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There have been significant advances in capturing gigapixel panoramas (GPP). However, solutions for viewing GPPs on head-mounted displays (HMDs) are lagging: an immersive experience requires ultra-fast rendering while directly loading a GPP onto the GPU is infeasible due to limited texture memory capacity. In this paper, we present a novel out-of-core rendering technique that supports not only classic panning, tilting, and zooming but also dynamic refocusing for viewing a GPP on HMD. Inspired by the network package transmission mechanisms in distributed visualization, our approach employs hierarchical image tiling and on-demand data updates across the main and the GPU memory. We further present a multi-resolution rendering scheme and a refocused light field rendering technique based on RGBD GPPs with minimal memory overhead. Comprehensive experiments demonstrate that our technique is highly efficient and reliable, able to achieve ultra-high frame rates ( $> 50$ > 50 fps) even on low-end GPUs. With an embedded gaze tracker, our technique enables immersive panorama viewing experiences with unprecedented resolutions, field-of-view, and focus variations while maintaining smooth spatial, angular, and focal transitions.

Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the target is obtained. Antibody phage display has become the first and most widely used in vitro screening technology. Phage display derivatives play an important role in the diagnosis and treatment of diseases. This article reviews the phage display system of phage display technology, the size and classification of antibody libraries and their applications, and discusses the application prospects and challenges of phage display technology. This thesis lays the foundation for the theoretical and experimental research of bacteriophages.

Biopanning, a common affinity selection approach in phage display, has evolved numerous ligands for diagnosis, imaging, delivery, and therapy applications. However, traditional biopanning has suffered from time-consuming processes, highly-repetitive procedures and labor-intensive manual operation. Herein, a highly integrated and automated biopanning platform (Auto-Panning) is proposed. Based on digital microfluidics (DMF), biopanning processes are integrated on a chip with highly reproducible, precise, automated liquid manipulation. Therefore, 3 rounds of Auto-Panning can be accomplished within 16 h, instead of nearly a week of complicated manual operations. Auto-Panning has been used to evolve a specific peptide against cancer biomarker EphA2 with excellent cellular penetrating ability and significant invasion suppression biofunction, successfully demonstrating the practicality of the platform. Overall, as an automated programmable molecular screening platform, Auto-Panning will further promote the discovery and applications of novel ligands.

The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications.

Although some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction.

PURPOSE: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met. METHODS: A phage library, with a functional size of 5.5 × 10(10) individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques. RESULTS: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (K(aff): 0.3 × 10(9) M(−1)), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%). CONCLUSION: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00334-z) contains supplementary material, which is available to authorized users.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαβ–Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Improving biocompatibility of metallic alloy biomaterials has been of great interest to prevent implant associated-diseases, such as stent thrombosis. Herein a simple and efficient procedure was designed to biofunctionalize a biomaterial surface by isolating a SUS316L stainless steel binding peptide. After three rounds of phage panning procedure, 12 mer peptide (SBP-A; VQHNTKYSVVIR) was identified as SUS316L-binding peptide. The SBP-A peptide formed a stable bond to a SUS316L modified surface and was not toxic to HUVECs. The SBP-A was then used for anti-ICAM antibody modification on SUS316L to construct a vascular endothelial cell-selective surface. The constructed surface dominantly immobilized vascular endothelial cells to smooth muscle cells, demonstrating that the SBP-A enabled simple immobilization of biomolecules without disturbing their active biological function. The SUS316L surface was successfully biofunctionalized using the novel isolated peptide SBP-A, showing its potential as an ideal interface molecule for stent modification. This is the first report of material binding peptide-based optimal surface functionalization to promote endothelialisation. This simple and efficient biofunctionalization procedure is expected to contribute to the development of biocompatible materials.

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naïve phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional ‘bulk’ amplification.Abstract FigureFind Ligands in DropletsCanonical phage display selection of ligands for breast cancer cells, which uses bulk amplification (BA) of phage library, reproducibly identified peptide ligands from a ~0.0001% sub-population of the library, which harbors fast-growing phage. Replacing BA by emulsion-amplification (EmA) altered the selection landscape and yielded cell-binding ligands not accessible to conventional phage-display select

This protocol is a comprehensive guide to phage display based selection of virus neutralizing VH antibody domains. It details three optimized parts including 1) Construction of large size (theoretically > 1011) naïve human antibody heavy chain domain library, 2) SARS-COV-2 antigen expression and stable cell line construction, and 3) Library panning for selection of SARS-CoV-2 specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy., Graphical Abstract

Scaling is the major operation performed in Transformation of images. The Scaling is an important operation for resizing and reshaping the images that are in digital form. Various operations can be performed with digital images out of which the shrinking and zooming are the most widely operations by any type of users in the world. The other name for shrinking is sub sampling and the zooming operation is also named as Oversampling. The purpose of zooming operation is to extend or enlarge the image in order to have a clear and efficient view. Zooming operations are mostly performed in our mobile for viewing the images in our gallery and this operation is the most frequently performed operation by the mobile users. By considering all these factors, graphical images are the most widely agent to convey any information to the transmitter in order to know the position, size of an object. With this in concern, we try to focus on graphical image zooming as it is very important process in image processing. In this paper, we have used Fractional Replication for Image Zooming that is processed by copying and replicating the pixels from the base image based on the inexact spatial correspondence between the base image and zoom image. And also, the pixel address of the zoom image is calculated fractionally from the pixel address of the base image. We have implemented all the zooming operations like Zoom out in subject of Y-Axis, Zoom out in subject of X-Axis, Zoom in subject of Y Axis, Zoom in subject of X Axis, Left Side Panning, Right Side Panning, Top Zoom Panning and Bottom Zoom Panning for a graphical image using C programming language. Here we attempt to process the various Zooming operation analysis of the graphical image.

Hydrologists use various machine learning approaches for segmenting water bodies for carrying out various panning activities. Conventional approaches are mainly clustering or classification based a...

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

Selective modification of natural proteins is a daunting methodological challenge and a stringent test of selectivity and reaction scope. There is a continued need for new reactivity and new selectivity concepts. Transition metals exhibit a wealth of unique reactivity that is orthogonal to biological reactions and processes. As such, metal-based methods play an increasingly important role in bioconjugation. This Review examines metal-based methods as well as their reactivity and selectivity for the functionalization of natural proteins and peptides.

This paper introduces a novel technique for smooth and efficient zooming and panning based on dynamical systems in hyperbolic space. Unlike the technique of van Wijk and Nuij, the animations produced by our technique are smooth at the endpoints and when interrupted by a change of target. To analyze the results of our technique, we introduce world/screen diagrams, a novel technique for visualizing zooming and panning animations.

The automatic estimation of the various types of camera motion (e.g., traveling, panning, rolling, zoom…) that are present in videos represents an important challenge for automatic video indexing. Previous research works are mainly based on optical flow estimation and analysis. In this paper, we propose a different, deep learning-based approach that makes it possible to classify the videos according to the type of camera motion. The proposed method is inspired from action recognition approaches and exploits 3D convolutional neural networks with residual blocks. The performances are objectively evaluated on challenging videos, involving blurry frames, fast/slow motion, poorly textured scenes. The accuracy rates obtained (with an average score of 94%) demonstrate the robustness of the proposed model.

Phage display technology (PD) is a powerful technique for the generation of tumor‑targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell‑based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell‑based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast‑binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor‑targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML‑binding phages during panning. Furthermore, suggesting a common design‑related mechanism using a 'single‑pot' PD library, such as the well‑known Tomlinson single‑chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast‑binding phage particles were isolated and soluble scFv‑Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv‑Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.

Macadamia nut contains important food allergens that potentially cause allergic reactions with severe adverse effects in infants and adults. Reliable and accurate detection of macadamia is critical to avoid allergic reactions. However, knowledge on macadamia allergen is scarce and a reliable detection method has not been reported, yet. In this study, an unbiased immunization and selection strategy was employed to select nanobodies (Nbs) recognizing specifically macadamia allergen, as well as to establish a detection method to unveil a macadamia protein contamination. An alpaca was immunized with a crude protein extract of macadamia followed by construction of a Nb library from its lymphocytes. The panning and screening of this immune Nb repertoire resulted in the selection of six target-specific Nbs. Nb-mediated immuno-capturing combined with mass spectrometry allowed us to identify the target as the macadamia vicilin-like antimicrobial peptides 2-3 (MiAMP2), a novel food allergenic protein abbreviated as Mac i 1. Later on, an immunoassay of a heterologous sandwich ELISA method based on the selected Nb-pairs was established, providing a linear response in the range of 0.442-2,800 μg/mL and with a limit of detection of 27.1 ng/mL. The dedicated immunoassay has been verified by detecting the antigen spiked in food samples. Our study provided evidence for the successful application of the unprejudiced strategy to retrieve Nbs against a priori undefined macadamia allergen. These target-specific Nbs were used to design a highly reliable and effective immunoassay.

Zearalenone (ZEN), a type of non-steroidal estrogenic mycotoxin, is mainly produced by several species of Fusarium molds. It is ubiquitous in contaminated grains and grain products all over the world, posing a serious threat to animal and human health. This study aims to screen the mimotopes of ZEN from a phage display random 12-mer peptide library and explore their immunogenicity. A monoclonal antibody (mAb) against ZEN was employed as the target for mimotope selection from a phage display random peptide library. After four rounds of panning, six mimotopes that could specifically bind to ZEN mAb were obtained. In order to explore the immunogenicity of these mimotopes, Balb/c mice were immunized with phages Z8, Z21, Z35, Z8:Z21:Z35(1:1:1) and the conjugate of ZEN-bovine serum albumin (ZEN-BSA), respectively. The titers of antibodies in the mice immunized with mimotopes were 1:3200 (Z8), 1:3200 (Z21), 1:6400 (Z35), 1:6400 (1:1:1 mixture of Z8, Z21 and Z35), and the binding between serum antibodies and ZEN-OVA could be blocked by ZEN standards. These results demonstrated that the mimotopes of ZEN could induce specific antibodies against ZEN, suggesting that these displayed peptides were immunogenic.

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-μM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

Molecular evolution is an important step in the development of therapeutic antibodies. However, the current method of affinity maturation is overly costly and labor-intensive because of the repetitive mutation experiments needed to adequately explore sequence space. Here, we employed a long short term memory network (LSTM)—a widely used deep generative model—based sequence generation and prioritization procedure to efficiently discover antibody sequences with higher affinity. We applied our method to the affinity maturation of antibodies against kynurenine, which is a metabolite related to the niacin synthesis pathway. Kynurenine binding sequences were enriched through phage display panning using a kynurenine-binding oriented human synthetic Fab library. We defined binding antibodies using a sequence repertoire from the NGS data to train the LSTM model. We confirmed that likelihood of generated sequences from a trained LSTM correlated well with binding affinity. The affinity of generated sequences are over 1800-fold higher than that of the parental clone. Moreover, compared to frequency based screening using the same dataset, our machine learning approach generated sequences with greater affinity.

To do away with the time consuming, error prone and expert dependent gold detection and localization in the current gold panning procedure, automatic image-based techniques are considered in this paper. Identifying and locating gold particles during the gold panning process is a fundamental process in gold extraction. In the automation of the gold panning process and robotic handling it is important to identify and locate the gold particles in the images captured by the image sensor. Image segmentation is a vital step in image simplification, image understanding and object detection. Image segmentation is the process of identifying and extracting homogeneous regions (segments) in an image satisfying a homogeneity criterion based on features formulated from spectral components of the image. Three image thresholding techniques were tested and evaluated on sample gold panning images. Color image thresholding in the CIELAB color space performed better in detecting and locating gold particles in an image. The proposed method will serve as a front-end technique for an automated gold panning system as it will automate the visual feature identification of gold particles and aid in the control of the handling system of the gold particles during the panning process.

In this paper, we present an end-to-end application to perform automatic multiple player detection, unsupervised labelling, and a semi-automatic approach to finding homographies. We incorporate dense optical flow for modelling camera movement and user-assisted calibration on automatically chosen key-frames. Players detection is performed with a pre-trained YOLOv3 detector and player labelling is done using features in HSV colorspace. The experimental results demonstrate that our method is reliable with generating heatmaps from players’ positions in case of moderate camera movement. Major limitations of proposed method are the necessity of manual calibration of characteristic frames, inaccuracy with fast camera movements, and small tolerance of vertical camera movement.

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.

Next-generation sequences (NGS) dataset of nanobody (Nb) clones in a phage display library (PDL) is of immense value as it serves in many different ways, such as: i). estimating the library size, ii). improving selection and identification of Nbs, iii). informing about frequency of V gene families, diversity and length of CDRs, iv). high resolution analysis of natural and synthetic libraries, etc. [1], [2], [3]. We used a fraction of our previously constructed PDL of Nbs derived from an E. coli lipopolysaccharide-immunized Indian desert camel in order to obtain the dataset of NGS reads of Nbs. The cryo-preserved transformants library was revived to extract the Nb-encoding VHH (inserts)-pHEN4 (vector) DNA pool. The DNA sample was used for amplifying VHH pool by PCR [6]. The VHH amplicons band was gel-purified and subjected to NGS using Illumina MiSeqTM platform. 'Nextra XT micro V2 Index' kit was used for the Nb library DNA sample sequencing, with the adaptors: 'i7' (N706: TAGGCATG) and 'i5' (S517: GCGTAAGA). The raw data comprised of a total read count of 182146 (matched= 179591; unmatched=2555), with average read length of 130.33 bases and a total of 23.74 Mb. Of 179591 matched reads, 142004 were paired reads and 37587 broken paired reads. The raw data of NGS reads was submitted to NCBI Sequence Reads Archive accessible at URL: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA516512 (dataset ref. [7]), and after analysis deposited in Mendeley Datasets repository, which is accessible at URL: [https://data.mendeley.com/datasets/4rsz3snvk5/3] (dataset ref. [8]). The sequence reads were analyzed by bioinformatics tools [9], [10], [11], [12]. The assembled consensus contigs revealed Nb orthologs of diverse Ag-specificities, including those isolated by conventional panning and Sanger-sequenced functional Nbs. Contig 1 CDR1-3 matched to those of anti-Trypanosoma evansi RoTat1.2 variant surface glycoprotein (VSG), while Contig 2 CDR1-3 matched to those of anti-LPS Nb clones isolated from the library. Contig 3 was however incomplete and lacked CDR3. Despite lacking the depth, the NGS data is a useful guide for selection of antigen-specific Nbs from the library, as demonstrated by anti-T. evansi VSG Nbs, and provides templates for Nb-based diagnostic reagents and therapeutic agents.

The pandemic caused by emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a global public health threat. Illustrating human antibody responding to viral antigen could potentially provide valuable information for basic research and clinical diagnosis. The antibody can be used as a complement to the viral detection for the rapid diagnosis of infected patients. Compared with spike protein (SP), nucleocapsid protein (NP) is normally conserved and highly immunogenic in many coronavirus members. As a major antigen, NP is a potential target for the diagnosis of SARS-CoV-2 infection. Here, we constructed a combinatorial fragment of antigen-binding (Fab)antibody phage library based on peripheral blood-derived from five coronavirus disease 2019 (COVID-19) infected donors. From the library, 159 Fab antibodies were obtained and identified by panning with NP. Among them, 16 antibodies were evaluated for their binding properties and epitopes recognition. Among these 16 antibodies, two well-paired antibodies were finally screened out for SARS-CoV-2 diagnosis by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. Our works may provide a potential resource for the clinical diagnosis of SARS-CoV-2 infection.

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

Significance Effective therapies are urgently needed for COVID-19. We rapidly (within a week) identified a fully human monoclonal germline-like antibody (ab1) from phage-displayed libraries that potently inhibited mouse ACE2-adapted SARS-CoV-2 replication in wild-type BALB/c mice and native virus in transgenic mice expressing human ACE2 as well as in hamsters when administered before virus challenge. It was also effective when administered after virus infection of hamsters, although at lower efficacy than when used prophylactically. Ab1 was highly specific and did not bind to human cell membrane-associated proteins. It also exhibited good developability properties including complete lack of aggregation. Ab1 has potential for prophylaxis and therapy of COVID-19 alone or in combination with other agents., Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

Traditional antibody generation, using either phage display or animal immunization, relies on purified antigens. Many membrane proteins, such as G protein-coupled receptors, solute carriers, or ion channels, are important drug targets but very challenging for the formation of antibodies due to the difficulty of protein purification. Whole-cell panning is an alternative approach for generating antibodies without the need for antigen purification. However, it often suffers from background interference and therefore requires extensive screening with low success rates. Here, we develop a new phage selection method, dubbed affinity-tag-guided proximity selection (A-GPS), to efficiently isolate specific antibodies directly from the antigen-presenting cells. By engineering a genetically fused affinity tag for the target antigen, A-GPS confines the proximity labeling reaction near the target antigen and preferentially enriches the phage bound to the target antigen. Using surface-presented GFP on human cells as a model antigen, we demonstrated that A-GPS successfully enriched the antigen-specific clones in two rounds of selection. Among the 46 randomly picked clones, >95% of clones showed great affinity and specificity for GFP over the background of HEK293T surface proteins. One of the best clones expressed as a Fab fragment showed subnanomolar binding affinity for GFP. This clone was successfully applied to common biological applications, such as immunofluorescence and flow cytometry, reflecting the usefulness of A-GPS for generating commercial-grade antibodies.

Developability assessment of therapeutic mAb candidates before entering CMC development mitigates the risk of later failure because of manufacturing and stability issues. For mAbs derived from library based screenings, such evaluation starts with the first panning and ends with the selection of a lead candidate. This candidate should show, amongst others, high affine target binding and beneficial conformational as well as chemical stability. In addition, colloidal stability, reflected by the self-interaction propensity, should be superior in order to reduce aggregate formation and unacceptably high viscosity at elevated protein concentrations. Here, we present a study demonstrating the application of self-interaction bio-layer interferometry (SI-BLI) in a developability assessment, including the evaluation of preformulations. We reveal that the formulation rankings based on SI-BLI, DLS and viscosity measurements correlate. SI-BLI provides a deeper understanding of influencing factors on mAb self-interaction such as ionic strength or cation species. The attractive mAb self-interaction propensity was significantly more suppressed by Mg2+ compared to Na+. SI-BLI can be performed in high throughput with minimal material and sample preparation needs. Therefore, it can be applied in early stages of developability assessment going beyond the use of a platform formulation and a small number of analysis, to screen more parameters before proceeding with candidate selection and further extensive development.

The emergence of coronavirus disease 2019 (COVID-19) pandemic led to an urgent need to develop therapeutic interventions. Among them, neutralizing antibodies play crucial roles for preventing viral infections and contribute to resolution of infection. Here, we describe the generation of antibody libraries from 17 different COVID-19 recovered patients and screening of neutralizing antibodies to SARS-CoV-2. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Then the positive clones were sequenced and reconstituted into whole human IgG for epitope binning assays. After that, all 19 IgG were classified into 6 different epitope groups or Bins. Although all these antibodies were shown to have ability to bind RBD, the antibodies in Bin2 have more superiority to inhibit the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 can also strongly bind with mutant RBDs (W463R, R408I, N354D, V367F and N354D/D364Y) derived from SARS-CoV-2 strain with increased infectivity, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, these neutralizing antibodies strongly restrict the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, these antibodies can even block the entry of live SARS-CoV-2 into cells at only 12.5 nM. These results suggest that these neutralizing human antibodies from the patient-derived antibody libraries have the potential to become therapeutic agents against SARS-CoV-2 and its mutants in this global pandemic.

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.

G-protein-coupled receptor (GPCR) drug research is presently hindered by the technical challenges associated with generating purified receptors. Consequently, the application of critical modern discovery technologies has been limited, and the vast untapped opportunity for new GPCR-directed medicines is not being realised. A simple but transformative solution is to purify receptors without removing them from their native phospholipid environment by using polymer lipid particle (PoLiPa) technology, with reagents such as styrene-maleic acid co-polymer (SMA). Compared with contemporary detergent-based and stabilising mutagenesis methods, the PoLiPa approach is simple and generic and, therefore, offers huge advantages, with the potential to revolutionise GPCR research by facilitating the availability of the purified receptors that are required for structural biology, biophysical, and panning technologies.

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

The COVID-19 pandemic is spreading rapidly, highlighting the urgent need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We describe here the development of a phage-displayed single-domain antibody library by grafting naïve CDRs into framework regions of an identified human germline IGHV allele. This enabled the isolation of high-affinity single-domain antibodies of fully human origin. The panning using SARS-CoV-2 RBD and S1 as antigens resulted in the identification of antibodies targeting five types of neutralizing or non-neutralizing epitopes on SARS-CoV-2 RBD. These fully human single-domain antibodies bound specifically to SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of them were found to potently neutralize pseudotyped and live virus, and therefore may represent promising candidates for prophylaxis and therapy of COVID-19. This study also reports unique immunogenic profile of SARS-CoV-2 RBD compared to that of SARS-CoV and MERS-CoV, which may have important implications for the development of effective vaccines against SARS-CoV-2.

Trans-2-nonenal is a fatty aldehyde with a long-chain, containing nine carbons. It was known that trans-2-nonenal is a cause of the odor associated with aging, and mainly found from the ages of 40 years. Phage display was a famous laboratory technique for proteins, peptides, and DNA interaction using bacteriophages to connect proteins with the genetic information. In this study, we have carried out random phage display screening to identify specific binding peptides for trans-2-nonenal. Trans-2-nonenal was directly coated on the plate. To select the phages against trans-2-nonenal, the random phages were attached to trans-2-nonenal and selected by 1–5 panning steps. Individual DNA of phage after 5 panning was analyzed to find the specific peptide sequence against trans-2-nonenal. The affinity of positive and negative binding phages was verified by ELISA assay using M13 phage-specific antibody. Among the phages, the phage with sequence of AHKSKLHQHVMFGGG (called as P4) in the end of tail, has shown the highest response. To explore a role of the peptide selected in sequence analysis and ELISA assay, the peptide was connected to magnetic beads. The peptide-coated beads were treated within trans-2-nonenal: treatment of P4 peptide shows significant decrease of trans-2-nonenal compared to negative peptide. Based on our results, it is suggested that the peptide, which is selected by phage display, could be used for the removal of trans-2-nonenal and odor associated with aging.

This article describes a procedure for isolating T cell subpopulations using various methods including indirect panning and immunopanning by microarray. In these methods, cells are selected by their capacity to bind to antibody-coated plates (or slides) on the basis of particular cell-surface markers. Such methods can be superior to the antibody/complement lysis method (Alternate Protocol), as they can select additional cell population for analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of T cell populations by indirect panning Basic Protocol 2: Immunopanning with microarray Alternate Protocol: Isolation of T cell populations by antibody/complement-mediated cytotoxicity.

Objective To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. Results We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics. Electronic supplementary material The online version of this article (10.1186/s13104-018-3955-8) contains supplementary material, which is available to authorized users.

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery.

Replates are a new way to experience replays of sporting events. We use computer vision tools, such as camera tracking and background subtraction, to align all frames in the original video with regard to each other. Using this alignment, we create a single plate as it would be seen from a wide-angle static camera. Key players and events are then reinserted into this arena using an interactive tool, recovering the big picture that is typically lost due to camera panning and zoom. Replates are played in continuous loop. We allow replate authors to include multiple instances of each player, offset by regular intervals, so viewers can quickly and repeatedly inspect their favorite moments. Authors can also choose from a variety of effects that help them construct a narrative of their favourite plays. The interactive system gives immediate feedback allowing authors to adjust the rendered replates interactively. Replates take a fraction of the size of the original input video, and produce an effect reminiscent of video game reenactments of famous sporting events. The footage, however, is real.

Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies.

Aims To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. Methods and results Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. Conclusions The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. Significance and impact of the study The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries.

The 3-D reconstruction involves the construction of a 3-D model from a set of images. The 3-D reconstruction has varied uses that include 3-D printing, the generation of 3-D models that can be shared through social media, and more. The 3-D reconstruction involves complex computations in mobile phones that must determine the pose estimation. The pose estimation involves the process of transforming a 2-D object into 3-D space. Once the pose estimation is done, then the mesh generation is performed using the graphics processing unit. This helps render the 3-D object. The competitive advantages of using hardware processors are to accelerate the intensive computation using graphics processors and digital signal processors. The stated problem that this technical paper addresses is the need for a reliable automated test for the 3-D reconstruction feature. The solution to this problem involved the design and development of an automated test system using a programmable robotic arm and rotor for precisely testing the quality of 3-D reconstruction features. The 3-D reconstruction testing involves using a robotic arm lab to accurately test the algorithmic integrity and end-to-end validation of the generated 3-D models. The robotic arm can move the hardware at different panning speeds, specific angles, fixed distances from the object, and more. The ability to reproduce the scanning at a fixed distance and the same panning speed helps to generate test results that can be benchmarked by different software builds. The 3-D reconstruction also requires a depth sensor to be mounted onto the device under examination. We use this robotic arm lab for functional, high performance, and stable validation of the 3-D reconstruction feature. This paper addresses the computer vision use case testing for 3-D reconstruction features and how we have used the robotic arm lab for automating these use cases.