Antibody-assisted selective isolation of Purkinje cell nuclei from mouse cerebellar tissue.

We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.

Antibody-Decorated Nanoplatform to Reprogram Macrophage and Block Immune Checkpoint LSECtin for Effective Cancer Immunotherapy.

Repolarizing tumor-associated macrophages (TAMs) into tumor-inhibiting M1 macrophages has been considered a promising strategy for enhanced cancer immunotherapy. However, several immunosuppressive ligands (e.g., LSECtin) can still be highly expressed on M1 macrophages, inducing unsatisfactory therapeutic outcomes. We herein developed an antibody-decorated nanoplatform composed of PEGylated iron oxide nanoparticles (IONPs) and LSECtin antibody conjugated onto the surface of IONPs via the hydrazone bond for enhanced cancer immunotherapy. After intravenous administration, the tumor microenvironment (TME) pH could trigger the hydrazone bond breakage and induce the disassociation of the nanoplatform into free LSECtin antibodies and IONPs. Consequently, the IONPs could repolarize TAMs into M1 macrophages to remodel immunosuppressive TME and provide an additional anticancer effect via secreting tumoricidal factors (e.g., interlukin-12). Meanwhile, the LSECtin antibody could further block the activity of LSECtin expressed on M1 macrophages and relieve its immunosuppressive effect on CD8+ T cells, ultimately leading to significant inhibition of tumor growth.

Geometric epitope and paratope prediction.

MOTIVATION: Identifying the binding sites of antibodies is essential for developing vaccines and synthetic antibodies. In this article, we investigate the optimal representation for predicting the binding sites in the two molecules and emphasize the importance of geometric information. RESULTS: Specifically, we compare different geometric deep learning methods applied to proteins' inner (I-GEP) and outer (O-GEP) structures. We incorporate 3D coordinates and spectral geometric descriptors as input features to fully leverage the geometric information. Our research suggests that different geometrical representation information is useful for different tasks. Surface-based models are more efficient in predicting the binding of the epitope, while graph models are better in paratope prediction, both achieving significant performance improvements. Moreover, we analyze the impact of structural changes in antibodies and antigens resulting from conformational rearrangements or reconstruction errors. Through this investigation, we showcase the robustness of geometric deep learning methods and spectral geometric descriptors to such perturbations. AVAILABILITY AND IMPLEMENTATION: The python code for the models, together with the data and the processing pipeline, is open-source and available at https://github.com/Marco-Peg/GEP.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Antibody design using deep learning: from sequence and structure design to affinity maturation.

Deep learning has achieved impressive results in various fields such as computer vision and natural language processing, making it a powerful tool in biology. Its applications now encompass cellular image classification, genomic studies and drug discovery. While drug development traditionally focused deep learning applications on small molecules, recent innovations have incorporated it in the discovery and development of biological molecules, particularly antibodies. Researchers have devised novel techniques to streamline antibody development, combining in vitro and in silico methods. In particular, computational power expedites lead candidate generation, scaling and potential antibody development against complex antigens. This survey highlights significant advancements in protein design and optimization, specifically focusing on antibodies. This includes various aspects such as design, folding, antibody-antigen interactions docking and affinity maturation.

More Velcro for the TGF-ß1 straitjacket: A new antibody straps latent TGF-ß1 to the matrix.

In this issue of Science Signaling, Jackson et al. present a new antibody strategy to-quite literally-strap transforming growth factor-ß1 (TGF-ß1) to latent complexes in the extracellular matrix. The antibody has no effect on latent TGF-ß1 presented on the surface of immune cells and thus allows targeting of the detrimental effects of TGF-ß1 in fibrosis without affecting its beneficial immune-suppressing activities.

Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling.

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

Colorimetric Immunoassays with Boronic Acid-Decorated, Peroxidase-like Metal-Organic Frameworks as the Carriers of Antibodies and Enzymes.

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity.

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

Exploiting the Role of Features for Antigens-Antibodies Interaction Site Prediction.

Antibodies are a class of proteins that recognize and neutralize pathogens by binding to their antigens. They are the most significant category of biopharmaceuticals for both diagnostic and therapeutic applications. Understanding how antibodies interact with their antigens plays a fundamental role in drug and vaccine design and helps to comprise the complex antigen binding mechanisms. Computational methods for predicting interaction sites of antibody-antigen are of great value due to the overall cost of experimental methods. Machine learning methods and deep learning techniques obtained promising results.In this work, we predict antibody interaction interface sites by applying HSS-PPI, a hybrid method defined to predict the interface sites of general proteins. The approach abstracts the proteins in terms of hierarchical representation and uses a graph convolutional network to classify the amino acids between interface and non-interface. Moreover, we also equipped the amino acids with different sets of physicochemical features together with structural ones to describe the residues. Analyzing the results, we observe that the structural features play a fundamental role in the amino acid descriptions. We compare the obtained performances, evaluated using standard metrics, with the ones obtained with SVM with 3D Zernike descriptors, Parapred, Paratome, and Antibody i-Patch.

Highly Specific Aptamer-Antibody Birecognized Sandwich Module for Ultrasensitive Detection of a Low Molecular Weight Compound.

Herein, the aptamer-antibody sandwich module was first introduced to accurately recognize a low molecular weight compound (mycotoxin). Impressively, compared with the large steric hindrance of a traditional dual-antibody module, the aptamer-antibody sandwich with low Gibbs free energy and a low dissociation constant has high recognition efficiency; thus, it could reduce false positives and false negatives caused by a dual-antibody module. As a proof of concept, a sensitive electrochemiluminescence (ECL) biosensor was constructed for detecting mycotoxin zearalenone (ZEN) based on an aptamer-antibody sandwich as a biological recognition element and porous ZnO nanosheets (Zn NSs) supported Cu nanoclusters (Cu NCs) as the signal transduction element, in which the antibody was modified on the vertex of a tetrahedral DNA nanostructure (TDN) with a rigid structure to increase the kinetics of target recognition for promoting the detection sensitivity. Moreover, the Cu NCs/Zn NSs exhibited an excellent ECL response that was attributed to the aggregation-induced ECL enhancement through electrostatic interactions. The sensing platform achieved trace detection of ZEN with a low detection limit of 0.31 fg/mL, far beyond that of the enzyme-linked immunosorbent assay (ELISA, the current rapid detection method) and high-performance liquid chromatography (HPLC, the national standard detection method). The strategy has great application potential in food analysis, environmental monitoring, and clinical diagnosis.

Oriented Antibody Coupling to an Antifouling Polymer Using Glycan Remodeling for Biosensing by Particle Motion.

Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.

Efficient generation of a stable CHO-K1 cell line overexpressing the human water channel aquaporin-5 as tool to generate therapeutic antibodies.

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren's syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

An Analytical Protocol for Detecting Antibody Titer Levels in Serum/Saliva by Indirect Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) detects qualitatively and quantitatively the presence of antibodies or antigens in a sample. Due to its simplicity, high sensitivity, and user-friendliness, the test is widely used in laboratory research, clinical diagnoses, and food testing. This chapter describes the indirect semiquantitative ELISA protocol used to monitor antibody levels in animals and analyze the titer levels of specific antibodies against a target antigen in serum and saliva.

Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.

Structural Characterization of Peptide Antibodies.

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery. To better understand the underlying mechanisms of antibody-antigen interaction, here, we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Cytochemical and Histochemical Staining with Peptide Antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/absorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Functionalized magnetic nanoparticles remove donor-specific antibodies (DSA) from patient blood in a first ex vivo proof of principle study.

The presence of donor-specific antibodies (DSA) such as antibodies directed against donor class I human leucocyte antigen (e.g., HLA-A) is a major barrier to kidney transplant success. As a proof of concept, functionalized magnetic nanoparticles have been designed to eliminate DSA from saline, blood and plasma of healthy donors and sensitized patients. Specific HLA-A1 protein was covalently bound to functionalized cobalt nanoparticles (fNP), human serum albumin (HSA) as control. fNP were added to anti-HLA class I-spiked saline, spiked volunteers' whole blood, and to whole blood and plasma of sensitized patients ex vivo. Anti-HLA-A1 antibody levels were determined with Luminex technology. Antibodies' median fluorescent intensity (MFI) was defined as the primary outcome. Furthermore, the impact of fNP treatment on blood coagulation and cellular uptake was determined. Treatment with fNP reduced MFI by 97 ± 2% and by 94 ± 4% (p < 0.001 and p = 0.001) in spiked saline and whole blood, respectively. In six known sensitized anti-HLA-A1 positive patients, a reduction of 65 ± 26% (p = 0.002) in plasma and 65 ± 33% (p = 0.012) in whole blood was achieved. No impact on coagulation was observed. A minimal number of nanoparticles was detected in peripheral mononuclear blood cells. The study demonstrates-in a first step-the feasibility of anti-HLA antibody removal using fNP. These pilot data might pave the way for a new personalized DSA removal technology in the future.