Antigen-antibody Reaction Research Articles

Rapid, high-sensitivity detection of biomolecules using dual-comb biosensing

Rapid, sensitive detection of biomolecules is important for biosensing of infectious pathogens as well as biomarkers and pollutants. For example, biosensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still strongly required for the fight against coronavirus disease 2019 (COVID-19) pandemic. Here, we aim to achieve the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein antigen by enhancing the performance of optical biosensing based on optical frequency combs (OFC). The virus-concentration-dependent optical spectrum shift produced by antigen–antibody interactions is transformed into a photonic radio-frequency (RF) shift by a frequency conversion between the optical and RF regions in the OFC, facilitating rapid and sensitive detection with well-established electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation with a dual-comb configuration enables the very small change in the virus-concentration-dependent signal to be extracted from the large, variable background signal caused by temperature disturbance. The achieved performance of dual-comb biosensing will greatly enhance the applicability of biosensors to viruses, biomarkers, environmental hormones, and so on.

Development of a Multiplex HIV/TB Diagnostic Assay Based on the Microarray Technology.

Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen-antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.

Unleashing the power of complement activation: unraveling renal damage in human anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (GBM) disease is a rare but life-threatening autoimmune disorder characterized by rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. Renal biopsies of anti-GBM patients predominantly show linear deposition of IgG and complement component 3 (C3), indicating a close association between antigen-antibody reactions and subsequent complement activation in the pathogenesis of the disease. All three major pathways of complement activation, including the classical, lectin, and alternative pathways, are involved in human anti-GBM disease. Several complement factors, such as C3, C5b-9, and factor B, show a positive correlation with the severity of the renal injury and act as risk factors for renal outcomes. Furthermore, compared to patients with single positivity for anti-GBM antibodies, individuals who are double-seropositive for anti-neutrophil cytoplasmic antibody (ANCA) and anti-GBM antibodies exhibit a unique clinical phenotype that lies between ANCA-associated vasculitis (AAV) and anti-GBM disease. Complement activation may serve as a potential "bridge" for triggering both AAV and anti-GBM conditions. The aim of this article is to provide a comprehensive review of the latest clinical evidence regarding the role of complement activation in anti-GBM disease. Furthermore, potential therapeutic strategies targeting complement components and associated precautions are discussed, to establish a theoretical basis for complement-targeted therapies.

Determination of the epitopic peptides of fig mosaic virus and the single-chain variable fragment antibody by mass spectrometry

The study of antibody-antigen interactions, through epitope mapping, enhances our understanding of antibody neutralization and antigenic determinant recognition. Epitope mapping, employing monoclonal antibodies and mass spectrometry, has emerged as a rapid and precise method to investigate viral antigenic determinants. In this report, we propose an approach to improve the accuracy of epitopic peptide interaction rate recognition. To achieve this, we investigated the interaction between the nucleocapsid protein of fig mosaic virus (FMV-NP) and single-chain variable fragment antibodies (scFv-Ab). These scFv-Ab maintain high specificity similar to whole monoclonal antibodies, but they are smaller in size. We coupled this with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The experimental design involved using two different enzymes to digest FMV-NP separately. The resulting peptides were then incubated separately with the desired scFv-Ab at different incubation times and antibody concentrations. This allowed us to monitor the relative rate of epitopic peptide interaction with the antibody. The results demonstrated that, at a 1:1 ratio and after 2 h of interaction, the residues 122–136, 148–157, and 265–276 exhibited high-rate epitopic peptide binding, with reductions in peak intensity of 78%, 21%, and 22%, respectively. Conversely, the residues 250–264 showed low-rate binding, with a 15% reduction in peak intensity. This epitope mapping approach, utilizing scFv-Ab, two different enzymes, and various incubation times, offers a precise and dependable analysis for monitoring and recognizing the binding kinetics of antigenic determinants. Furthermore, this method can be applied to study any kind of antigens.

Unlocking the Potential of Molecularly Imprinted Polydopamine in Sensing Applications.

Molecularly imprinted polymers (MIPs) are synthetic receptors that mimic the specificity of biological antibody-antigen interactions. By using a "lock and key" process, MIPs selectively bind to target molecules that were used as templates during polymerization. While MIPs are typically prepared using conventional monomers, such as methacrylic acid and acrylamide, contemporary advancements have pivoted towards the functional potential of dopamine as a novel monomer. The overreaching goal of the proposed review is to fully unlock the potential of molecularly imprinted polydopamine (MIPda) within the realm of cutting-edge sensing applications. This review embarks by shedding light on the intricate tapestry of materials harnessed in the meticulous crafting of MIPda, endowing them with tailored properties. Moreover, we will cover the diverse sensing applications of MIPda, including its use in the detection of ions, small molecules, epitopes, proteins, viruses, and bacteria. In addition, the main synthesis methods of MIPda, including self-polymerization and electropolymerization, will be thoroughly examined. Finally, we will examine the challenges and drawbacks associated with this research field, as well as the prospects for future developments. In its entirety, this review stands as a resolute guiding compass, illuminating the path for researchers and connoisseurs alike.

Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry.

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

Antibody designing against IIIabc junction (JIIIabc) of HCV IRES through affinity maturation; RNA-Antibody docking and interaction analysis.

Hepatitis C virus is a single-stranded RNA based virus which can cause chronic HCV and hepatocellular carcinoma. HCV genotype 3a has relatively higher rate of fibrosis progression, prevalence of steatosis and incidence of HCC. Despite HCVs variation in genomic sequence, the 5' untranslated region containing internal ribosome entry site (IRES) is highly conserved among all genotypes. It is responsible for translation and initiation of the viral protein. In present study, IRES was targeted by designing variants of reported antigen binding fragment (Fab) through affinity maturation approach. Affinity maturation strategy allowed the rational antibody designing with better biophysical properties and antibody-antigen binding interactions. Complementarity determining regions of reported Fab (wild type) were assessed and docked with IRES. Best generated model of Fab was selected and subjected to alanine scanning Three sets of insilico mutations for variants (V) designing were selected; single (1-71), double (a-j) and triple (I-X). Redocking of IRES-Fab variants consequently enabled the discovery of three variants exhibiting better docking score as compared to the wild type Fab. V1, V39 and V4 exhibited docking scores of -446.51, -446.52 and-446.29 kcal/mol respectively which is better as compared to the wild type Fab that exhibited the docking score of -351.23 kcal/mol. Variants exhibiting better docking score were screened for aggregation propensity by assessing the aggregation prone regions in Fab structure. Total A3D scores of wild type Fab, V1, V4 and V39 were predicted as -315.325, -312.727, -316.967 and -317.545 respectively. It is manifested that solubility of V4 and V39 is comparable to wild type Fab. In future, development and invitro assessment of these promising Fab HCV3 variants is aimed.

Optimization of therapeutic antibodies for reduced self-association and non-specific binding via interpretable machine learning.

Antibody development, delivery, and efficacy are influenced by antibody-antigen affinity interactions, off-target interactions that reduce antibody bioavailability and pharmacokinetics, and repulsive self-interactions that increase the stability of concentrated antibody formulations and reduce their corresponding viscosity. Yet identifying antibody variants with optimal combinations of these three types of interactions is challenging. Here we show that interpretable machine-learning classifiers, leveraging antibody structural features descriptive of their variable regions and trained on experimental data for a panel of 80 clinical-stage monoclonal antibodies, can identify antibodies with optimal combinations of low off-target binding in a common physiological-solution condition and low self-association in a common antibody-formulation condition. For three clinical-stage antibodies with suboptimal combinations of off-target binding and self-association, the classifiers predicted variable-region mutations that optimized non-affinity interactions while maintaining high-affinity antibody-antigen interactions. Interpretable machine-learning models may facilitate the optimization of antibody candidates for therapeutic applications.

Antibody Cross-Reactivity in Auto-Immune Diseases.

Autoimmunity is defined by the presence of antibodies and/or T cells directed against self-components. Although of unknown etiology, autoimmunity commonly is associated with environmental factors such as infections, which have been reported to increase the risk of developing autoimmune diseases. Occasionally, similarities between infectious non-self and self-tissue antigens may contribute to immunological cross-reactivity in autoimmune diseases. These reactions may be interpreted as molecular mimicry, which describes cross-reactivity between foreign pathogens and self-antigens that have been reported to cause tissue damage and to contribute to the development of autoimmunity. By focusing on the nature of antibodies, cross-reactivity in general, and antibody-antigen interactions, this review aims to characterize the nature of potential cross-reactive immune reactions between infectious non-self and self-tissue antigens which may be associated with autoimmunity but may not actually be the cause of disease onset.

From Cereal Grains to Immunochemistry-What Role Have Antibodies Played in the History of the Home Pregnancy Test.

Today, the home pregnancy test is the most frequently performed laboratory test for self-diagnosis (home diagnostic test). It is also the first laboratory test that has been adapted for self-use at home. This is probably because women have always wanted to know the answer to the question: "Am I pregnant or not?" and always preferred to know the answer to this question intimately and in a discreet way. The history of the pregnancy test is also an interesting example of how the discovery of antibodies and the development of in vitro diagnostic methods based on the antigen-antibody reaction were important for the development of laboratory and clinical diagnostics. Immunodiagnostic techniques (based on the antigen-antibody reaction) are currently the basis of modern specialist laboratory diagnostics, which is essential in clinical diagnosis. The history of the pregnancy test is an interesting one and dates back to ancient times. A pregnancy test is defined as a procedure intended to reveal the presence or absence of pregnancy. Nowadays, every pregnancy test is based on the detection of human chorionic gonadotropin (hCG) in urine or blood. Human chorionic gonadotropin is secreted by the placenta right after a fertilized egg cell implants in the uterus and can be detected in both the urine and blood of pregnant women. Urine pregnancy tests are convenient for self-use at home. Blood tests are performed in medical laboratories. Specialized laboratory methods not only detect hCG but also determine the concentration of this hormone. However, both of these methods are highly accurate and common. Throughout the ages, many different methods were used to detect pregnancy at the earliest stage. Grain, wine, and various small animals were used as research tools. These were both long-term and often unreliable; most were based on folk beliefs and superstitions. Animal pregnancy tests were the first biological tests used in this field. This was a significant advance in the accurate detection of relatively early pregnancy. Animal tests in modern times are considered cruel and inhumane, no matter how reliable their results can be. Their place is now taken by much more specific, more sensitive, and definitely more ethical immunochemical tests. The pregnancy test and the methods to find out whether a woman is pregnant have gone through massive transformations, from bioassays using plants to bioassays on animals to advanced immunochemical techniques and biosensors. Modern pregnancy tests are not invasive and are very sensitive. Nowadays, it takes only about 3 min to know the answer to the question: "Am I pregnant or not?". However, it was not always as simple as it is today. This manuscript aims to show the important role played by antibodies in the development of laboratory and clinical diagnostics in the example of the interesting history of the pregnancy test.

Adsorption and Electropolymerization of p-Aminophenol Reduces Reproducibility of Electrochemical Immunoassays

Electrochemical immunoassays are a class of biosensors that achieve high specificity, low detection limits, and low cost attributed to strong antibody-antigen interactions and inexpensive electrochemical instrumentation. Electrochemical assays typically use an enzyme/substrate pair to enable electrochemical signal production. p-Aminophenyl phosphate is a commonly used enzymatic substrate which is hydrolyzed into p-aminophenol (PAP)by alkaline phosphatase. PAP exhibits low oxidation potential and electrochemical reversibility, making it an appealing candidate for assay design. The electrochemical oxidative signal from PAP is increased by its adsorption to the electrode. This is further confirmed by the prevention of PAP adsorption via self-assembled monolayers on gold electrodes reducing PAP’s oxidative current. On bare screen-printed electrodes (SPE), PAP adsorption results in oxidative current variability depending on the electroactive surface area of the working electrode. This variability could not be remedied by cleaning and reusing the same gold SPE. However, adsorption of PAP displays concentration dependence, as the decrease ofthe PAP concentration to 3.8 μM greatly improved the consistency of the measurements. PAP use is also limited in continuous monitoring applications as multiple PAP oxidations on the same electrode caused polymerization. The results from this study suggest that the use of PAP production in immunoassays with SPEs must be fine-tuned and electrodes must be cleaned or disposed of between measurements.

Transitioning from Supramolecular Chemistry to Molecularly Imprinted Polymers in Chemical Sensing.

This perspective article focuses on the overwhelming significance of molecular recognition in biological processes and its emulation in synthetic molecules and polymers for chemical sensing. The historical journey, from early investigations into enzyme catalysis and antibody-antigen interactions to Nobel Prize-winning breakthroughs in supramolecular chemistry, emphasizes the development of tailored molecular recognition materials. The discovery of supramolecular chemistry and molecular imprinting, as a versatile method for mimicking biological recognition, is discussed. The ability of supramolecular structures to develop selective host-guest interactions and the flexible design of molecularly imprinted polymers (MIPs) are highlighted, discussing their applications in chemical sensing. MIPs, mimicking the selectivity of natural receptors, offer advantages like rapid synthesis and cost-effectiveness. Finally, addressing major challenges in the field, this article summarizes the advancement of molecular recognition-based systems for chemical sensing and their transformative potential.

Semiconductor-based biosensor exploiting competitive adsorption with charged pseudo-target molecules for monitoring 5-fluorouracil concentration in human serum

Rapid and simple monitoring of anticancer drug concentrations in blood is important for improving the efficacy of chemotherapeutic cancer treatment. In this study, we demonstrated the detection of 5-fluorouracil (5-FU) in human serum using a field-effect transistor (FET) biosensor with competitive adsorption between uncharged anticancer drugs and charged pseudo-targets. The target 5-FU and 5-FU-modified bovine serum albumin (BSA/5-FU) were competitively adsorbed on the FET sensor surface using antigen-antibody reaction with antigen-binding fragment (Fab) receptor. FET responses to the target 5-FU concentrations were obtained via changes in the amount of negatively charged BSA/5-FU captured by Fab molecules. In addition, the influence of serum components could be suppressed by diluting the samples with a nonionic surfactant solution. As a result, the FET biosensing system was capable of quantitatively detecting 5-FU concentrations in serum. Therefore, the semiconductor-based sensing system could enable the adjustment of anticancer drug dosages and potentially lead to improvements in anticancer drug therapy.

Parameter estimation and identifiability analysis for a bivalent analyte model of monoclonal antibody-antigen binding

Surface plasmon resonance (SPR) is an extensively used technique to characterize antigen-antibody interactions. Affinity measurements by SPR typically involve testing the binding of antigen in solution to monoclonal antibodies (mAbs) immobilized on a chip and fitting the kinetics data using 1:1 Langmuir binding model to derive rate constants. However, when it is necessary to immobilize antigens instead of the mAbs, a bivalent analyte (1:2) binding model is required for kinetics analysis. This model is lacking in data analysis packages associated with high throughput SPR instruments and the packages containing this model do not explore multiple local minima and parameter identifiability issues that are common in non-linear optimization. Therefore, we developed a method to use a system of ordinary differential equations for analyzing 1:2 binding kinetics data. Salient features of this method include a grid search on parameter initialization and a profile likelihood approach to determine parameter identifiability. Using this method we found a non-identifiable parameter in data set collected under the standard experimental design. A simulation-guided improved experimental design led to reliable estimation of all rate constants. The method and approach developed here for analyzing 1:2 binding kinetics data will be valuable for expeditious therapeutic antibody discovery research.

Advances, limitations, and considerations on the use of vibrational spectroscopy towards the development of management decision tools in food safety.

Food safety and food security are two of the main concerns for the modern food manufacturing industry. Disruptions in the food supply and value chains have created the need to develop agile screening tools that will allow the detection of food pathogens, spoilage microorganisms, microbial contaminants, toxins, herbicides, and pesticides in agricultural commodities, natural products, and food ingredients. Most of the current routine analytical methods used to detect and identify microorganisms, herbicides, and pesticides in food ingredients and products are based on the use of reliable and robust immunological, microbiological, and biochemical techniques (e.g. antigen-antibody interactions, extraction and analysis of DNA) and chemical methods (e.g. chromatography). However, the food manufacturing industries are demanding agile and affordable analytical methods. The objective of this review is to highlight the advantages and limitations of the use of vibrational spectroscopy combined with chemometrics as proxy to evaluate and quantify herbicides, pesticides, and toxins in foods.

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

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

An Ultrasensitive Norfentanyl Sensor Based on a Carbon Nanotube-Based Field-Effect Transistor for the Detection of Fentanyl Exposure.

The opioid crisis is a worldwide public health crisis that has affected millions of people. In recent years, synthetic opioids, primarily illicit fentanyl, have become the primary driver of overdose deaths. There is a great need for a highly sensitive, portable, and inexpensive analytical tool that can quickly indicate the presence and relative threat of fentanyl. In this work, we develop a semiconductor enriched (sc-) single-walled carbon nanotube (SWCNT)-based field-effect transistor (FET) biosensor functionalized with norfentanyl antibodies for the sensitive detection of norfentanyl, the primary inactive metabolite of fentanyl, in urine samples. Different sensor configurations were explored in order to obtain the most optimized sensing results. Moreover, by employing the "reduced" antibody, we achieved orientated immobilization of the norfentanyl antibody and thus brought the antigen-antibody interaction closer to the sensor surface, further improving the sensitivity. The reported norfentanyl biosensors have a limit of detection in the fg/mL region in both calibration samples and synthetic urine samples, showing ultrasensitivity and high reliability.

Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity.

Conventional serum antibody titer, which expresses antibody level, does not provide antigen binding avidity of the variable region of the antibody, which is essential for the defense response to infection. Here, we quantified anti-SARS-CoV-2 antibody binding avidity to the receptor-binding domain (RBD) by competitive binding-inhibition activity (IC50) between SARS-CoV-2 S1 antigen immobilized on the DCP microarray and various RBD doses added to serum and expressed as 1/IC50 nM. The binding avidity analyzed under equilibrium conditions of antigen-antibody binding reaction is different from the avidity index measured with the chaotropic agent, such as urea, under nonequilibrium and short-time conditions. Quantitative determination of the infection-protection potential of antibodies was assessed by ABAT (antigen binding avidity antibody titer), which was calculated by the quantity (level) × quality (binding avidity) of antibodies. The binding avidity correlated strongly (r = 0.811) with cell-based virus-neutralizing activity. Maturation of the protective antibody induced by repeated vaccinations or SARS-CoV-2 infection was classified into three categories of ABAT, such as an initial, low, and high ABAT. Antibody maturity correlated with the clinical severity of COVID-19. Once a mature high binding avidity was achieved, it was maintained for at least 6-8 months regardless of the subsequent change in the antibody levels.

The Method and Study of Detecting Phenanthrene in Seawater Based on a Carbon Nanotube-Chitosan Oligosaccharide Modified Electrode Immunosensor.

Phenanthrene (PHE), as a structurally simple, tricyclic, polycyclic aromatic hydrocarbon (PAHs), is widely present in marine environments and organisms, with serious ecological and health impacts. It is crucial to study fast and simple high-sensitivity detection methods for phenanthrene in seawater for the environment and the human body. In this paper, a immunosensor was prepared by using a multi-wall carbon nanotube (MWCNTs)-chitosan oligosaccharide (COS) nanocomposite membrane loaded with phenanthrene antibody. The principle was based on the antibody-antigen reaction in the immune reaction, using the strong electron transfer ability of multi-walled carbon nanotubes, coupled with chitosan oligosaccharides with an excellent film formation and biocompatibility, to amplify the detection signal. The content of the phenanthrene in seawater was studied via differential pulse voltammetry (DPV) using a potassium ferricyanide system as a redox probe. The antibody concentration, pH value, and probe concentration were optimized. Under the optimal experimental conditions, the response peak current of the phenanthrene was inversely proportional to the concentration of phenanthrene, in the range from 0.5 ng·mL-1 to 80 ng·mL-1, and the detection limit was 0.30 ng·mL-1. The immune sensor was successfully applied to the detection of phenanthrene in marine water, with a recovery rate of 96.1~101.5%, and provided a stable, sensitive, and accurate method for the real-time monitoring of marine environments.

Performance Optimization of a Microfluidic Virus Detection Cartridge: A Numerical and Experimental Study.

Detection and imaging of viruses in a complex solution is particularly significant for virology and requires a comprehensive understanding of biosensors. While lab-on-a-chip systems are used in virus detection as biosensors, analysis and optimization of these systems are especially challenging due to the size of the system to be used in the certain application. The system of interest for virus detection is required to be cost efficient and is also needed to be able to easily operable with a simple setup. Moreover, the detailed analysis of these microfluidic systems should be made with precision in order to predict the capabilities and the efficiency of the system accurately. This paper reports on the use of a common commercial computational fluid dynamics (cfd) software for the analysis of a microfluidic lab-on-a-chip virus detection cartridge. This study evaluates the problems commonly encountered during microfluidic applications of cfd softwares particularly in the area of reaction modeling of the antigen-antibody interaction. cfd analysis is later validated and combined with experiments to optimize the amount of dilute solution used in the tests. Thereafter, the geometry of the microchannel is also optimized and optimal test conditions are set for a cost efficient and effective virus detection kit using light microscopy.