Detection Of Specific Proteins Research Articles

Multiplexed direct detection of barcoded protein reporters on a nanopore array.

Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila.

In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM. However, this technique still requires the generation of new molecular constructions. Thus, we decided to develop a versatile method that would take advantage of the numerous GFP-tagged proteins already existing and create a tool combining a nanobody anti-GFP (GBP) with APEX. This GBP-APEX tool allows a simple and efficient detection of any GFP fusion proteins without the needs of specific antibodies nor the generation of additional constructions. We have shown the feasibility and efficiency of this method to detect various proteins in Drosophila ovarian follicles such as nuclear proteins, proteins associated with endocytic vesicles, plasma membranes or nuclear envelopes. Lastly, we expressed this tool in Drosophila with the UAS/GAL4 system that enables spatiotemporal control of the protein detection.

ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism of ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.

New Impedimetric Sandwich Immunosensor for Ultrasensitive and Highly Specific Detection of Spike Receptor Binding Domain Protein of SARS-CoV-2.

An impedance sensing platform-combined conducting nanocomposite layer was fabricated to develop an effective and rapid method for detection of coronavirus infection (COVID-19) specific spike receptor binding domain (RBD) protein, a precious antigen marker of COVID-19 disease. Coronavirus infection has spread globally and swiftly with major impacts on health, economy, and quality of life of communities. Fast and reliable detection of COVID-19 is a very significant issue for the effective treatment of this bad illness. For this aim, first, an Epoxy functional group-substituted thiophene monomer was synthesized and electrodeposited on a single-use indium tin oxide (ITO) platform in the presence of acetylene black by employing a cyclic voltammetry technique; thus, a conducting nanocomposite (C-NC) layer with high conductivity was obtained. This composite was electrodeposited for the first time on the ITO surface to generate a facile and cost-effective impedimetric biosensor. In addition, this composite provided proper attachment points for antibody binding and also supported the biosensor construction. The immuno-specific biointeractions between anti-RBD and RBD proteins hampered the electron transfer between the ITO substrate surface and electrolyte, and this reaction caused variations in impedance signals, and these signals were proportional to the immobilized RBD antigen amounts. The as-prepared immunosensor showed a wide linear dynamic range (0.0012-120 pg/mL), an ultra-low detection limit of 0.58 fg/mL with added superiorities of great selectivity, suitable repeatability, multiple reusability, and excellent reproducibility.

Protein Labeling and Crosslinking by Covalent Aptamers.

We developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated through introduction of a proximity-driven electrophile at specific nucleotide sites. Using thrombin as a proof-of-concept, we demonstrate that covalent aptamers can selectively transfer a variety of functional handles and/or irreversibly crosslink to the target protein. This approach offers broad programmability and high target specificity. Furthermore, it addresses issues common to aptamers such as instability towards endogenous nucleases and residence times during target engagement. Covalent aptamers are new tools that enable specific protein modification and sensitive protein detection. Moreover, they provide prolonged, nuclease-resistant enzyme inhibition.

Protein Labeling and Crosslinking by Covalent Aptamers

AbstractWe developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated through introduction of a proximity‐driven electrophile at specific nucleotide sites. Using thrombin as a proof‐of‐concept, we demonstrate that covalent aptamers can selectively transfer a variety of functional handles and/or irreversibly crosslink to the target protein. This approach offers broad programmability and high target specificity. Furthermore, it addresses issues common to aptamers such as instability towards endogenous nucleases and residence times during target engagement. Covalent aptamers are new tools that enable specific protein modification and sensitive protein detection. Moreover, they provide prolonged, nuclease‐resistant enzyme inhibition.

A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.

Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-β-d-thiogalactoside (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti‐sFGL1 mAb followed by detection with anti‐sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is 35 pg/ml with recombinant sFLG1. Besides, it does not show cross‐reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.

Immobilization of Nanobodies with Vapor-Deposited Polymer Encapsulation for Robust Biosensors.

To produce next-generation, shelf-stable biosensors for point-of-care diagnostics, a combination of rugged biomolecular recognition elements, efficient encapsulants, and innocuous deposition approaches is needed. Furthermore, to ensure that the sensitivity and specificity that are inherent to biological recognition elements are maintained in solid-state biosensing systems, site-specific immobilization chemistries must be invoked such that the function of the biomolecule remains unperturbed. In this work, we present a widely applicable strategy to develop robust solid-state biosensors using emergent nanobody (Nb) recognition elements coupled with a vapor-deposited polymer encapsulation layer. As compared to conventional immunoglobulin G antibodies, Nbs are smaller (12-15 kDa as opposed to ~150 kDa), have higher thermal stability and pH tolerance, boast greater ease of recombinant production, and are capable of binding antigens with high affinity and specificity. Photoinitiated chemical vapor deposition affords thin, protective polymer barrier layers over immobilized Nb arrays that allow for retention of Nb activity and specificity after both storage under ambient conditions and complete desiccation. Most importantly, we also demonstrate that vapor-deposited polymer encapsulation of Nb arrays enables specific detection of target proteins in complex heterogeneous samples, such as unpurified cell lysate, which is otherwise challenging to achieve with bare Nb arrays.

Development of Monoclonal Antibodies against Pea Globulins for Multiplex Assays Targeting Legume Proteins.

Legume proteins are widely used as food ingredients, but only some (soybean, lupin, and peanut) must be declared under consumer safety regulations to protect allergy sufferers. It is not yet mandatory to declare pea proteins as allergens even though they are predicted to be allergenic based on cross-reactivity in sensitized people. The processing of legume proteins can modify their allergenic properties and hence the need for specific and precise methods for the detection of all major legume allergens. There are many commercially available tests for known food allergens but not for ingredients that are yet to be classified as allergenic. We therefore generated sets of pea-specific antibodies targeting globulins to be used in a multiplex assay for the simultaneous detection of soybean, lupin, peanut, and pea proteins. We focused on the 7S globulin family, which is the least conserved among the four legumes, allowing the specific detection of proteins from each species. Having confirmed the specificity and sensitivity of the multiplex assay, we evaluated different processing steps for proteins rich in pea globulins to demonstrate the impact of food processing on antibody binding. Our sensitive multiplex assay provides a fast and reliable method for the specific detection of soybean, lupin, peanut, and pea allergens and is therefore ideal for food safety and authenticity testing applications.

Randomly positioned gold nanoparticles as fluorescence enhancers in apta-immunosensor for malaria test

A plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays.Graphical abstract

Fluorescent Signaling of Molecularly Imprinted Nanogels Prepared via Postimprinting Modifications for Specific Protein Detection

Previous reports have stated the advantages of postimprinting modification (PIM) to develop multifunctional molecularly imprinted polymers (MIPs). However, existing technologies have only been applied on bulk‐ and film‐based MIPs. Herein, the fluorescent signaling of nanogels (NGs) for protein detection is demonstrated. The NGs are prepared by molecular imprinting and PIM. A bivalent functional monomer, 4‐[2‐(N‐methacrylamido)ethylaminomethyl] benzoic acid (FM1), comprising a benzoic acid moiety for interaction with proteins and secondary amine group for incorporating fluorescent reporter molecules is used. Human serum albumin (HSA), a marker protein for human health, serves as the model protein. HSA‐imprinted NGs (MIP‐NGs) are prepared by emulsifier‐free precipitation polymerization of FM1, N‐isopropylacrylamide, 2‐methacryloxyethyl phosphorylcholine, and N,N′‐methylenebis acrylamide in the presence of HSA. After the removal of HSA, a fluorescent dye (ATTO 647 N) is treated with amino groups on FM1 residue as the PIM; this yields fluorescent‐signaling MIP‐NGs (FL‐MIP‐NGs) of ≈20 nm in diameter. The FL‐MIP‐NG‐based sensing system detects HSA in a real sample by selectively reading out the HSA‐binding events as high‐affinity fluorescence changes (Kd = 2.5 × 10−9 M). The proposed MIP‐NGs functionalized via PIMs serve as advanced functional materials for sensing biorelated compounds.

A Proteomic- and Bioinformatic-Based Identification of Specific Allergens from Edible Insects: Probes for Future Detection as Food Ingredients.

The increasing development of edible insect flours as alternative sources of proteins added to food and feed products for improving their nutritional value, necessitates an accurate evaluation of their possible adverse side-effects, especially for individuals suffering from food allergies. Using a proteomic- and bioinformatic-based approach, the diversity of proteins occurring in currently consumed edible insects such as silkworm (Bombyx mori), cricket (Acheta domesticus), African migratory locust (Locusta migratoria), yellow mealworm (Tenebrio molitor), red palm weevil (Rhynchophorus ferrugineus), and giant milworm beetle (Zophobas atratus), was investigated. Most of them consist of phylogenetically-related protein allergens widely distributed in the different groups of arthropods (mites, insects, crustaceans) and mollusks. However, a few proteins belonging to discrete protein families including the chemosensory protein, hexamerin, and the odorant-binding protein, emerged as proteins highly specific for edible insects. To a lesser extent, other proteins such as apolipophorin III, the larval cuticle protein, and the receptor for activated protein kinase, also exhibited a rather good specificity for edible insects. These proteins, that are apparently missing or much less represented in other groups of arthropods, mollusks and nematods, share well conserved amino acid sequences and very similar three-dimensional structures. Owing to their ability to trigger allergic responses in sensitized people, they should be used as probes for the specific detection of insect proteins as food ingredients in various food products and thus, to assess their food safety, especially for people allergic to edible insects.

Development of a target identification approach using native mass spectrometry

A key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

Еffect of different breeds on the protein profile in ram seminal plasma

In this study the individual profiles of seminal plasma proteins (SPP) in rams of three breeds – Merino, Pleven Blackhead and Ile de France - were analysed. The study was carried out with three rams at 3, 6 and 10 years of age, grown and fed under similar conditions. Eighteen ejaculates (6 ejaculates from each ram) were evaluated by Sperm Class Analyzer. The total SPP concentration was measured spectrophotometrically. The separation and characterisation of SPP was performed by HPLC and one dimensional polyacrylamide gel electrophoresis (SDS-PAGE). There were no significant differences between the characteristics of ejaculates and the main kinematic parameters of the sperm in the breeds studied. Chromatograms showed specific profiles with 9, 10 and 11 protein peaks for Merino, Pleven Blackhead and Ile de France breeds, respectively. The total SPP concentration was the highest in the Pleven Blackhead breed and the lowest in Ile de France breed. The major parts of SPP in the three breeds were identical. The seminal plasma of Merino breed contained proteins with molecular mass of 30.3 kDa, 15.7 kDa and 15.2 kDa that were not present in the other two breeds. In the Ile de France and Pleven Blackhead samples only, two proteins with molecular masses of 39.7 kDa and 21.1 kDa, were observed. In conclusion, the detection of specific proteins can be used as a biological marker for sheep breed identification.

Simple Western: Bringing the Western Blot into the Twenty-First Century.

The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.

Evaluation of A Rapid Visual Pregnancy (Bio- RPD) test for detection of Pregnancy Specific Protein B (PSPB) in Cow Serum

The early pregnancy diagnosis allows optimizing production and timely management correction, with a greater reproductive output of livestock. The BioPRYN Rapid Visual Pregnancy Test® from Bio tracking USA consists of an ELISA for visual reading which does not require the use of readers in the laboratory, with satisfactory pregnancy diagnoses at 28 days of pregnancy in heifers and 30 days in cows. Therefore, the aim of this study is to determine the accuracy of this rapid test (Bio-RPD) based on visual observation of color change and Optical Density (OD) using BioPRYN Flex ELISA. For this purpose, 92 serum samples from cows were tested. The numbers of positive pregnant samples obtained with Biorapid (Bio-RPD) were 63 (68.5 %) while with BioPRYN Flex ELISA was 66 (71.7%). The numbers of negative not pregnant samples were 25 (27.2%) with Bio-RPD while they were 26 (28.2%0) with BioPRYN ELISA. Three samples were recorded as recheck with Bio-RPD and they were pregnant with BioPRYN Flex. Only one sample was identified as pregnant by Bio-RPD (1.1%) while it is not pregnant by BioPRYN Flex ELISA (false positive). Test results were showing that accuracy of the BioPRYN Visual Pregnancy test kit with sensitivity of 99% compared to BioPRYN ELISA Pregnancy test kit. In conclusion, we recommend that the Bio-RPD visual test could be used as a rapid accurate tool for pregnancy diagnosis on farms which has no laboratory ELISA equipment or trans rectal ultrasonography.

Japanese Food Allergy-Labeling System and Comparison with the International Experience; Detection and Thresholds.

In the Japanese allergy-labeling system, food labeling is mandated for 7 specific ingredients (egg, cow’s milk, wheat, buckwheat, peanut, shrimp, and crab) and recommended for 21 food ingredients in reference to case numbers of actual illness and the degree of seriousness. To monitor the validity of the labeling system, official methods for the detection of specific ingredient proteins in processed foods were developed. The official methods consist of ELISA methods for screening, and western blot methods for egg and milk, and PCR methods for wheat, buckwheat, peanut, shrimp/prawn, and crab as confirmation tests. The official methods consist of ELISA methods for screening, and western blot methods for egg and milk, and PCR methods for wheat, buckwheat, peanut, shrimp/prawn, and crab as confirmation tests. Threshold amounts (a few mg/kg) for labeling were set based on the approach of the analytical detections. Any foods containing protein allergens should be labeled if these contain allergens at greater than 10 ppm (mg/kg). Validation protocol criteria were established to standardize the Japanese official method. Food Safety Commission of Japan conducted a risk assessment of egg as a specific ingredient and judged that current labeling system for foods containing allergens is generally appropriate for “eggs”. In the future, it is important to accumulate necessary scientific knowledge in order to carry out food health impact assessment including further refinement. The Japanese experience and knowledge of food allergy-labeling system would contribute to harmonize international labeling guidelines to protect allergic consumers globally.

Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF.

Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.

Development of novel monoclonal antibodies and immunoassays for sensitive and specific detection of SULF1 endosulfatase

BackgroundCell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. MethodsWe have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. ResultsThis study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. ConclusionsThis study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. General significanceNew tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application.

The Latest Achievements in the Construction of Influenza Virus Detection Aptasensors.

Aptamers are short fragments of nucleic acids, DNA or RNA that have the ability to bind selected proteins with high specificity and affinity. These properties allow them to be used as an element of biosensors for the detection of specific proteins, including viral ones, which makes it possible to design valuable diagnostic tools. The influenza virus causes a huge number of human and animal deaths worldwide every year, and contributes to remarkable economic losses. In addition, in 2020, a new threat appeared—the SARS-Cov-2 pandemic. Both disease entities, especially in the initial stage of infection, are almost identical in terms of signs and symptoms. Therefore, a diagnostic solution is needed that will allow distinguishing between both pathogens, with high sensitivity and specificity; it should be cheap, quick and possible to use in the field, for example, in a doctor’s office. All the mentioned properties are met by aptasensors in which the detection elements are specific aptamers. We present here the latest developments in the construction of various types of aptasensors for the detection of influenza virus. Aptasensor operation is based on the measurement of changes in electric impedance, fluorescence or electric signal (impedimetric, fluorescence and electrochemical aptasensors, respectively); it allows both qualitative and quantitative determinations. The particularly high advancement for detecting of influenza virus concerns impedimetric aptasensors.