Fluorescence-based Immunoassay Research Articles

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

Fluorescence-Based Lateral Flow Immunoassay for Quantification of Infliximab: Analytical and Clinical Performance Evaluation.

Therapeutic drug monitoring of infliximab (IFX) can improve treatment outcomes; however, the temporal gap between drug concentration monitoring and subsequent availability restricts its practical application. To address this issue, an automated monitoring method, AFIAS IFX, was developed to rapidly and accurately analyze IFX concentration in blood. The analytical and clinical performances of this method were assessed to establish its clinical utility. The analytical performance of AFIAS IFX was evaluated according to Clinical and Laboratory Standard Institute guidelines. For clinical validation, AFIAS IFX was compared with 3 established enzyme-linked immunosorbent assay kits (LISA TRACKER, RIDASCREEN, and ImmunoGuide) using 100 consecutive samples from 28 patients treated with IFX. Passing-Bablok regression and Bland-Altman analyses were performed to compare the methods. The detection and quantification limits of AFIAS IFX were 0.12 and 0.20 mcg/mL, respectively. Furthermore, AFIAS IFX analyzed samples within 10 minutes for concentrations up to 50 mcg/mL, exhibiting reproducibility (coefficient of variation [CV] ≤7.8%) and accuracy (recovery 98%-101%) with serum, plasma, and whole blood samples. Clinically, it exhibited a good correlation with the 3 established enzyme-linked immunosorbent assay kits. For patients treated with Remicade (IFX), the Passing-Bablok regression slope was 1.001-1.259, with a mean difference of -1.48 to 0.28 mcg/mL. For patients treated with CT-P13, the Passing-Bablok regression slope was 0.974-1.254, with a mean difference of -2.44 to 0.15 mcg/mL. AFIAS IFX, a novel fluorescence-based lateral flow assay, exhibited excellent performance in analyzing IFX trough levels and is a potentially powerful tool for therapeutic drug monitoring in clinical settings, with opportunities for further development.

Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS<3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2= 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Using Temporally and Spatially Resolved Measurements to Improve the Sensitivity of Fluorescence-Based Immunoassays.

Detecting low concentrations of biomarkers is essential in clinical laboratories. To improve analytical sensitivity, especially in identifying fluorescently labeled molecules, typical optical detection systems, consisting of a photodetector or camera, utilize time-resolved measurements. Taking a different approach, magnetic modulation biosensing (MMB) is a novel technology that combines fluorescently labeled probes and magnetic particles to create a sandwich assay with the target molecules. By concentrating the target molecules and then using time-resolved measurements, MMB provides the rapid and highly sensitive detection of various biomarkers. Here, we propose a novel signal-processing algorithm that enhances the detection and estimation of target molecules at low concentrations. By incorporating both temporally and spatially resolved measurements using human interleukin-8 as a target molecule, we show that the new algorithm provides a 2-4-fold improvement in the limit of detection and an ~25% gain in quantitative resolution.

Immunoassays: Analytical and Clinical Performance, Challenges, and Perspectives of SERS Detection in Comparison with Fluorescent Spectroscopic Detection.

Immunoassays (IAs) with fluorescence-based detection are already well-established commercialized biosensing methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). Immunoassays with surface-enhanced Raman spectroscopy (SERS) detection have received significant attention from the research community for at least two decades, but so far they still lack a wide clinical commercial application. This review, unlike any other review that we have seen, performs a three-dimensional performance comparison of SERS IAs vs. fluorescence IAs. First, we compared the limit of detection (LOD) as a key performance parameter for 30 fluorescence and 30 SERS-based immunoassays reported in the literature. We also compared the clinical performances of a smaller number of available reports for SERS vs. fluorescence immunoassays (FIAs). We found that the median and geometric average LODs are about 1.5-2 orders of magnitude lower for SERS-based immunoassays in comparison to fluorescence-based immunoassays. For instance, the median LOD for SERS IA is 4.3 × 10-13 M, whereas for FIA, it is 1.5 × 10-11 M. However, there is no significant difference in average relative standard deviation (RSD)-both are about 5-6%. The analysis of sensitivity, selectivity, and accuracy reported for a limited number of the published clinical studies with SERS IA and FIA demonstrates an advantage of SERS IA over FIA, at least in terms of the median value for all three of those parameters. We discussed common and specific challenges to the performances of both SERS IA and FIA, while proposing some solutions to mitigate those challenges for both techniques. These challenges include non-specific protein binding, non-specific interactions in the immunoassays, sometimes insufficient reproducibility, relatively long assay times, photobleaching, etc. Overall, this review may be useful for a large number of researchers who would like to use immunoassays, but particularly for those who would like to make improvements and move forward in both SERS-based IAs and fluorescence-based IAs.

High-sensitive detection and quantitation of thyroid-stimulating hormone (TSH) from capillary/fingerstick and venepuncture whole-blood using fluorescence-based rapid lateral flow immunoassay (LFIA)

BackgroundIn the last decade, point of care testing (POCT) such as lateral flow immunoassays (LFIA) were developed for rapid TSH measurement. Most of these TSH-LFIAs are designed for qualitative measurements (i.e., if TSH values > 5, or >15 IU/L) and as screening tests for primary hypothyroidism in children and adults. Serum or plasma, but not venepuncture whole-blood or fingerstick/capillary, are usually used to quantify TSH accurately. Studies on performance evaluation of TSH-LFIAs POCT using venepuncture or fingerstick whole-blood are limited. Additionally, limited studies evaluated the performance and validity of TSH-LFIAs POCT compared to valid and reliable reference methods. To our knowledge, this is the first study to evaluate three different blood withdrawal techniques for evaluating POCT of TSH. AimWe aim to evaluate the performance of a new fluorescence-based LFIA and its Finecare™ fluorescent reader for quantitative measurement of TSH from a fingerstick, venepuncture whole-blood, and serum. Methods102 fingerstick, venepuncture whole-blood, and serum samples (with normal and abnormal TSH values) were analyzed by Finecare™ Rapid Quantitative LFIA test and Roche cobas® e 601 as a reference test. ResultsUsing serum, when compared to cobas® e 601 reference method, Finecare™ showed high sensitivity [90.5 % (69.6–98.8)] and specificity [96.3 % (89.6–99.2)] for diagnosis of thyroid abnormalities (<0.35 or >4.5 mIU/L). The actual test values (mIU/L) of Finecare™ showed excellent agreement (Cohen's Kappa = 0.85) and strong correlation (r = 0.93, p < 0.0001) with cobas® e 601. Using venepuncture whole-blood samples, Finecare™ showed similar results to serum with high sensitivity [95.2 % (76.2–99.9)], specificity [97.5 % (91.4–99.7)], excellent agreement (Cohen's Kappa = 0.91), and very strong correlation (r = 0.95, p < 0.0001) with cobas® e 601. These results suggest that Finecare™ can be used for quantitative measurement of TSH using serum or venepuncture whole-blood. These key performance indicators were slightly decreased when fingerstick whole-blood samples were used: sensitivity [85.7 %(63.7–97)], specificity [90.0 %,(81.5–96)], good agreement (Cohen's Kappa = 0.7) and very strong correlation (r = 0.9, p < 0.0001) with cobas® e 601. A subgroup analysis of abnormal TSH samples revealed a strong and significant correlation between the reference, Finecare™ whole-blood (r = 0.692; p = 0.0015), and fingerstick test Finecare™ (r = 0.66; p = 0.0025). A very strong correlation was also observed between cobas® e 601 serum and Finecare™ serum (r = 0.88; p < 0.0001). Conclusion: In comparison to the reference assay, our study demonstrates that Finecare™ exhibits high sensitivity, specificity, agreement, and a strong correlation. These findings provide evidence that Finecare™ is a reliable, valid, and accurate point-of-care test for TSH screening and quantitative measurement, especially in non- or small laboratory settings.

B-083 Novel High Affinity Monoclonal Antibodies for Specific Detection of Neurofilament Light Protein in Serum and Cerebrospinal Fluid

Abstract Background Neurofilaments are very important protein components of axons, and they maintain the function of nervous system. Cerebrospinal fluid (CSF) and serum neurofilament levels elevate due to damages in nervous system making neurofilaments promising biomarkers for neural diseases and injuries, such as Alzheimer’s disease, traumatic brain injury and Multiple sclerosis. Based on their size, neurofilaments are divided in groups: Neurofilament light (NfL), neurofilament medium (NfM), neurofilament heavy (NfH), α-internexin, and peripherin. Of these, NfL is considered to be the most promising biomarker since it is the most abundant form of neurofilaments. Methods We have developed four mouse monoclonal antibodies against human NfL. These antibodies, designated as Anti-h NfL 12601, 12603, 12604 or 12605, were tested as pairs in sandwich fluorescence-based immunoassay (FIA) with NfL spiked serum and buffer to determine linear measuring ranges and the matrix effect. Furthermore, the ability of the antibodies to measure NfL in clinical samples was tested in sandwich FIA by testing 10 CSF samples in the range of 0 to 5 083 pg/mL. The antibodies were also compared against commercially available reference antibodies in FIA. Kinetic parameters were determined with bio-layer interferometry (BLI). Results In sandwich FIA, the widest linear measuring range of 10 to 40 960 pg/mL of recombinant human NfL spiked in normal serum was achieved with antibody pair 12603 and 12604. In contrast, the commercial reference antibody pair achieved a linear measuring range of 2560 to 40 960 pg/mL. Matrix effect caused by serum was negligible. With clinical samples, the best correlation with the reference method (NF-light CSF ELISA from Uman Diagnostics) with a coefficient of correlation of 0.986 was achieved with antibody pair 12604 and 12601. This pair also showed a good correlation with the commercial reference pair with a coefficient of correlation of 0.984. In kinetics measurements, none of four antibodies showed dissociation indicating very high affinity. The fastest association of 4.0 × 105 1/Ms was achieved with antibody 12605. Conclusion These results demonstrate that the newly developed mouse monoclonal NfL antibodies are promising tools for development of diagnostic immunoassays measuring NfL levels in human serum and CSF samples. Comparison against commercial reference antibodies indicates that higher NfL detection sensitivity and wider detection ranges can be achieved with antibody pairs developed in this study. Additionally, the kinetic measurement results forecast these antibodies to be suitable for fast reaction kinetic assay formats, such as lateral flow assays, which might become a key resolution in acute neural injury diagnostics.

Isolation and functional analysis of phage-displayed antibody fragments targeting the staphylococcal superantigen-like proteins.

Staphylococcus aureus produces numerous virulence factors that manipulate the immune system, helping the bacteria avoid phagocytosis. In this study, we are investigating three immune evasion molecules called the staphylococcal superantigen-like proteins 1, 5, and 10 (SSL1, SSL5, and SSL10). All three SSLs inhibit vital host immune processes and contribute to S. aureus immune evasion. This study aimed to identify single-chain variable fragment (scFvs) antibodies from synthetic antibody phage libraries, which can recognize either of the three SSLs and could block the interaction between the SSLs and their respective human targets. The antibodies were isolated after three rounds of panning against SSL1, SSL5, and SSL10, and their ability to bind to the SSLs was studied using a time-resolved fluorescence-based immunoassay. We successfully obtained altogether 44 unique clones displaying binding activity to either SSL1, SSL5, or SSL10. The capability of the SSL-recognizing scFvs to inhibit the SSLs' function was tested in an MMP9 enzymatic activity assay, a P-selectin glycoprotein ligand 1 competitive binding assay, and an IgG1-mediated phagocytosis assay. We could show that one scFv was able to inhibit SSL1 and maintain MMP9 activity in a concentration-dependent manner. Finally, the structure of this inhibiting scFv was modeled and used to create putative scFv-SSL1-complex models by protein-protein docking. The complex models were subjected to a 100-ns molecular dynamics simulation to assess the possible binding mode of the antibody.

Optically Responsive Protein Coating of DNA Origami for Triggered Antigen Targeting.

DNA nanostructures have emerged as modular building blocks in several research fields including biomedicine and nanofabrication. Their proneness to degradation in various environments has led to the development of a variety of nature-inspired protection strategies. Coating of DNA origami nanostructures with proteins can circumvent degradation and alter their properties. Here, we have used a single-chain variable antibody fragment and serum albumin to construct positively charged and stimuli-responsive protein-dendron conjugates, which were complexed with DNA origami through electrostatic interactions. Using a stepwise assembly approach, the coated nanostructures were studied for their interaction with the corresponding antigen in fluorescence-based immunoassays. The results suggest that the antibody–antigen interaction can be disturbed by the addition of the bulky serum albumin. However, this effect is fully reversible upon irradiation of the structures with an optical stimulus. This leads to a selective dissociation of the serum albumin from the nanostructure due to cleavage of a photolabile group integrated in the dendron structure, exposing the antibody fragment and enabling triggered binding to the antigen, demonstrating that serum albumin can be considered as an externally controlled “camouflaging” agent. The presented stimuli-responsive complexation approach is highly versatile regarding the choice of protein components and could, therefore, find use in DNA origami protection, targeting, and delivery as well as their spatiotemporal control.

Use of Polymer Micropillar Arrays as Templates for Solid-Phase Immunoassays.

We investigate the use of periodic micropillar arrays produced by high-fidelity microfabrication with cyclic olefin polymers for solid-phase immunoassays. These three-dimensional (3D) templates offer higher surface-to-volume ratios than two-dimensional substrates, making it possible to attach more antibodies and so increase the signal obtained by the assay. Micropillar arrays also provide the capacity to induce wicking, which is used to distribute and confine antibodies on the surface with spatial control. Micropillar array substrates are modified by using oxygen plasma treatment, followed by grafting of (3-aminopropyl)triethoxysilane for binding proteins covalently using glutaraldehyde as a cross-linker. The relationship between microstructure and fluorescence signal was investigated through variation of pitch (10–50 μm), pillar diameter (5–40 μm), and pillar height (5–57 μm). Our findings suggest that signal intensity scales proportionally with the 3D surface area available for performing solid-phase immunoassays. A linear relationship between fluorescence intensity and microscale structure can be maintained even when the aspect ratio and pillar density both become very high, opening the possibility of tuning assay response by design such that desired signal intensity is obtained over a wide dynamic range compatible with different assays, analyte concentrations, and readout instruments. We demonstrate the versatility of the approach by performing the most common immunoassay formats—direct, indirect, and sandwich—in a qualitative fashion by using colorimetric and fluorescence-based detection for a number of clinically relevant protein markers, such as tumor necrosis factor alpha, interferon gamma (IFN-γ), and spike protein of severe acute respiratory syndrome coronavirus 2. We also show quantitative detection of IFN-γ in serum using a fluorescence-based sandwich immunoassay and calibrated samples with spike-in concentrations ranging from 50 pg/mL to 5 μg/mL, yielding an estimated limit of detection of ∼1 pg/mL for arrays with high micropillar density (11561 per mm2) and aspect ratio (1:11.35).

Performance evaluation of novel fluorescent-based lateral flow immunoassay (LFIA) for rapid detection and quantification of total anti-SARS-CoV-2 S-RBD binding antibodies in infected individuals

BackgroundA vast majority of the commercially available lateral flow immunoassay (LFIA) is used to detect SARS-CoV-2 antibodies qualitatively. Recently, a novel fluorescence-based lateral flow immunoassay (LFIA) test was developed for quantitative measurement of the total binding antibody units (BAUs) (BAU/mL) against SARS-CoV-2 spike protein receptor-binding domain (S-RBD). AimThis study aimed to evaluate the performance of the fluorescence LFIA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113). MethodsPlasma from 150 reverse trancriptase–PCR (RT-PCR)-confirmed positive individuals and 100 prepandemic samples were tested by FincareTM to access sensitivity and specificity. For qualitative and quantitative validation of the FinCareTM measurements, BAU/mL results of FinCareTM were compared with results of 2 reference assays: the surrogate virus-neutralizing test (sVNT, GenScript Biotech, USA) and the VIDAS®3 automated assay (BioMérieux, France). ResultsFinecareTM showed 92% sensitivity and 100% specificity compared with PCR. Cohen's Kappa statistic denoted moderate and excellent agreement with sVNT and VIDAS®3, with values being 0.557 (95% CI: 0.32–0.78) and 0.731 (95% CI: 0.51–0.95), respectively. A strong correlation was observed between FinecareTM/sVNT (r = 0.7, p < 0.0001) and FinecareTM/VIDAS®3 (r = 0.8, p < 0.0001). ConclusionFinecareTM is a reliable assay and can be used as a surrogate to assess binding and neutralizing antibody response after infection or vaccination, particularly in none or small laboratory settings.

Entrapping gold nanoparticles in membranes for simple-to-use enhanced fluorescence detection of proteins

Quantifying proteins under different physiological and pathological conditions can give important insights into human health and disease, since proteins are the functional components of cells. In order to be able to use protein expression levels to diagnose diseases, sensitive protein quantification techniques are required, because some proteins can be present in low concentrations in biofluids at early stages of a disease. Here, a novel and simple-to-implement signal enhancement method for fluorescence-based protein detection is presented, in which an immunoassay was conducted on a nitrocellulose membrane, and a solution of gold nanoparticles was then pipetted onto the membrane before signal acquisition with a fluorescence microscope. The gold nanoparticles were entrapped and adsorbed into the 3D membrane matrix upon drying due to the hydrophobic interactions with the membrane. Through optimizing the concentration and size of the nanoparticles and comparison of different membranes, we were able to achieve metal enhanced fluorescence (MEF). This new gold nanoparticle-based MEF method, together with the 3D membrane platform that improves antibody and gold nanoparticle binding capacity, provided higher signal intensity and assay sensitivity for fluorescence-based protein quantification. Fluorescently-labeled IgG protein was spiked in human plasma with different concentrations, and the lower limit of detection was determined to be 29 ng/mL, three orders of magnitude lower than that of conventional 2D assays and one order of magnitude lower than that of 3D membrane assays without applying gold nanoparticles. This method offers a simple way of improving the sensitivity of fluorescence-based immunoassays that can be easily adopted in a biological lab with basic lab set up. Furthermore, it can be potentially extended to the fluorescence detection of other analytes beyond proteins.

Fluorescent Immunoassays for Detection and Quantification of Cardiac Troponin I: A Short Review.

Cardiovascular diseases are considered one of the major causes of human death globally. Myocardial infarction (MI), characterized by a diminished flow of blood to the heart, presents the highest rate of morbidity and mortality among all other cardiovascular diseases. These fatal effects have triggered the need for early diagnosis of appropriate biomarkers so that countermeasures can be taken. Cardiac troponin, the central key element of muscle regulation and contraction, is the most specific biomarker for cardiac injury and is considered the “gold standard”. Due to its high specificity, the measurement of cardiac troponin levels has become the predominant indicator of MI. Various forms of diagnostic methods have been developed so far, including chemiluminescence, fluorescence immunoassay, enzyme-linked immunosorbent assay, surface plasmon resonance, electrical detection, and colorimetric protein assays. However, fluorescence-based immunoassays are considered fast, accurate and most sensitive of all in the determination of cardiac troponins post-MI. This review represents the strategies, methods and levels of detection involved in the reported fluorescence-based immunoassays for the detection of cardiac troponin I.

Key role of the CCR2-CCL2 axis in disease modification in a mouse model of tauopathy

BackgroundFor decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4+ T cells, including FOXP3+ regulatory CD4+ T cells.MethodsWe used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment.ResultsPerformance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect.ConclusionsThe CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, and the T-cell chemoattractant, Cxcl12.

A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 min. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.

Fluorescence Based on Surface Plasmon Coupled Emission for Ultrahigh Sensitivity Immunoassay of Cardiac Troponin I.

This work demonstrates the quantitative assay of cardiac Troponin I (cTnI), one of the key biomarkers for acute cardiovascular diseases (the leading cause of death worldwide) using the fluorescence-based sandwich immune reaction. Surface plasmon coupled emission (SPCE) produced by non-radiative coupling of dye molecules with surface plasmons being excitable via the reverse Kretschmann format is exploited for fluorescence-based sandwich immunoassay for quantitative detection of cTnI. The SPCE fluorescence chip utilizes the gold (2 nm)-silver (50 nm) bimetallic thin film, with which molecules of the dye Alexa 488 (conjugated with detection antibodies) make a near field coupling with the plasmonic film for SPCE. The experimental results find that the SPCE greatly improves the sensitivity via enhancing the fluorescence signal (up to 50-fold) while suppressing the photo-bleaching, permitting markedly enhanced signal-to-noise ratio. The limit of detection of 21.2 ag mL−1 (atto-gram mL−1) is obtained, the lowest ever reported to date amid those achieved by optical technologies such as luminescence and label-free optical sensing techniques. The features discovered such as ultrahigh sensitivity may prompt the presented technologies to be applied for early diagnosis of cTnI in blood, particularly for emergency medical centers overloaded with patients with acute myocardial infarction who would suffer from time-delayed diagnosis due to insufficient assay device sensitivity.

A highly sensitive immunofluorescence sensor based on bicolor upconversion and magnetic separation for simultaneous detection of fumonisin B1 and zearalenone.

Mycotoxins cause significant harm to human health, so it is imperative to develop a highly sensitive and easy-to-operate method for the detection of mycotoxins. Herein, a fluorescence-based magnetic separation immunoassay for simultaneous detection of mycotoxins fumonisin B1 and zearalenone is established. The method employed high fluorescent upconversion-nanoparticles(UCNPs) conjugated with biotinylated antigens as upconversion fluoroscent probes. Magnetic nanoparticles(MNPs) immobilized with monoclonal antibodies are used as immune-capture probes. Highly sensitive detection of FB1 and ZEN was achieved based on the luminescence properties of UCNPs and the separation effects of MNPs. The results showed a robust linear correlation between the enhanced fluorescence emission intensity and the logarithmic concentrations of FB1 and ZEN under the optimal conditions (R2(FB1) = 0.9965, R2(ZEN) = 0.9976), and the linear ranges were 0.05-5 ng mL-1. Furthermore, the limits of detection (LOD) were 0.016 ng mL-1 for FB1 and 0.012 ng mL-1 for ZEN. The standard addition method was used to determine the content of FB1 and ZEN in the samples to evaluate the accuracy of the process. The average recoveries were 89.48% to 113.69% and 85.97% to 113.82%, respectively. Compared with the other five mycotoxins, this method had high selectivity. It is expected that the multi-component simultaneous detection can be further realized by using the multicolor labeling characteristics of UCNPs.

Fluorescence-based quantitative platform for ultrasensitive food allergen detection: From immunoassays to DNA sensors.

Food allergies are global health issue with an increasing prevalence that affect food safety; hence, food allergen detection, labeling, and management are considered to be important priorities in the food industry. In this critical review, we provide a comprehensive overview of several fluorescence-based platforms based on different biorecognition ligands, such as antibodies, DNA, aptamers, and cells, for food allergen quantification. Traditional analytical methods are generally unsuitable for food manufacturers to accomplish the real-time identification of food allergens in food products. Therefore, it is important to develop simple, rapid, inexpensive, accurate, and sensitive methods to improve user accessibility. A fluorescence-based quantitative platform provides an excellent detection platform for food allergens because of its high sensitivity. This review summarizes the traditional antibody-based fluorescent techniques for food allergen detection, such as the time-resolved fluoroimmunoassay , immunofluorescence imaging, fluorescence enzyme-linked immune sorbent assay, flow injection fluoroimmunoassay, and fluorescence immunosensors. However, these methods suffer from disadvantages such as the significant rate of false-positive and false-negative results due to antibody cross-reactivity with nontarget food components in the complex food matrix and epitope degradation during food processing. Hence, different types of fluorescence-based immunoassays are suitable for standardization and quantification of allergens in fresh foods. In addition, we summarize new fluorescence-based quantitative platforms, including fluorescence genosensors, fluorescence cell sensors, and fluorescence aptamer sensors. With the advantages of high sensitivity and simple operation, fluorescence biosensors will have great potential in the future and could provide portable methods for multiallergen real-time detection in complex food systems.

Rhombic-like Al nanosupporter-based fluorescent immunochromatographic assay for the sensitive detection of tetracycline

Fluorescence-based lateral flow immunoassay (FLFIA) is a prospective technology in detecting tetracycline (TET). However, the existing fluorescent materials (FMs) often require cross-linkers to bind with antibodies (Abs) to form FLFIA FM-Ab probe, which complicates the modification process and may affect the fluorescence intensity of FMs. Herein, the rhombic-like Al nanosupporter (RAlNS) was prepared via a one-step hydrothermal method and used for designing fluorescence nanosupporter for FLFIA to detect TET in real samples. The RAlNS not only possessed excellent water stability, strong fluorescence intensity and favorable thermal stability, but also could adsorb Abs to obtain FMs-Ab probe by simple centrifugation. Under optimal conditions, the limit of detection (LOD) of RAlNS-FLFIA was 0.0516 ng/mL (n = 3), which was 18 times lower than traditional colloidal gold-based LFIA. Meanwhile, the proposed sensor also exhibited high specificity for TET against other antibiotics. Furthermore, the RAlNS-FLFIA was successfully applied to the detection of TET in beef, chicken, milk and honey samples, separately, which demonstrated a significant recovery of 80.40–112.05%. The novel RAlNS-FLFIA provided great potential for the detection of various analytes in food analysis.

Fluorescence-based immunoassay for the detection of Xanthomonas oryzae pv. oryzae in rice leaf

The identification of rice bacterial leaf blight disease requires a simple, rapid, highly sensitive, and quantitative approach that can be applied as an early detection monitoring tool in rice health. This paper highlights the development of a turn-off fluorescence-based immunoassay for the early detection of Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative bacterium that causes rice bacterial leaf blight disease. Antibodies against Xoo bacterial cells were produced as specific bio-recognition molecules and the conjugation of these antibodies with graphene quantum dots and gold nanoparticles was performed and characterized, respectively. The combination of both these bio-probes as a fluorescent donor and metal quencher led to changes in the fluorescence signal. The immunoreaction between AntiXoo-GQDs, Xoo cells, and AntiXoo-AuNPs in the immuno-aggregation complex led to the energy transfer in the turn-off fluorescence-based quenching system. The change in fluorescence intensity was proportional to the logarithm of Xoo cells in the range of 100–105 CFU mL−1. The limit of detection was achieved at 22 CFU mL−1 and the specificity test against other plant disease pathogens showed high specificity towards Xoo. The detection of Xoo in real plant samples was also performed in this study and demonstrated satisfactory results.