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Abstract Background Gold nanorods (GNRs) display unique capacity to absorb and scatter near infrared light, which arises from their peculiar composition of surface plasmon resonances. For this reason, GNRs have become an innovative material of great hope in nanomedicine, in particular for imaging and therapy of cancer, as well as in photonic sensing of biological agents and toxic compounds for e.g. biomedical diagnostics, forensic analysis and environmental monitoring. As the use of GNRs is becoming more and more popular, in all these contexts, there is emerging a latent need for simple and versatile protocols for their modification with targeting units that may convey high specificity for any analyte of interest of an end-user. Results We introduce protein G-coated GNRs as a versatile solution for the oriented immobilization of antibodies in a single step of mixing. We assess this strategy against more standard covalent binding of antibodies, in terms of biocompatibility and efficiency of molecular recognition in buffer, serum and plasma, in the context of the development of a direct immunoenzymatic assay. In both cases, we estimate an average of around 30 events of molecular recognition per particle. In addition, we disclose a convenient protocol to store these particles for months in a freezer, without any detrimental effect. Conclusions The biocompatibility and efficiency of molecular recognition is similar in either case of GNRs that are modified with antibodies by covalent binding or oriented immobilization through protein G. However, protein G-coated GNRs are most attractive for an end-user, owing to their unique versatility and ease of bioconjugation with antibodies of her/his choice.

Alzheimer's disease is one of the most common causes of dementia. It is believed that the aggregation of short Aβ‐peptides to form oligomeric and protofibrillar amyloid assemblies plays a central role for disease‐relevant neurotoxicity. In recent years, passive immunotherapy has been introduced as a potential treatment strategy with anti‐amyloid antibodies binding to Aβ‐amyloids and inducing their subsequent degradation by the immune system. Although so far mostly unsuccessful in clinical studies, the high‐dosed application of the monoclonal antibody Aducanumab has shown therapeutic potential that might be attributed to its much greater affinity to Aβ‐aggregates vs monomeric Aβ‐peptides. In order to better understand how Aducanumab interacts with aggregated Aβ‐forms compared to monomers, we have generated structural model complexes based on the known structure of Aducanumab in complex with an Aβ2 − 7‐eptitope. Structural models of Aducanumab bound to full‐sequence Aβ1 − 40‐monomers, oligomers, protofilaments and mature fibrils were generated and investigated using extensive molecular dynamics simulations to characterize the flexibility and possible additional interactions. Indeed, an aggregate‐specific N‐terminal binding motif was found in case of Aducanumab binding to oligomers, protofilaments and fibrils that is located next to but not overlapping with the epitope binding site found in the crystal structure with Aβ2 − 7. Analysis of binding energetics indicates that this motif binds weaker than the epitope but likely contributes to Aducanumab's preference for aggregated Aβ‐species. The predicted aggregate‐specific binding motif could potentially serve as a basis to reengineer Aducanumab for further enhanced preference to bind Aβ‐aggregates vs monomers. [ABSTRACT FROM AUTHOR]

We had previously shown that three anti–Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti–Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti– Zaire ebolavirus sdAb and a totally specific anti– Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus– specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus –specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes—"paratope duality." One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting. Image 1 • Differential specificities of anti–Ebola virus nucleoprotein nanobodies are explained. • Frameworks enable atypical "side-on" binding of antigens by nanobodies. • Atypical paratopes are recontoured to accommodate nonconcave epitopes. • Typical and atypical nanobody approaches are not necessarily mutually exclusive. • Paratope duality allows the simultaneous engagement of two nonoverlapping epitopes. [ABSTRACT FROM AUTHOR]

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.

Abstract: Deciphering antibody‐protein antigen recognition is of fundamental and practical significance. We constructed an antibody structural dataset, partitioned it into human and murine subgroups, and compared it with nonantibody protein‐protein complexes. We investigated the physicochemical properties of regions on and away from the antibody‐antigen interfaces, including net charge, overall antibody charge distributions, and their potential role in antigen interaction. We observed that amino acid preference in antibody‐protein antigen recognition is entropy driven, with residues having low side‐chain entropy appearing to compensate for the high backbone entropy in interaction with protein antigens. Antibodies prefer charged and polar antigen residues and bridging water molecules. They also prefer positive net charge, presumably to promote interaction with negatively charged protein antigens, which are common in proteomes. Antibody‐antigen interfaces have large percentages of Tyr, Ser, and Asp, but little Lys. Electrostatic and hydrophobic interactions in the Ag binding sites might be coupled with Fab domains through organized charge and residue distributions away from the binding interfaces. Here we describe some features of antibody‐antigen interfaces and of Fab domains as compared with nonantibody protein‐protein interactions. The distributions of interface residues in human and murine antibodies do not differ significantly. Overall, our results provide not only a local but also a global anatomy of antibody structures. [ABSTRACT FROM AUTHOR]

Previously, we developed a method to predict epitopes on a protein recognized by specific antibodies. In this study, we have applied this method to identify the epitopes of the human papillomavirus 16 (HPV16) L1 capsomer that is bound by monoclonal antibodies U4, AE3 and AG7. Initially, the method was validated by the identification of epitopes of HPV16 L1 capsomer that bind to antibody U4. Our predicted epitopes were in agreement with the cryto-electron microscopy (cryto-EM) structure of the complex. The method was then used to predict the epitopes of HPV16 L1 binding of antibodies AE3 and AG7. Our calculations indicated that antibody AE3 binds to the HPV16 L1 capsomer at two different regions. Firstly, the region recognized by antibody U4 and secondly, the region recognized by antibody V5, which have been shown in the cryto-EM structure of the V5 and HPV16 L1 complex. In comparison, the antibody AG7 binds to the capsomer only at the epitopes bound by antibody U4. Therefore, antibody AE3 is predicted to have higher affinity than antibody AG7 and could be used for developing highly efficient anti-HPV monoclonal antibodies in the clinical treatment of HPV infections. [ABSTRACT FROM AUTHOR]

Background: Gold nanorods (GNRs) display unique capacity to absorb and scatter near infrared light, which arises from their peculiar composition of surface plasmon resonances. For this reason, GNRs have become an innovative material of great hope in nanomedicine, in particular for imaging and therapy of cancer, as well as in photonic sensing of biological agents and toxic compounds for e.g. biomedical diagnostics, forensic analysis and environmental monitoring. As the use of GNRs is becoming more and more popular, in all these contexts, there is emerging a latent need for simple and versatile protocols for their modification with targeting units that may convey high specificity for any analyte of interest of an end-user. Results: We introduce protein G-coated GNRs as a versatile solution for the oriented immobilization of antibodies in a single step of mixing. We assess this strategy against more standard covalent binding of antibodies, in terms of biocompatibility and efficiency of molecular recognition in buffer, serum and plasma, in the context of the development of a direct immunoenzymatic assay. In both cases, we estimate an average of around 30 events of molecular recognition per particle. In addition, we disclose a convenient protocol to store these particles for months in a freezer, without any detrimental effect. Conclusions: The biocompatibility and efficiency of molecular recognition is similar in either case of GNRs that are modified with antibodies by covalent binding or oriented immobilization through protein G. However, protein G-coated GNRs are most attractive for an end-user, owing to their unique versatility and ease of bioconjugation with antibodies of her/his choice. [ABSTRACT FROM AUTHOR]

Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen complexes with improved properties. This review summarizes recent progress in the field of in silico design of antibodies, including antibody structure modeling, antibody-antigen complex prediction, antibody stability evaluation, and allosteric effects in antibodies and functions. We listed the cases in which these methods have helped experimental studies to improve the affinities and physicochemical properties of antibodies. We emphasized how the molecular dynamics unveiled the allosteric effects during antibody-antigen recognition and antibody-effector recognition.

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3. [ABSTRACT FROM AUTHOR]

Abstract: A copper-free “click” reaction between an azido-terminated self-assembled monolayer (SAM) chemisorbed on planar gold-coated glass sensors and an alkynyl Fischer carbene complex yielded functionalized surfaces onto which facile and swift grafting of amine-containing molecules was achieved via aminolysis of the Fischer carbene moieties. The course of this process was conveniently monitored by Polarization-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS), contact angle measurements and X-ray photoelectron spectroscopy (XPS). A study of the parameters involved in the covalent grafting of the model protein bovine serum albumin (BSA) to the Fischer carbene modified SAM was carried out. As an application, the bacterial toxin staphylococcal enterotoxin A (SEA) produced by some pathogenic strains of Staphylococcus aureus was immobilized on the gold chips and immunocapture of a polyclonal antibody raised against SEA was detected by PM-IRRAS so as to ultimately construct an optical immunosensor for the detection of this toxin in food samples. [Copyright &y& Elsevier]

Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations, we show that a single static X-ray structure is not sufficient to identify determinants of antibody-antigen recognition. Here, we investigate antibodies that undergo substantial conformational changes upon antigen binding and have been classified as difficult cases in an extensive benchmark for antibody-antigen docking. We present thermodynamics and transition kinetics of these conformational rearrangements and show that paratope states can be used to improve antibody-antigen docking. By using the unbound antibody X-ray structure as starting structure for molecular dynamics simulations, we retain a binding competent conformation substantially different to the unbound antibody X-ray structure. We also observe that the kinetically dominant antibody paratope conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that paratope states in solution can improve antibody-antigen docking and structure prediction. [Display omitted] • Predicting antibody-antigen complexes is a major challenge in computational biology • Biophysical properties of antibodies are governed by their dynamic character • Dominant antibody paratope states coincide with the binding competent conformation • Paratope ensembles in solution improve structure prediction and docking Describing an antibody's binding site using only one single static structure limits the understanding and characterization of the antibody's function and binding properties. Fernández-Quintero et al., show that paratope ensembles in solution can be successfully used to improve structure prediction and enhance the predictivity of antibody-antigen docking. [ABSTRACT FROM AUTHOR]

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays. [ABSTRACT FROM AUTHOR]

Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen complexes with improved properties. This review summarizes recent progress in the field of in silico design of antibodies, including antibody structure modeling, antibody-antigen complex prediction, antibody stability evaluation, and allosteric effects in antibodies and functions. We listed the cases in which these methods have helped experimental studies to improve the affinities and physicochemical properties of antibodies. We emphasized how the molecular dynamics unveiled the allosteric effects during antibody-antigen recognition and antibody-effector recognition. [ABSTRACT FROM AUTHOR]