural infection and vaccination between October 2020 and September 2021 in the Czech Republic: a prospective national cohort study

ObjectiveExamine changes in SARS-CoV-2 seropositivity before and during the national vaccination campaign in the Czech Republic.DesignProspective national population-based cohort study.SettingMasaryk University, RECETOX, Brno.Participants22 130 persons provided blood samples at two...

PSA reactivity in extracellular microvesicles to commercial immunoassays

AimsCharacterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods. Materials and methodsEVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA). ResultsEv-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80–4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+. ConclusionEVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.

Neutralizing Antibodies against SARS-CoV-2 Beta and Omicron Variants Inhibition Comparison after BNT162b2 mRNA Booster Doses with a New PETIA sVNT Assay.

Monitoring antibody response following SARS-CoV-2 vaccination is strategic, and neutralizing antibodies represent the gold standard. The neutralizing response to Beta and Omicron VOCs was evaluated versus the gold standard by a new commercial automated assay. Serum samples from 100 healthcare workers from the Fondazione Policlinico Universitario Campus Biomedico and the Pescara Hospital were collected. IgG levels were determined by chemiluminescent immunoassay (Abbott Laboratories, Wiesbaden, Germany) and serum neutralization assay as the gold standard. Moreover, a new commercial immunoassay, the PETIA test Nab (SGM, Rome, Italy), was used for neutralization evaluation. Statistical analysis was performed with R software, version 3.6.0. Anti-SARS-CoV-2 IgG titers decayed during the first ninety days after the vaccine second dose. The following booster dose significantly (p < 0.001) increased IgG levels. A correlation between IgG expression and neutralizing activity modulation was found with a significant increase after the second and the third booster dose (p < 0.05. Compared to the Beta variant of the virus, the Omicron VOC was associated with a significantly larger quantity of IgG antibodies needed to achieve the same degree of neutralization. The best Nab test cutoff for high neutralization titer (≥1:80) was set for both Beta and Omicron variants. This study correlates vaccine-induced IgG expression and neutralizing activity using a new PETIA assay, suggesting its usefulness for SARS-CoV2 infection management.

Ultrasensitive Homogeneous Detection of PCSK9 and Efficacy Monitoring of the PCSK9 Inhibitor Based on Proximity Hybridization-Dependent Chemiluminescence Imaging Immunoassay.

Accurate quantification of proprotein convertase subtilisin/kexin type 9 (PCSK9) in serum before and after the medication is helpful in grasping the evolution of PCSK9-related disease and evaluating the efficacy of PCSK9 inhibitors. Conventional approaches for PCSK9 quantification suffered from complicated operations and low sensitivity. By integrating stimuli-responsive mesoporous silica nanoparticles, dual-recognition proximity hybridization, and T7 exonuclease-assisted recycling amplification, a novel homogeneous chemiluminescence (CL) imaging approach was proposed for ultrasensitive and convenient immunoassay of PCSK9. Owing to the intelligent design and signal amplification property, the whole assay was conducted without separation and rinsing, significantly simplifying the procedure and eliminating the errors associated with the professional operation; meanwhile, it showed linear ranges over 5 orders of magnitude and detection limit as low as 0.7 pg mL-1. Parallel testing was allowed due to the imaging readout, which brought a maximum throughput of 26 tests h-1. The proposed CL approach was applied to analyze PCSK9 from hyperlipidemia mice before and after the intervention of the PCSK9 inhibitor. Serum PCSK9 levels in the model group and the intervention group could be distinguished efficiently. The results were reliable compared to commercial immunoassay results and histopathologic findings. Thus, it could facilitate the monitoring of the serum PCSK9 level and the lipid-lowering effect of the PCSK9 inhibitor, showing promising potential in bioanalysis and pharmaceuticals.

Cotinine as a Sentinel of Canine Exposure to Tobacco Smoke.

The adverse health effects of both active and passive tobacco smoke have been well-known in humans for a long time. It is presumable that even pets, which intimately share the owner's lifestyle, may be exposed to the same risks. This study aimed to detect and quantify cotinine (a metabolite of nicotine) in the serum and hair of dogs using a specific commercial ELISA immunoassay kit. A total of 32 dogs, 16 exposed and 16 unexposed to the owner's smoke, were enrolled. The cotinine concentration was higher in the exposed than the unexposed group in both matrices (p < 0.001), with greater values in serum than in hair (p < 0.001). Exposed bitches had higher hair cotinine than male dogs (p < 0.001). Conversely, serum and fur cotinine concentrations were lower in female than male dogs of the unexposed group (p < 0.01). The exposure intensity, age, and weight of the dogs did not affect cotinine concentrations. A cut-off value of 2.78 ng/mL and 1.13 ng/mL cotinine concentration in serum and fur, respectively, was estimated to distinguish between the exposed and unexposed dogs. Cotinine was confirmed as a valuable marker of passive smoking also in dogs. Although owners do not perceive secondhand smoke as a risk for their dogs, greater awareness should be advisable, especially in pregnant animals.

Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes.

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5-98.8%) and a mean Sp of 100% (95% CI = 92.0-100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3-99.5%) and a mean Sp of 90% (95% CI = 78.3-97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4-100%) and a mean specificity of 100% (95% CI = 100-100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies.

Residual Central Nervous System Immune Activation Is Not Prevented by Antiretroviral Therapy Initiated During Early Chronic HIV Infection.

Antiretroviral therapy (ART) initiated during acute infection can potentially impact the central nervous system (CNS) reservoir, but the differential long-term effects of ART initiation during early or late chronic infection are unknown. We included neuroasymptomatic people with human immunodeficiency virus (HIV) with suppressive ART initiated during chronic (>1 year since transmission) HIV with archived cerebrospinal fluid (CSF) and serum samples after 1 and/or ≥3 years of ART from a cohort study. CSF and serum neopterin was measured using a commercial immunoassay (BRAHMS, Germany). In total, 185 people with HIV (median, 79 [interquartile range, 55-128] months on ART) were included. A significant inverse correlation was found between CD4+ T-cell count and CSF neopterin only at baseline (r = -0.28, P = .002), but not after 1 (r = -0.026, P = .8) or ≥3 (r -0.063, P = .5) years of ART. No significant differences were seen in CSF or serum neopterin concentrations between different pretreatment CD4+ T-cell strata after 1 or ≥3 (median, 6.6) years of ART. In people with HIV initiating ART during chronic infection, occurrence of residual CNS immune activation was not correlated with pretreatment immune status, even when treatment was initiated at high CD4+ T-cell counts, suggesting that the CNS reservoir, once established, is not differentially affected by the timing of ART initiation during chronic infection.

P514 Serum levels of Klotho in inflammatory bowel disease: an exploratory study

Abstract Background Alpha klotho (α-Klotho) is an anti-aging protein recognized as a humoral factor with anti-oxidant and anti-inflammatory effects, showing a bidirectional relationship with inflammation, a main pathophysiologic factor in atherosclerosis. A decrease in Klotho has been associated to cardiovascular disease (CVD), but no previous studies have investigated the relationship between inflammatory bowel disease (IBD), a risk factor for CVD, and Klotho levels. Therefore, we assessed if the serum levels of Klotho differ between patients with IBD and controls, and how this protein is related to the clinical and laboratory characteristics of the disease, subclinical atherosclerosis and metabolic risk factors. Methods Cross-sectional multicentric study that included 251 individuals: 191 IBD patients and 60 healthy controls was conducted. Serum Klotho levels were determined by commercial ELISA immunoassay (IBL International GmbH Laboratories). In the IBD patients disease activity was evaluated using CDAI for Crohn’s disease, partial Mayo (pMayo) for ulcerative colitis, specific laboratory parameters, determination of the Systematic Coronary Risk Evaluation (SCORE), and the presence of subclinical atherosclerosis was assessed using carotid ultrasound for identifying carotid plaque and measurement of carotid intima-media thickness. Results Serum Klotho levels were significantly higher in the IBD group: 734.31 pg/ml vs. 484.17 pg/ml (p&amp;lt;0.001) compared with controls. In the IBD group, no significant differences in the serum Klotho levels were found between the type of IBD (p=0.911), time from diagnosis (p=0.126), disease activity: CDAI (p=0.277), pMayo (p=0.916), type of treatment (0.76), the presence of plaque (p=0.101) or the intima media thickness (p=0.429). However, a significant inverse correlation between serum Klotho and the SCORE was observed in IBD patients: r = -0.176 (p= 0.014) (figure 1) Conclusion Patients with IBD had higher Klotho levels than healthy controls, with an inverse and significant relationship with the SCORE in the IBD patients. Further studies are necessary to clarify the role of Klotho in IBD.

Cortisol Variations to Estimate the Physiological Stress Response in Horses at a Traditional Equestrian Event.

In many countries, horses remain involved in traditional equestrian events such as those celebrated in Menorca (Balearic Islands, Spain) every year since at least the 14th century. The present study aimed to evaluate the variations in salivary cortisol concentrations to estimate the physiological stress response in horses at the Menorca patronal festivals. Two different editions (years 2016 and 2018) of the festivals in honor of the Virgin of Grace in Maó (Menorca, Spain) were studied. Nineteen and seventeen Pure Breed Menorca stallions were included in the study, respectively. The stallions were aged between seven and twelve years. During celebrations, samples were collected before the start of the festivals between 8-9 a.m. and during the festivals at 8-9 p.m. On the second day of celebrations, the samples were collected at 8-9 a.m. and 3-4 p.m. Finally, on the day after the festivals, one sample was collected at 8-9 p.m. Additionally, a control group was sampled at 8-9 a.m., 3-4 p.m., and 8-9 p.m. Salivary cortisol concentrations were assessed by using a commercial enzyme immunoassay kit specially validated to quantify salivary cortisol in horses. Salivary cortisol concentrations did not show significant differences between sampling hours in the control group (p > 0.05). All the samples collected during festivals were significantly higher than samples of the control group (p < 0.05). Within the twenty-four hours after the end of the celebrations, cortisol concentrations returned to baseline levels and did not differ significantly from the control group (p > 0.05). Hence, the present study describes that the participation of the horses in these particular acts generate an acute and transitory stress response. Overall, the current work provides a reasonable basis for future research on the stress physiology and well-being of horses participating in traditional celebrations or similar events.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 μM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 μM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 μM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Affinity Exploration of SARS-CoV-2 RBD Variants to mAb-Functionalized Plasmonic Metasurfaces for Label-Free Immunoassay Boosting.

Plasmonic metasurfaces (PMs) functionalized with the monoclonal antibody (mAb) are promising biophotonic sensors for biomolecular interaction analysis and convenient immunoassay of various biomarkers, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Previous PM biosensing suffers from the slow affinity detection rate and lack of sufficient immunoassay studies on various SARS-CoV-2 variants. Here, we develop a high-efficiency affinity testing method based on label-free PM sensors with mAbs and demonstrate their binding characteristics to 12 spike receptor binding domain (RBD) variants of SARS-CoV-2. In addition to the research of plasmonic near-field influence on surface biomolecule sensing, we provide a comprehensive report about the Langmuir binding equilibrium of molecular kinetics between 12 SARS-CoV-2 RBD variants and mAb-functionalized PMs, which plays a crucial role in label-free immunosensing. A high-affinity mAb can be combined with the highly sensitive propagating plasmonic mode to boost the detection of SARS-CoV-2 variants. Owing to a better understanding of molecular dynamics on PMs, we develop an ultrasensitive biosensor of the SARS-CoV-2 Omicron variant. The experiments show great distinguishment of P < 0.0001 from respiratory diseases induced by other viruses, and the limit of detection is 2 orders smaller than the commercial colloidal gold immunoassay. Our study shows the label-free biosensing by low-cost wafer-scale PMs, which will provide essential information on biomolecular interaction and facilitate high-precision point-of-care testing for emerging SARS-CoV-2 variants in the future.

Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference.

Neutralizing antibodies (nAbs) are considered a valuable marker for measuring humoral immunity against SARS-CoV-2. However, live-virus neutralization tests (NTs) require high-biosafety-level laboratories and are time-consuming. Therefore, surrogate virus neutralization tests (sVNTs) have been widely applied, but unlike most anti-spike (S) antibody assays, NTs and sVNTs are not harmonized, requiring further evaluation and comparative analyses. This study compared seven commercial sVNTs and anti-S-antibody assays with a live-virus NT as a reference, using a panel of 720 single and longitudinal serum samples from 666 convalescent patients after SARS-CoV-2 infection. The sensitivity of these assays for detecting antibodies ranged from 48 to 94% after PCR-confirmed infection and from 56% to 100% relative to positivity in the in-house live-virus NT. Furthermore, we performed receiver operating characteristic (ROC) curve analyses to determine which immunoassays were most suitable for assessing nAb titers exceeding a specific cutoff (NT titer, ≥80) and found that the NeutraLISA and the cPass assays reached the highest area under the curve (AUC), exceeding 0.91. In addition, when the assays were compared for their correlation with nAb kinetics over time in a set of longitudinal samples, the extent of the measured decrease of nAbs after infection varied widely among the evaluated immunoassays. Finally, in vaccinated convalescent patients, high titers of nAbs exceeded the upper limit of the evaluated assays' quantification ranges. Based on data from this study, we conclude that commercial immunoassays are acceptable substitutes for live-virus NTs, particularly when additional adapted cutoffs are employed to detect nAbs beyond a specific threshold titer. IMPORTANCE While the measurement of neutralizing antibodies is considered a valuable tool in assessing protection against SARS-CoV-2, neutralization tests employ live-virus isolates and cell culture, requiring advanced laboratory biosafety levels. Including a large sample panel (over 700 samples), this study provides adapted cutoff values calculated for seven commercial immunoassays (including four surrogate neutralization assays and a protein-based microarray) that robustly correlate with specific titers of neutralizing antibodies.

Deciphering the Autoantibody Response to the OJ Antigenic Complex

(1) Background: Myositis specific antibodies (MSA) are important diagnostic biomarkers. Among the rarest and most challenging MSA are anti-OJ antibodies which are associated with anti-synthetase syndrome (ASS). In contrast to the other tRNA synthetases that are targets of ASS autoantibodies (e.g Jo-1, PL-7, PL-12, EJ, KS, Zo), OJ represents a macromolecular complex with several ribonucleoprotein subunits. Therefore, the choice of the antigen in autoantibody assays can be challenging. (2) Methods: We collected two independent cohorts with anti-OJ antibodies, one based on a commercial line immunoassay (LIA) (n = 39), the second based on protein immunoprecipitation (IP) (n = 15). Samples were tested using a particle-based multi-analyte technology (PMAT) system that allows for the simultaneous detection of antibodies to various autoantigens. For the detection of anti-OJ antibodies, two different antigens were deployed (KARS, IARS) on PMAT. The reactivity to the two antigens KARS and IARS was analyzed individually and combined in a score (sum of the median fluorescence intensities). (3) Results: In the cohort selection based on LIA, 3/39 (7.7%) samples were positive for anti-KARS and 7/39 (17.9%) for anti-IARS and 14/39 (35.9%) when the two antigens were combined. In contrast, in samples selected by IP the sensitivity of anti-KARS was higher: 6/15 (40.0%) samples were positive for anti-KARS, 4/15 (26.7%) for anti-IARS and 12/15 (80.0%) for the combination of the two antigens. 18/39 (46.2%) of the LIA samples generated a cytoplasmic IIF pattern (compatible with anti-synthetase antibodies), but there was no association with the antibody levels, neither with LIA nor with PMAT. (4) Conclusions: The combination of IARS and KARS might represent a promising approach for the detection of anti-OJ antibodies on a fully automated platform.