Current Immunoassays Research Articles

Multiple Reaction Monitoring-Mass Spectrometric Immunoassay Analysis of Parathyroid Hormone Fragments with Vitamin D Deficiency in Patients with Diabetes Mellitus.

Current immunoassay techniques for analyzing clinically relevant parathyroid hormone (PTH) circulating fragments cannot distinguish microheterogeneity among structurally similar molecular species. This hinders the identification of molecular species and the capture of target analyte information. Since structural modifications are important in disease pathways, mass spectrometry can detect, identify, and quantify heterogeneous ligands captured by antibodies. We aimed to create a sensitive and selective multiple reaction monitoring-mass spectrometric immunoassay analysis (MRM-MSIA)-based method for detecting and quantifying PTH fragments or proteoforms for clinical research. Our study established MRM transitions using triple-quadrupole tandem mass spectrometry for the signature peptides of five PTH fragments. This method was validated according to FDA guidelines, employing the mass spectrometric immunoassay (MSIA) protocol to bolster detection selectivity and sensitivity. This validated approach was applied by analyzing samples from type 2 diabetes mellitus (T2DM) patients with and without vitamin D deficiency. We found serum PTH fragments associated with vitamin D deficiency in patients with and without T2DM. We developed and validated the MRM-MSIA technique specifically designed for the detection and quantification (amino acid (aa38-44), (aa45-51), and (aa65-75)) of these fragments associated with vitamin D deficiency and T2DM. This study is the first to accurately quantify plasma PTH fragments using MRM-MSIA, demonstrating its potential for clinical diagnostics.

B-008 An Evaluation of the Analytical Performance of the New Beckman Coulter DxC 500i Clinical Analyzer

Abstract Background The DxC 500i Clinical Analyzer* is Beckman Coulter’s latest integrated Clinical Chemistry and Immunoassay analyzer. It provides reliability, scalability, and workflow efficiency via a flexible independent maintenance mode, increased uptime, and a dynamic sample handler to optimize sample management. Standard Beckman Coulter Chemistry and Immunoassay consumables and reagents ensure network standardization and commutability. The purpose of this study was to evaluate the analytical performance of the new DxC 500i analyzer and to compare the performance against the Beckman Coulter stand-alone Clinical Chemistry and Immunoassay analyzers on the market. Methods To assess performance over a range of assay methodologies, several DxC 500i Beckman Coulter Immunoassays and Chemistry assays were evaluated. Precision was assessed using human samples following CLSI EP05-A3 guidelines. Linear range was assessed following CLSI EP06-A guidelines. Method Comparison and Bias Estimation studies compared the DxC 500i Clinical Analyzer against the Access 2 Immunoassay System, the AU480 and the DxC 700 AU Clinical Chemistry analyzers, following CLSI EP09c-ED3 guidelines. To investigate carryover potential as samples are processed across the integrated system, a carryover study was designed. This focused on evaluating potential sample-to-sample carryover caused by the AU sample probe and its effects on immunoassay samples during reflex scenarios. Results DxC 500i Chemistry results: Glucose Serum (OSR6x21) Assessments of repeatability and within laboratory precision at multiple critical analyte concentrations showed total imprecision of <3% and within-run imprecision of <3%. The Glucose serum assay demonstrated acceptable linearity throughout the 10-800 mg/dL analytical measuring range. Method comparison studies compared the DxC 500i analyzer against the AU480 and DxC 700 AU systems. A Weighted Deming regression of serum patient samples (n>100 measured across the analytical range) yielded a 0.986 slope, 0.227 mg/dL intercept, and R=0.9999 correlation coefficient against the DxC 700 AU and a 0.995 slope, 0.356 mg/dL intercept, and R=0.9999 correlation coefficient against the AU480. DxC 500i Immunoassay results: Cortisol (33600) Beckman Coutler’s Cortisol assay exhibited total imprecision of <12%CV at <5 μg/dL or <10CV% at ≥5 μg/dL for serum. The Cortisol assay demonstrated acceptable linearity throughout the 0.4-60 μg/dL analytical measuring range. A method comparison study compared the DxC 500i against the Access 2 system. A Weighted Deming regression of serum patient samples (n>100 measured across the analytical range) yielded a 0.9785 slope, 0.2764 μg/dL intercept, and R=0.9932 correlation coefficient. DxC 500i Carryover Assessment The Total βhCG (5th IS) assay was used to evaluate the potential of chemistry analyzer to immunoassay analyzer sample carryover. The study used High hCG serum samples (>1,000,000 mIU/mL) and Low hCG serum samples (4-6 mIU/mL). The DxC 500i exhibited a cumulative carryover of ≤2 parts per million (PPM) for serum. Conclusions These studies demonstrate excellent analytical performance for the new Beckman Coulter DxC 500i Clinical Analyzer* and confirm comparable performance to current Beckman Coulter standalone Clinical Chemistry and Immunoassay analyzers on the market. *Pending clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use in the U.S. Product availability and regulatory status depends on country registration per applicable regulations.

Despite the improved clinical sensitivity of the Roche benzodiazepines II assay it cannot replace mass spectrometry in all patient populations

IntroductionBenzodiazepines are frequently prescribed and misused therefore urine drug screening (UDS) is performed in many patient populations. Most current benzodiazepine immunoassays have poor sensitivity, particularly for detecting the metabolites of newer benzodiazepines such as lorazepam in urine. ObjectivesWe aimed to verify the clinical performance of the new qualitative Roche Benzodiazepines II (BNZ2) immunoassay, as well as compare its performance to the Roche Benzodiazepines Plus (BENZ) assay in two patient populations: UDS in the emergency department (ED) and compliance monitoring. MethodsAn initial verification study was performed, selecting for samples containing clonazepam and lorazepam metabolites. Performance of the BNZ2 and BENZ assays was compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the reference method. Sensitivity, specificity, false positive rate (FPR) and false negative rate (FNR) were determined. ResultsWe verified the performance claims in the initial verification and demonstrated similar precision, with coefficient of variations (CVs) of 12.8% and 7.7% for negative and positive controls, respectively. Furthermore, we observed higher clinical sensitivity and lower FNR with the BNZ2 assay in both the ED and compliance monitoring populations due to improved cross-reactivity for lorazepam and clonazepam metabolites. Despite these improvements, the BNZ2 assay was unable to detect 27% of specimens positive by LC-MS/MS, including specimens from patients using benzodiazepines without prescription. DiscussionDue to its improved performance and rapid turnaround time, the BNZ2 assay should be implemented for UDS in the ED. However, the assay should not replace LC-MS/MS testing for compliance monitoring, as unsuspected benzodiazepine use may go undetected.

Development of NULISAseq™ 250-Plex Inflammation Panel for Immune Profiling of Autoimmune Diseases

Abstract Comprehensive profiling of cytokines and chemokines in blood can provide deeper insights into the mechanisms underlying this highly complex and heterogeneous group of diseases. However, many of these proteins are present at very low concentrations in plasma, below the limit of detection of current immunoassays. We recently developed a novel automated multiplex immunoassay technology, NULISA™, capable of attomolar-level sensitivity and high levels of multiplexing. Here we report the development of a 250-plex inflammation-focused panel targeting a broad range of cytokines/chemokines and other important inflammation and immune response-related proteins. We conducted a pilot study to assess the utility of this panel for autoimmune disease research. We analyzed plasma samples from patients with rheumatoid arthritis (RA), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis (UC), and compared to healthy donors. Linear model analysis identified differentially abundant proteins associated with diseases including many low-abundance targets with important roles in autoimmune diseases, such as IL4, IL5, IL20, IL17A, IL17F, IL33, and IL2RB. In summary, with the ultrahigh sensitivity and the most comprehensive inflammatory cytokine/chemokine panel, the NULISA™ Platform promises to be a powerful discovery tool for autoimmune disease research, which may lead to new diagnostic biomarkers and therapeutic targets.

Microfluidic devices integrated with plasmonic nanostructures for sensitive fluorescent immunoassays.

The robust identification and quantification of various biomarkers is of utmost significance in clinical diagnostics and precision medicine. Fluorescent immunoassays are widely used and considered as a gold standard for biomarker detection due to their high specificity and accuracy. However, current commercial immunoassay tests suffer from limited detection sensitivity and complicated, labor-intensive operation procedures, making them impractical for point-of-care diagnosis, particularly in resource-limited regions. Recently, microfluidic immunoassay devices integrated with plasmonic nanostructures have emerged as a powerful tool for sensitive detection of biomarkers, addressing specific issues, such as integration schemes, easy operation, multiplexed detection, and sensitivity enhancement. In this paper, we provide a discussion on the recent advances in the plasmonic nanostructures integrated with microfluidic devices for fluorescent immunoassays. We shed light on the nanofabrication strategies and various fluidic designs for rapid, sensitive, and highly efficient sensing of antigens. Finally, we share our perspectives on the potential directions of these integrated devices for practical applications.

The Cross Reactivity of Cannabinoid Analogs (Delta-8-THC, Delta-10-THC and CBD), their metabolites, and Chiral Carboxy HHC metabolites in Urine of Six Commercially Available Homogeneous Immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to Delta-9-tetrahydrocannabinol (Delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to Delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids - Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay - Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC - Thermo Fisher Scientific, ONLINE DAT Cannabinoid II - Roche Diagnostics, and Syva EMIT®II Plus - Siemens Healthineers) were evaluated at two different cutoff concentrations: 50ng/mL and 20 or 25ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and Delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross reactivity was evaluated by preparing each analyte at 20, 50, 100, and 1000ng/mL in urine. Analytes that did not cross react at 1000ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: Delta-8-THC, 11-OH-Delta-8-THC, 11-COOH-Delta-8-THC, 6-OH-CBD, 7-OH-CBD, all Delta-10-THC and HHC carboxylic acid chiral analogs, and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A, and olivetolic acid.

Miniaturized microarray-format digital ELISA enabled by lithographic protein patterning

The search for reliable protein biomarker candidates is critical for early disease detection and treatment. However, current immunoassay technologies are failing to meet increasing demands for sensitivity and multiplexing. Here, the authors have created a highly sensitive protein microarray using the principle of single-molecule counting for signal amplification, capable of simultaneously detecting a panel of cancer biomarkers at sub-pg/mL levels. To enable this amplification strategy, the authors introduce a novel method of protein patterning using photolithography to subdivide addressable arrays of capture antibody spots into hundreds of thousands of individual microwells. This allows for the total sensor area to be miniaturized, increasing the total possible multiplex capacity. With the immunoassay realized on a standard 75x25 mm form factor glass substrate, sample volume consumption is minimized to <10 μL, making the technology highly efficient and cost-effective. Additionally, the authors demonstrate the power of their technology by measuring six secretory factors related to glioma tumor progression in a cohort of mice. This highly sensitive, sample-sparing multiplex immunoassay paves the way for researchers to track changes in protein profiles over time, leading to earlier disease detection and discovery of more effective treatment using animal models.

Late-night salivary cortisol cut-offs for diagnosis of Cushing syndrome using second-generation electrochemiluminescence immunoassay kits.

Late-night salivary cortisol (LNSC) is a simple and reliable screening test for Cushing syndrome (CS). With improved analytical performance of the current second-generation electrochemiluminescence immunoassay (ECLIA; Elecsys Cortisol-II; Roche Diagnostics), there is a need to revisit the LNSC cut-offs, especially in a South-Asian population. To derive LNSC cut-offs for diagnosis of CS using second-generation ECLIA kits. Diagnostic accuracy study. We prospectively recruited 155 controls aged 18-60 years, including, normal-weight (body mass index [BMI] < 25 kg/m2 and no hypertension or diabetes [n = 53]) and overweight/obese (BMI 25-30 kg/m2 and hypertension and/or diabetes [n = 52] or BMI ≥ 30 kg/m2 with/without comorbidities [n = 50]) participants. All participants submitted LNSC samples collected at home; overweight/obese controls additionally underwent dexamethasone suppression test to exclude CS. We also reviewed records of adults with endogenous CS (cases, n = 92) and a valid LNSC result using the same method. The 95th percentile for LNSC in controls was 6.76 nmol/L. The mean ± SD LNSC levels were 40.47 ± 49.63 nmol/L in cases and 3.37 ± 1.18 nmol/L in controls (p < 0.001). Receiver operating characteristic (ROC) analysis showed excellent diagnostic performance of LNSC for CS, with area under curves (AUCs) of 0.994 (cases vs. all controls) and 0.993 (cases vs. overweight/obese controls), respectively. The best diagnostic performance was achieved at cut-offs ≥6.73 nmol/L (sensitivity: 97.8%, specificity: 94.8%) and ≥7.26 nmol/L (sensitivity: 97.8%, specificity: 95.1%), respectively. LNSC measured using second-generation ECLIA demonstrated high diagnostic accuracy for CS. Based on this study, we propose a LNSC cutoff ≥6.73 nmol/L to diagnose CS.

Evaluation of a Delta-9-Tetrahydrocannabinol Carboxylic Acid (Δ9-THC-COOH) Immunoassay and a Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection of Delta-8-Tetrahydrocannabinol Carboxylic Acid (Δ8-THC-COOH).

Delta-8 tetrahydrocannabinol (Δ8-THC) is a naturally occurring or synthetically prepared cannabinoid that elicits psychological and physiological experiences commonly reported for its more infamous isomer, delta-9 tetrahydrocannabinol (Δ9-THC). Unlike Δ9-THC, Δ8-THC products are generally legal under federal law and there has been a rise in their usage. One of the main targets for detection and quantitation of Δ9-THC is its inactive metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH). This study evaluated the ability of the currently used Δ9-THC-COOH immunoassay and gas chromatography-mass spectrometry (GC-MS) methods to detect 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THC-COOH) and distinguish it from Δ9-THC-COOH. The EMIT II Plus® Cannabinoid immunoassay for Δ9-THC-COOH with a cutoff of 20 ng/mL showed positive results for Δ8-THC-COOH with concentrations of 30 ng/mL or higher. Although many of the ion fragments generated by mass spectrometry were found to overlap between the 2 compounds, the GC-MS method presently used to quantify Δ9-THC-COOH separated the 2 compounds sufficiently to identify them independently by relative retention time. Current immunoassays and GC-MS methods should be evaluated for the ability to detect and distinguish the presence of Δ8-THC-COOH.

Dissolution-Enhanced Luminescence Enhanced Digital Microfluidics Immunoassay for Sensitive and Automated Detection of H5N1.

Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.

Nanophotonic immunoarray with electrochemically roughened surfaces for handheld detection of secreted PD-L1 to predict immuno-oncology efficacy.

The analysis of secreted protein biomarkers can be a useful non-invasive method of predicting or monitoring cancer therapeutic response. The increased level of soluble programmed cell death protein ligand 1 (sPD-L1) is a promising predictive biomarker for selecting patients who are likely to respond to immune checkpoint immunotherapy. The current established immunoassay for secreted protein analysis is enzyme-linked immunosorbent assay (ELISA). Yet, ELISA is generally still liable to limited detection sensitivity and restricted to bulky chromogenic readout equipment. Herein, we present a designed nanophotonic immunoarray sensor which achieved sPD-L1 analysis at high-throughput, enhanced detection sensitivity and portability. The key benefits of our nanophotonic immunoarray sensor are (i) high-throughput surface-enhanced Raman scattering (SERS) analysis of multiple samples on a singular platform; (ii) improved sPD-L1 detection sensitivity at 1 pg mL-1 (by two orders of magnitude as compared to ELISA) via electrochemically roughened gold sensor surfaces; (iii) fit for handheld SERS detection with miniaturized equipment footprint. We evaluated the analytical performance of the nanophotonic immunoarray sensor and successfully demonstrated quantitative sPD-L1 detection in a cohort of contrived human plasma samples.

Improved Sensitivity and Wide Range Detection of Small Analytes Using a Two-Antigen-Combined Competitive Immunoassay.

Competitive immunoassays have unique advantages in the detection of small molecules and are widely used in clinical practice. However, the concentrations of some analytes usually vary greatly among different populations, which makes it difficult to balance the sensitivity and detection range of competitive immunoassays. Studies have shown that using haptens with weaker affinity for specific antibodies as competitive antigens can help improve the sensitivity of the method. Here, we developed a competitive light initiated chemiluminescence assay based on the combination of antigens with different affinities, which has high sensitivity and wide detection range. As a proof of concept, estradiol was used as the analyte. After the mixing ratio was optimized, the two labeled haptens played different competitive roles due to the different concentrations of estradiol to be tested, which improved the sensitivity of estradiol detection, while ensuring a certain detection range. The limit of detection of this method was 5.30 pg/mL, which is lower than most current estradiol immunoassay kits. Good linearity (R 2 = 0.9902) was obtained between estradiol concentrations of 17.07-2376.22 pg/mL. This study provides a new solution for the detection of small molecule biomarkers with a large concentration span, which also has considerable potential in other immunological detection methods.

Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer’s disease continuum

BackgroundAlzheimer’s disease is characterized by an abnormal increase of phosphorylated tau (pTau) species in the CSF. It has been suggested that emergence of different pTau forms may parallel disease progression. Therefore, targeting multiple specific pTau forms may allow for a deeper understanding of disease evolution and underlying pathophysiology. Current immunoassays measure pTau epitopes separately and may capture phosphorylated tau fragments of different length depending on the non-pTau antibody used in the assay sandwich pair, which bias the measurement.MethodsWe developed the first antibody-free mass spectrometric method to simultaneously measure multiple phosphorylated epitopes in CSF tau: pT181, pS199, pS202, pT205, pT217, pT231, and pS396. The method was first evaluated in biochemically defined Alzheimer’s disease and control CSF samples (n = 38). All seven pTau epitopes clearly separated Alzheimer’s disease from non-AD (p < 0.001, AUC = 0.84–0.98). We proceeded with clinical validation of the method in the TRIAD (n = 165) and BioFINDER-2 cohorts (n = 563), consisting of patients across the full Alzheimer’s disease continuum, including also young controls (< 40 years), as well as patients with frontotemporal dementia and other neurodegenerative disorders.ResultsIncreased levels of all phosphorylated epitopes were found in Alzheimer’s disease dementia and Aβ positron emission tomography-positive patients with mild cognitive impairment compared with Aβ-negative controls. For Alzheimer’s disease dementia compared with Aβ-negative controls, the best biomarker performance was observed for pT231 (TRIAD: AUC = 98.73%, fold change = 7.64; BioFINDER-2: AUC = 91.89%, fold change = 10.65), pT217 (TRIAD: AUC = 99.71%, fold change = 6.33; BioFINDER-2: AUC = 98.12%, fold change = 8.83) and pT205 (TRIAD: AUC = 99.07%, fold change = 5.34; BioFINDER-2: AUC = 93.51%, fold change = 3.92). These phospho-epitopes also discriminated between Aβ-positive and Aβ-negative cognitively unimpaired individuals: pT217 (TRIAD: AUC = 83.26, fold change = 2.39; BioFINDER-2: AUC = 91.05%, fold change = 3.29), pT231 (TRIAD: AUC = 86.25, fold change = 3.80; BioFINDER-2: AUC = 78.69%, fold change = 3.65) and pT205 (TRIAD: AUC = 71.58, fold change = 1.51; BioFINDER-2: AUC = 71.11%, fold change = 1.70).ConclusionsWhile an increase was found for all pTau species examined, the highest fold change in Alzheimer’s disease was found for pT231, pT217 and pT205. Simultaneous antibody-free measurement of pTau epitopes by mass spectrometry avoids possible bias caused by differences in antibody affinity for modified or processed forms of tau, provides insights into tau pathophysiology and may facilitate clinical trials on tau-based drug candidates.

Reporter Molecules Embedded Au@Ag Core-Shell Nanospheres as SERS Nanotags for Cardiac Troponin I Detection

Rapid and accurate detection of acute myocardial infarction can improve patients' chances of survival. Cardiac troponin I (cTn I) is an important diagnostic biomarker for acute myocardial infarction. However, current immunoassays are insufficient to accurately measure cTn I, as they have limited detection sensitivity and are time-consuming. Surface-enhanced Raman scattering (SERS) is a brilliant fingerprints diagnostic technique characterised by ultrasensitivity, fast response, and qualitative and quantitative analysis capabilities. In this study, reporter molecules (4-Mercaptobenzoic acid, 4-MBA) embedded Au@Ag core-shell nanospheres as SERS nanotags were prepared for the detection of cTn I. As the Raman reporters were embedded between the core and the shell, they could be protected from the external environment and nanoparticle aggregation. Excellent SERS performances were obtained due to the enhanced local electromagnetic field in the gap of core and shell metals. In a standard phosphate buffered saline (PBS) environment, the limit of detection for cTn I was 0.0086 ng mL-1 (8.6 ppt) with a good linear relationship. The excellent Raman detection performance was attributed to the localized surface plasmon resonance effect and strong electromagnetic field enhancement effect produced by the gap between the Au core and the Ag shell. The SERS nanotags we prepared were facile to synthesize, and the analysis procedure could be completed quickly (15 min), which made the detection of cTn I faster. Therefore, the proposed SERS nanotags have significant potential to be a faster and more accurate tool for acute myocardial infarction diagnostics.

Enhanced Label-Free Nanoplasmonic Cytokine Detection in SARS-CoV-2 Induced Inflammation Using Rationally Designed Peptide Aptamer.

Rapid and precise serum cytokine quantification provides immense clinical significance in monitoring the immune status of patients in rapidly evolving infectious/inflammatory disorders, examplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. However, real-time information on predictive cytokine biomarkers to guide targetable immune pathways in pathogenic inflammation is critically lacking, because of the insufficient detection range and detection limit in current label-free cytokine immunoassays. In this work, we report a highly sensitive localized surface plasmon resonance imaging (LSPRi) immunoassay for label-free Interleukin 6 (IL-6) detection utilizing rationally designed peptide aptamers as the capture interface. Benefiting from its characteristically smaller dimension and direct functionalization on the sensing surface via Au-S bonding, the peptide-aptamer-based LSPRi immunoassay achieved enhanced label-free serum IL-6 detection with a record-breaking limit of detection down to 4.6 pg/mL, and a wide dynamic range of ∼6 orders of magnitude (values from 4.6 to 1 × 106 pg/mL were observed). The immunoassay was validated in vitro for label-free analysis of SARS-CoV-2 induced inflammation, and further applied in rapid quantification of serum IL-6 profiles in COVID-19 patients. Our peptide aptamer LSPRi immunoassay demonstrates great potency in label-free cytokine detection with unprecedented sensing capability to provide accurate and timely interpretation of the inflammatory status and disease progression, and determination of prognosis.

Current immunoassays and detection of antibodies elicited by Omicron SARS-CoV-2 infection.

The present study aimed to determine whether current commercial immunoassays are adequate for detecting anti-Omicron antibodies. We analyzed the anti-SARS-CoV-2 antibody response of 23 unvaccinated individuals 1-2 months after an Omicron infection. All blood samples were tested with a live virus neutralization assay using a clinical Omicron BA.1 strain and four commercial SARS-CoV-2 immunoassays. We assessed three anti-Spike immunoassays (SARS-CoV-2 IgG II Quant [Abbott S], Wantaï anti-SARS-CoV-2 antibody ELISA [Wantaï], Elecsys Anti-SARS-CoV-2 S assay [Roche]) and one anti-Nucleocapsid immunoassay (Abbott SARS-CoV-2 IgG assay [Abbott N]). Omicron neutralizing antibodies were detected in all samples with the live virus neutralization assay. The detection rate of the Abbott S, Wantai, Roche, and Abbott N immunoassays were 65.2%, 69.6%, 86.9%, and 91.3%, respectively. The sensitivities of Abbott S and Wantai immunoassays were significantly lower than that of the live virus neutralization assay (p = 0.004, p = 0.009; Fisher's exact test). Antibody concentrations obtained with anti-S immunoassays were correlated with Omicron neutralizing antibody concentrations. These data provide clinical evidence of the loss of performance of some commercial immunoassays to detect antibodies elicited by Omicron infections. It highlights the need to optimize these assays by adapting antigens to the circulating SARS-CoV-2 strains.

Apolipoprotein L1 is increased in frontotemporal lobar degeneration post-mortem brain but not in ante-mortem cerebrospinal fluid

AimsFrontotemporal Dementia (FTD) is caused by frontal-temporal lobar degeneration (FTLD), characterized mainly by brain protein aggregates of tau (FTLD-Tau) or TDP-43 (FTLD-TDP). The clinicopathological heterogeneity makes ante-mortem diagnosis of these pathological subtypes challenging. Our proteomics study showed increased Apolipoprotein L1 (APOL1) levels in CSF from FTD patients, which was prominently expressed in FTLD-Tau. We aimed to understand APOL1 expression in FTLD post-mortem brain tissue and to validate its potential as a CSF biomarker for FTD and its pathological subtypes. MethodsAPOL1 levels were analyzed in the frontal cortex of FTLD (including FTLD-Tau and FTLD-TDP) and non-demented controls by immunohistochemistry (FTLD total = 18 (12 FTLD-Tau and 6 FTLD-TDP); controls = 9), western blot (WB), and a novel prototype ELISA (FTLD total = 44 (21 FTLD-Tau and 23 FTLD-TDP); controls = 9). The association of APOL1 immunoreactivity with phosphorylated Tau (pTau) and TDP-43 (pTDP-43) immunoreactivity was assessed. CSF APOL1 was analyzed in confirmed FTD patients (n = 27, including 12 FTLD-Tau and 15 FTLD-TDP) and controls (n = 15) using the same ELISA. ResultsAPOL1 levels were significantly increased in FTLD post-mortem tissue compared to controls as measured by immunohistochemistry, WB, and ELISA. However, no differences between the pathological subtypes were observed. APOL1 immunoreactivity was present in neuronal and glial cells but did not co-localize with pTau or pTDP-43. CSF APOL1 levels were comparable between FTD patients and controls and between pathological subtypes. ConclusionAPOL1 is upregulated in FTLD pathology irrespective of the subtypes, indicating a role of this novel protein in FTD pathophysiology. The APOL1 levels detected in brain tissue were not mirrored in the CSF, limiting its potential as a specific FTD biofluid-based biomarker using our current immunoassay.

Evaluation of a Multiplex Bead Assay against Single-Target Assays for Detection of IgG Antibodies to SARS-CoV-2.

ABSTRACTSerological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected.IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.

Spectral image contrast-based flow digital nanoplasmon-metry for ultrasensitive antibody detection

BackgroundGold nanoparticles (AuNPs) have been widely used in local surface plasmon resonance (LSPR) immunoassays for biomolecule sensing, which is primarily based on two conventional methods: absorption spectra analysis and colorimetry. The low figure of merit (FoM) of the LSPR and high-concentration AuNP requirement restrict their limit of detection (LOD), which is approximately ng to μg mL−1 in antibody detection if there is no other signal or analyte amplification. Improvements in sensitivity have been slow in recent for a long time, and pushing the boundary of the current LOD is a great challenge of current LSPR immunoassays in biosensing.ResultsIn this work, we developed spectral image contrast-based flow digital nanoplasmon-metry (Flow DiNM) to push the LOD boundary. Comparing the scattering image brightness of AuNPs in two neighboring wavelength bands near the LSPR peak, the peak shift signal is strongly amplified and quickly detected. Introducing digital analysis, the Flow DiNM provides an ultrahigh signal-to-noise ratio and has a lower sample volume requirement. Compared to the conventional analog LSPR immunoassay, Flow DiNM for anti-BSA detection in pure samples has an LOD as low as 1 pg mL−1 within only a 15-min detection time and 500 μL sample volume. Antibody assays against spike proteins of SARS-CoV-2 in artificial saliva that contained various proteins were also conducted to validate the detection of Flow DiNM in complicated samples. Flow DiNM shows significant discrimination in detection with an LOD of 10 pg mL−1 and a broad dynamic detection range of five orders of magnitude.ConclusionTogether with the quick readout time and simple operation, this work clearly demonstrated the high sensitivity and selectivity of the developed Flow DiNM in rapid antibody detection. Spectral image contrast and digital analysis further provide a new generation of LSPR immunoassay with AuNPs.Graphical

Development of Generic Immuno-Magnetic Bead-Based Enzyme-Linked Immunoassay for Ustiloxins in Rice Coupled with Enrichment.

Ustiloxins are a group of mycotoxins produced by rice false smut pathogen. Previous studies have shown that the false smut balls contain six types of ustiloxins, and these toxins are toxic to living organisms. Thus, immunoassay for on-site monitoring of ustiloxins in rice is urgently required. The current immunoassays are only for detecting single ustiloxin, and they cannot meet the demand for synchronous and rapid detection of the group toxins. Therefore, this study designed and synthesized a generic antigen with ustiloxin G as material based on the common structure of the mycotoxins. Ustiloxin G was conjugated to two carrier proteins including bovine serum albumin (BSA) and ovalbvmin (OVA) by carbon diimide method. The mice were immunized with ustiloxin-G-BSA to generate the antibody serum, which was further purified to obtain the generic antibody against ustiloxins. The conjugated ustiloxin G-OVA and generic antibodies were used for establishing the enzyme-linked immunosorbent assay (ELISA) for ustiloxin detection and optimizing experiment conditions. The characterization of the antibody showed that the semi-inhibitory concentrations (IC50) of ustiloxin A, B, and G were 0.53, 0.34, and 0.06 µg/mL, respectively, and that their corresponding cross-reactivities were 11.9%, 18.4%, and 100%, respectively. To increase ELISA detection efficiency, generic antibody was combined with magnetic beads to obtain sensitive and class-specific immune-magnetic beads. Based on these immuno-magnetic beads, a high-efficiency enzyme-linked immunoassay method was developed for ustiloxin detection, whose sensitivity to ustiloxin A, B, and G was improved to 0.15 µg/mL, 0.14 µg/mL, and 0.04 µg/mL, respectively. The method accuracy was evaluated by spiking ustiloxin G as standard, and the spiked samples were tested by the immune-magnetic bead-based ELISA. The result showed the ustiloxin G recoveries ranged from 101.9% to 116.4% and were accepted by a standard HPLC method, indicating that our developed method would be promising for on-site monitoring of ustiloxins in rice.