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The studies on the influence of organic co-solvents on the structure and function of antibodies are of key interest, especially in view of antibodies broad use as recognizing elements in different analytical systems. Here we studied the effect of co-solvent 2,2,2-trifluoroethanol (TFE) on the ability of anti-ovalbumin monoclonal antibodies to interact with its specific antigen. Antibody affinity to antigen and the rate constants of antibody binding to immobilized antigen were analyzed. Changes in antibody reactivity with incubation time which depended on TFE concentration and temperature were revealed. When treatment of antibodies with TFE was carried out at 0°C, we observed nonlinear, non-monotonous changes of antibody reactivity with initial fast decrease and substantial increase as incubation continued that may be related to the loss of antigen binding reactivity by some part of antibodies at the start but its restoration when the incubation proceeds.

The newly emerged SARS‐CoV‐2 causing coronavirus disease (COVID‐19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2′ site, by the TMPRSS2 protease. S2′ cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2′ site to be conserved among all the SARS‐CoV‐2 variants, we chose an S2′ epitope encompassing the S2′ cleavage site and generated single‐chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2′ epitope was determined. Incidentally, S2′ epitope in the scFv bound structure adopts an alpha‐helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody‐S2′ epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2′ cleavage competitively. PDB Code(s): 7YUE; [ABSTRACT FROM AUTHOR]

Antibodies represent key effectors of the adaptive immune system. The specificity of antibodies is an established hallmark of the immune response. However, a certain proportion of antibodies exhibit limited promiscuity or multireactivity. Germline antibodies display plasticity which imparts multispecificity to enhance the antibody repertoire. Surprisingly, even affinity matured antibodies display such plasticity and multireactivity enabling their binding to more than one antigen. We propose that antibody multispecificity is a physiological requirement to expand the antibody repertoire at the germline level and to tolerate plasticity in antigens at the mature level. This property of the humoral immune response may attenuate the ability of infectious RNA viruses such as influenza, HIV and SARS-CoV-2 to acquire mutations that render resistance to neutralizing antibodies. • Antibodies (Abs) ensure specificity and fidelity of immune response. • Multispecificity is a physiological requirement of the immune system. • Ability of germline Abs to recognize distinct antigens enhances Ab diversity. • Multispecificity of mature Abs may retard evolution of highly mutable pathogens. [ABSTRACT FROM AUTHOR]

[Display omitted] • The pathogenic role of anti-phospholipase A2 receptor antibodies in primary MN has been well-established. • Macrocarpal B was identified as the most potent inhibitor between PLA2R and anti-PLA2R IgG interaction via ELISA. • Macrocarpal B displayed a reproducible inhibition of PLA2R and anti-PLA2R IgG interaction via surface plasmon resonance. • Macrocarpal B demonstrated selective binding to the PLA2R antigen, but not to the antibody. • Macrocarpal B attenuated membranous nephropathy plasma-mediated podocyte injuries. The pathogenic role of anti-phospholipase A2 receptor (PLA2R) antibodies in primary membranous nephropathy (MN) has been well-established. This study aimed to identify potential small-molecule inhibitors against the PLA2R-antibody interaction, offering potential therapeutic benefits. A comprehensive screening of over 4000 small-molecule compounds was conducted by ELISA to assess their inhibitory effects on the binding between the immobilized full-length extracellular PLA2R and its antibodies. The affinity of anti-PLA2R IgG from MN patients and the inhibitory efficacy of each compound were evaluated via surface plasmon resonance (SPR). Human podocyte injuries were analyzed using CCK-8 assay, wound healing assay, western blot analysis, and immunofluorescence, after exposure to MN plasma +/- blocking compound. Fifteen compounds were identified as potential inhibitors, demonstrating inhibition rates >20 % for the PLA2R-antibody interaction. Anti-PLA2R IgG exhibited a consistent affinity among patients (K D = 10−8 M). Macrocarpal B emerged as the most potent inhibitor, reducing the antigen–antibody interaction by nearly 30 % in a dose-dependent manner, comparable to the performance of the 31-mer peptide from the CysR domain. Macrocarpal B bound to the immobilized PLA2R with an affinity of 1.47 × 10−6 M, while showing no binding to anti-PLA2R IgG. Human podocytes exposed to MN plasma showed decreased podocin expression, impaired migration function, and reduced cell viability. Macrocarpal B inhibited the binding of anti-PLA2R IgG to podocytes and reduced the cellular injuries. [ABSTRACT FROM AUTHOR]

Development of vaccines and therapeutic antibodies to deal with infectious and other diseases are the most perceptible scientific interventions that have had huge impact on public health including that in the current Covid-19 pandemic. From inactivation methodologies to reverse vaccinology, vaccine development strategies of 21st century have undergone several transformations and are moving towards rational design approaches. These developments are driven by data as the combinatorials involved in antigenic diversity of pathogens and immune repertoire of hosts are enormous. The computational prediction of epitopes is central to these developments and numerous B-cell epitope prediction methods developed over the years in the field of immunoinformatics have contributed enormously. Most of these methods predict epitopes that could potentially bind to an antibody regardless of its type and only a few account for antibody class specific epitope prediction. Recent studies have provided evidence of more than one class of antibodies being associated with a particular disease. Therefore, it is desirable to predict and prioritize ‘peptidome’ representing B-cell epitopes that can potentially bind to multiple classes of antibodies, as an open problem in immunoinformatics. To address this, AbCPE, a novel algorithm based on multi-label classification approach has been developed for prediction of antibody class(es) to which an epitope can potentially bind. The epitopes binding to one or more antibody classes (IgG, IgE, IgA and IgM) have been used as a knowledgebase to derive features for prediction. Multi-label algorithms, Binary Relevance and Label Powerset were applied along with Random Forest and AdaBoost. Classifier performance was assessed using evaluation measures like Hamming Loss, Precision, Recall and F1 score. The Binary Relevance model based on dipeptide composition, Random Forest and AdaBoost achieved the best results with Hamming Loss of 0.1121 and 0.1074 on training and test sets respectively. The results obtained by AbCPE are promising. To the best of our knowledge, this is the first multi-label method developed for prediction of antibody class(es) for sequential B-cell epitopes and is expected to bring a paradigm shift in the field of immunoinformatics and immunotherapeutic developments in synthetic biology. The AbCPE web server is available at http://bioinfo.unipune.ac.in/AbCPE/Home.html.

Abstract Background Computational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances. Results Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method. Conclusion The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, “optimized” methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.

A compact S-tapered fibre biosensor functionalised with class I hydrophobin HGFI found in Grifola frondosa has been proposed for the first time to accrue label-free detection of reaction between goat–anti-rabbit immunoglobulin G (IgG) (GAR, antibody) and rabbit–anti-hemagglutinin IgG (R, antigen). The HGFI nanolayer self-assembled on fibre surface provides a distinguished analytical platform to implement biocompatible binding owing to its prominent amphiphilicity and remarkable optical as well as biochemical properties. Water contact angle measurements, atomic force microscopy, and immunofluorescence assay are utilised to characterize the modification properties of siliconised substrate surface with HGFI. Functionalisation of the S-tapered fibre is achieved by exploiting self-assembly of HGFI and immobilisation of GAR for specific R detection. HGFI with a concentration of 300 μg/ml allows the formation of a self-assembled amphipathic film on fibre surface, which can adsorb antibody smoothly with such desirable merits as ease of operation, fast response, good stability, good repeatability, and no side effects. Immunoassay experiments are conducted based on dip wavelengths interrogation of the interferometric transmission spectrum. In this study, we propose a novel antigen detection scheme, which could be extended for the detection of other biomolecules owing to its high integration, good specificity, real-time detection, and simple detection scheme.

A compact S-tapered fibre biosensor functionalised with class I hydrophobin HGFI found in Grifola frondosa has been proposed for the first time to accrue label-free detection of reaction between goat–anti-rabbit immunoglobulin G (IgG) (GAR, antibody) and rabbit–anti-hemagglutinin IgG (R, antigen). The HGFI nanolayer self-assembled on fibre surface provides a distinguished analytical platform to implement biocompatible binding owing to its prominent amphiphilicity and remarkable optical as well as biochemical properties. Water contact angle measurements, atomic force microscopy, and immunofluorescence assay are utilised to characterize the modification properties of siliconised substrate surface with HGFI. Functionalisation of the S-tapered fibre is achieved by exploiting self-assembly of HGFI and immobilisation of GAR for specific R detection. HGFI with a concentration of 300 μg/ml allows the formation of a self-assembled amphipathic film on fibre surface, which can adsorb antibody smoothly with such desirable merits as ease of operation, fast response, good stability, good repeatability, and no side effects. Immunoassay experiments are conducted based on dip wavelengths interrogation of the interferometric transmission spectrum. In this study, we propose a novel antigen detection scheme, which could be extended for the detection of other biomolecules owing to its high integration, good specificity, real-time detection, and simple detection scheme. [ABSTRACT FROM AUTHOR]

We present a nano couple platform for the detection of cardiac biomarker in which the pre-quenched fluorescence of monoclonal antibody conjugated nano fluorophore was lighted up successfully by the utilization of immunocomplex formation between antigen and antibody. Folic acid-capped fluorescent gold nanoclusters were surface-engineered with cardiac troponin-T(cTnT) specific monoclonal antibody through carbodiimide chemistry. Further, its fluorescence was quenched with a two-dimensional nanosheet made of MnO 2. This pre-quenched nano couple can detect the presence of cTnT selectively by giving an enhanced fluorescence signal proportional to the concentration of cTnT antigen. The specific detection was manifested by the specific antigen-antibody interaction forming an immunocomplex, disrupting the direct contact of cluster and two-dimensional sheet. • Target-specific antibody-tagged 0D nanoclusters / 2D nanosheets fluorescence probe for cardiac biomarker was developed. • A standard cardiac biomarker, cardiac troponin T (cTnT) antigen, was probed successfully. • The antibody-tagged gold nanoclusters/MnO 2 nano couple gave a stable fluorescence turn-on signal with cTnT antigen. • The probe successfully detected clinically relevant concentrations of cTnT antigen in aqueous media and human serum samples. • A low-cost paper-based assay strip with this probe successfully detected the presence of cTnT antigen. [ABSTRACT FROM AUTHOR]

The use of computational methods in drug discovery research has increased substantially in recent years. Computational chemistry techniques, such as quantum chemical calculations and molecular dynamics simulations, continue to be widely used. In this review, we focused on drug discovery-related studies that employ fragment molecular orbital methods. Furthermore, we focused on inhibitor discovery, protein-protein interaction analysis, including antigen-antibody interaction analysis, and integration with molecular dynamics simulations.

Background: Computational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances. Results: Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method. Conclusion: The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, "optimized" methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases. [ABSTRACT FROM AUTHOR]

Highlights • Novel macroporous polymer supports were developed and tested for protein microarray. • Effect of porous characteristics of matrix on analytical efficiency was established. • The developed biochips are suitable for the quantitative study of binding properties. • Novel method for the detection of acetylcholinesterase was suggested. • Developed biochips can be used for the determination of enzyme activity on a chip. Abstract A series of rigid macroporous polymer layers differed by hydrophobic–hydrophilic properties was synthesized in situ in preliminary fabricated wells and applied as the platforms for protein biochips. Scanning electron microscopy, etalon porosimetry and BET analysis were used for materials characterization. The comparison of analytical efficiency of the developed platforms allowed for the choice of the most optimal polymer, as well as the evaluation of impact of material porous properties. The quantitative parameters of affinity interaction between two different protein pairs were calculated depending on biochip characteristics using the developed analytical protocol. Moreover, the described biochips were successfully tested to detect acetylcholinesterase via catalytic reaction followed by the formation of fluoresceine as a product. Different parameters of enzymatic reaction were calculated for the reaction on a chip and compared to those established for in solution process. [ABSTRACT FROM AUTHOR]

A problem of similarity and differences between so-called polyreactive immunoglobulins (PRIGs) and natural antibodies (NAbs), capable of cross-reacting with some structurally dissimilar antigens, has been considered. The analysis of mechanisms of an unspecific interaction between PRIGs or NAbs and antigens evidences for the fact that essential differences exist between these substances. These differences permit classifying the abovementioned substances as different types of immunoglobulin molecules. The major difference between PRIGs and NAbs may include both the mechanisms of the above mentioned immunoglobulin molecules binding to antigens and their interaction affinity, as well as an absolutely different influence of some low-molecular substances on the efficiency of the interaction with antigens. Relying on the obtained data it can be assumed that, since PRIGs and NAbs have fundamental differences, they may perform not only similar but also different functions of the immune system.

Graphical abstract Abstract To detect biomolecules with low molecular weight by quartz crystal microbalance (QCM) method, it is necessary to improve its sensitivity. Here, we have investigated the performance of a QCM based biosensor coated with anodic aluminum oxide (AAO). AAO nanostructures were fabricated on a quartz crystal substrate by anodizing thin aluminum film in an oxalic acid solution. We obtain AAO nanostructures with nanohole diameter ranging from 40 to 80 nm by changing anodic conditions, where the pitch of the nanohole was maintained about 100 nm. The AAO thicknesses ranged from 200 to 1000 nm by changing the deposition time of the Al thin film. We demonstrated that the QCM-based sensor with an AAO nanostructure increased sensitivity with the surface area due to AAO nanostructure on the measuring an antigen-antibody interaction. Additionally, the calibration curve corresponding to the antigen-antibody interaction showed that the detection limits of the AAO-nanostructured and Au-thin-film QCM-based sensors were 0.20 and 0.48μg/mL, respectively. [ABSTRACT FROM AUTHOR]

Structural information pertaining to antigen-antibody interactions is fundamental in immunology, and benefits structure-based vaccine design. Modeling of antigen-antibody immune complexes from co-crystal structures or molecular docking simulations provides an extensive profile of the epitope at the interface; however, the key amino acids involved in the interaction must be further clarified, often through the use of experimental mutagenesis and subsequent binding assays. Here, we describe an in silico mutagenesis method to identify key sites at antigen-antibody interfaces, using significant increase in pH-dependency energy among saturated point mutations. Through a comprehensive analysis of the crystal structures of three antigen-antibody immune complexes, we show that a cutoff value of 1 kcal/mol of increased interaction energy provides good congruency with the experimental non-binding mutations conducted in vitro. This in silico mutagenesis strategy, in association with energy calculations, may provide an efficient tool for antibody-antigen interface analyses, epitope optimization, and/or conformation prediction in structure-based vaccine design. [ABSTRACT FROM AUTHOR]

Because antibodies have become an important therapeutic tool, rational antibody design is a challenging issue involving various science and technology fields. From the computational aspect, many types of design-assist methods have been developed, but their accuracy is not fully satisfactory. Because of recent advancements in computational power, molecular dynamics (MD) simulation has become a helpful tool to trace the motion of proteins and to characterize their properties. Thus, MD simulation has been applied to various systems involving antigen-antibody complexes and has been shown to provide accurate insight into antigen-antibody interactions and dynamics at an atomic resolution. Therefore, it is highly possible that MD simulation will play several roles complementing the conventional antibody design. In this review, we address several important features of MD simulation in the context of rational antibody design. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs), which are thought to be the main candidate for metastasis, are gaining importance owing to their potential impact on human health and public welfare. For capturing CTCs, antigen-antibody interaction has been used in which specific antigens expressed on cell surface of CTCs can be bound to antibodies immobilized on substrates. Conventional detection methods for CTCs have often suffered not only from relatively low antigen-antibody coupling efficiency but also from cumbersome fabrication processes of micro/nano-structures for CTCs capture. Herein, we report a facile, robust antibody-based CTCs detection technique using centrifugal force, which can enhance the capturing efficacy of CTCs. We validate chemical functionalization process of antibodies on a silica substrate by using atomic force microscopy. Furthermore, it turned out that the centrifugal force from a benchtop centrifuge is enough to produce a ~2.3-fold increase in capture yield of CTCs through enhancement of binding avidity between CTCs and the antibodies. Our result points out the great potential of our method for practical application in CTCs diagnostics and opens a new avenue for biological and chemical sensing. [ABSTRACT FROM AUTHOR]

The influence of twin 20, lysozyme and protamine on the capability of polyreactive immunoglobu­lins (PRIG) to attach to various antigens was investigated. Twin 20 can inhibit the binding of PRIG to antigens on immunological plates but lysozyme and protamine can enhance it. As far as the mixture of the optimal concentrations of lysozyme and protamine cannot increase PRIG-antigen interaction in comparison to the optimal dose of protamine, we have concluded that the mechanism of their effect on PRIG binding is similar. Of special interest­ is the fact that twin 20 at optimal concentration of lysozyme or protamine does not decrease PRIG binding to various antigens but, on the contrary, increases PRIG-antigen interaction.

The present study focus on optical sensing of breast cancer antigen 15.3 (CA 15.3) using cadmium sulphide quantum dot (CdS‐QD) in saline and serum samples spiked with antigen. The surface of CdS‐QD was modified by cysteamine capping followed by tagging of CA 15.3 antibody. The samples were characterised using UV‐visible absorption spectroscopy (UV‐VIS Spectroscopy), Fourier transform infrared spectroscopy (FTIR), high‐resolution transmission electron microscopy (HRTEM) attached with energy‐dispersive X‐ray spectroscopy, phase contrast inverted epi‐fluorescence microscopy and photoluminescence (PL) spectrophotometry (EDS). The CdS‐QD showed a mean diameter of 3.02 ± 0.6 nm. The complex formed after antigen‐antibody interaction resulted in distinguishable optical and fluorescence intensity with respect to varying concentration of antigen. The PL study revealed that CA 15.3 antibody labelled CdS QD can detect CA 15.3 tumour marker even at very low concentration of 0.002 KU/L with a constant response time of 15 min. This study clearly indicates that detection of CA 15.3 at low concentration is possible using surface modified CdS QD in serum samples and can find immense applications in biosensor development for detection of breast cancer marker similar to various automated detection kits available in market. [ABSTRACT FROM AUTHOR]

Cross-reactive and broadly neutralizing antibodies against surface proteins of diverse strains of rapidly evolving viral pathogens like SARS-CoV-2 can prevent infection and therefore are crucial for the development of effective universal vaccines. While antibodies typically incorporate mutations in their complementarity determining regions during affinity maturation, mutations in the framework regions have been reported as players in determining properties of broadly neutralizing antibodies against HIV and the Influenza virus. We propose an increase in the cross-reactive potential of CR3022 against the emerging SARS- CoV-2 variants of concern through enhanced conformational flexibility. In this study, we use molecular dynamics simulations, in silico mutagenesis, structural modeling, and docking to explore the role of light chain FWR mutations in CR3022, a SARS-CoV anti-spike (S)-protein antibody cross-reactive to the S-protein receptor binding domain of SARS-CoV-2. Our study shows that single substitutions in the light chain framework region of CR3022 with conserved epitopes across SARS-CoV strains allow targeting of diverse antibody epitope footprints that align with the epitopes of recently-categorized neutralizing antibody classes while enabling binding to more than one strain of SARS-CoV-2. Our study has implications for rapid and evolution-based engineering of broadly neutralizing antibodies and reaffirms the role of framework mutations in effective change of antibody orientation and conformation via improved flexibility.Communicated by Ramaswamy H. Sarma.

Assay artifacts were reported in plate-based immuoassays during the assessment of specific molecular interactions owing to the surface induced aggregation/conformational changes. To circumvent surface adsorption and associated artifacts, we used a solution competition ELISA by allowing unrestricted interaction between binding partners to occur in solution for better discrimination between epitopes with subtle differences. A difference of two orders of magnitude in binding to neutralizing antibodies for two truncated versions of the hepatitis E virus capsid protein was observed, while other assays showed comparable antigenicity with the same monoclonal antibodies. Discrimination of epitopes with high degree resemblance in analogous viral capsid proteins was demonstrated quantitatively based on their specific interactions. Therefore, the solution competition ELISA is a method of choice when the detection of subtle differences of two highly analogous proteins is desired. [ABSTRACT FROM AUTHOR]

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (Δ Cp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition. [ABSTRACT FROM AUTHOR]

In the following we give a short summary of examples for biosensor concepts in areas in which reduced graphene oxide-based electronic devices can be developed into new classes of biosensors, which are highly sensitive, label-free, disposable and cheap, with electronic signals that are easy to analyze and interpret, suitable for multiplexed operation and for remote control, compatible with NFC technology, etc., and in many cases a clear and promising alternative to optical sensors. The presented areas concern sensing challenges in medical diagnostics with an example for detecting general antibody-antigen interactions, for the monitoring of toxins and pathogens in food and feed stuff, exemplified by the detection of aflatoxins, and the area of smell sensors, which are certainly the most exciting development as there are very few existing examples in which the typically small and hydrophobic odorant molecules can be detected by other means. The example given here concerns the recording of a honey flavor (and a cancer marker for neuroblastoma), homovanillic acid, by the odorant binding protein OBP 14 from the honey bee, immobilized on the reduced graphene oxide gate of an FET sensor. [ABSTRACT FROM AUTHOR]

Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature–humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55–60 °C and 37–40 °C produced optimum nucleic acid hybridization and antigen–antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25 °C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings. [ABSTRACT FROM AUTHOR]

Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires. [ABSTRACT FROM AUTHOR]

To detect 2,4,6-trinitrotoluene (TNT) using a displacement method over a surface plasmon resonance immunosensor, we developed sensor surfaces modified with a poly-(N-vinylformamideco-vinylamine), which was synthesized from a monomer, N-vinylformamide, and was hydrolyzed to produce amine groups, and TNT analogs. Poly-(N-vinylformamide-co-vinylamine) that reacted with succinic acid was used as a matrix of a sensor surface for increasing the number of antibody binding sites. 2,4-Dinitrophenyl glycine, 3-nitrophenyl acetic acid, and 4-nitrophenyl acetic acid were immobilized on the polymer-modified sensor surfaces. We evaluated the suppression ability of the sensor surface for nonspecific adsorption and determined the limit of detection (LOD) for TNT. As a result, a sensor chip suppressed nonspecific adsorption for 1 mg/mL bovine serum albumin and lysozyme. TNT was measured by the displacement method, and the LOD achieved over a 3-nitrophenyl acetic acid-modified sensor chip was 0.9 ng/mL (ppb). [ABSTRACT FROM AUTHOR]

Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ 1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP 1 -, NIP 1 -, or NNP 1 -hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation. [ABSTRACT FROM AUTHOR]

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively., (© 2023 The Protein Society.)

Growing demands for ultrasensitive biosensing have led to the development of numerous signal-amplification strategies. Based on their unique properties (i.e., electro-conductivity, bio-compatibility and ease of synthesis), various iron magnetic nanoparticles (MNPs) have proved to be an excellent nanomaterial for applications in electrochemical biosensing. This review shows how iron MNPs have made significant contributions in the development of electrochemical nanobiosensors, including immuno-, enzyme, DNA and aptamer types. More importantly, we discuss in detail different aspects of the electrochemical biosensors (e.g., modes of magnetic particles, detection techniques, analytes and the corresponding sensitivity and sample matrix, and several prominent characteristics). Accordingly, we discuss research opportunities and future development trends in these areas. [ABSTRACT FROM AUTHOR]

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10-10 M and (1.39 ± 0.12) × 109 M-1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Hemagglutinin (HA) is the main surface glycoprotein of the influenza A virus. The H9N2 subtype influenza A virus is recognized as the most possible pandemic strain as it has crossed the species barrier, infecting swine and humans. We use terahertz spectroscopy to study the hydration shell formation around H9 subtype influenza A virus's HA protein (H9 HA) as well as the detection of antigen binding of H9 HA with the broadly neutralizing monoclonal antibody. We observe a remarkable concentration dependent nonlinear response of the H9 HA, which reveals the formation process of the hydration shell around H9 HA molecules. Furthermore, we show that terahertz dielectric properties of the H9 HA are strongly affected by the presence of the monoclonal antibody F10 and that the terahertz dielectric loss tangent can be used to detect the antibody binding at lower concentrations than the standard ELISA test. [ABSTRACT FROM AUTHOR]

ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial TyrL50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of TyrL50 pivoted toward PheF68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of PheF68. MD simulations predicted that the TyrL50-PheF68 interaction almost entirely dominated Fab-Fn3 binding, and Ala-substitution of TyrL50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala-substitution of TyrL50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

In the following we give a short summary of examples for biosensor concepts in areas in which reduced graphene oxide-based electronic devices can be developed into new classes of biosensors, which are highly sensitive, label-free, disposable and cheap, with electronic signals that are easy to analyze and interpret, suitable for multiplexed operation and for remote control, compatible with NFC technology, etc., and in many cases a clear and promising alternative to optical sensors. The presented areas concern sensing challenges in medical diagnostics with an example for detecting general antibody-antigen interactions, for the monitoring of toxins and pathogens in food and feed stuff, exemplified by the detection of aflatoxins, and the area of smell sensors, which are certainly the most exciting development as there are very few existing examples in which the typically small and hydrophobic odorant molecules can be detected by other means. The example given here concerns the recording of a honey flavor (and a cancer marker for neuroblastoma), homovanillic acid, by the odorant binding protein OBP 14 from the honey bee, immobilized on the reduced graphene oxide gate of an FET sensor.

Biology and medicine have seen great advancements in the development of nanobiosensors capable of characterizing and quantifying biomolecules. This review reports a systematic study of the usefulness of hydrogel nanoparticles (HNPs) in the different steps of the electroanalytical process developed in electrochemical biosensing systems. We illustrate the advantages offered by HNPs in detection of analytes with representative recent examples that highlight the scientific interest in widening the use of HNPs in electrochemical biosensing methods. We review different types of HNP-based electrochemical biosensors, such as enzyme, protein, and nucleic acids, in terms of their sensing performance and their potential for future development. [ABSTRACT FROM AUTHOR]