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1.
Methods Mol Biol ; 2564: 317-323, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107351

RESUMO

Protein-protein interactions play a vital role in the cellular physiology of living organisms. Among several available approaches, co-immunoprecipitation (co-IP) has emerged as a reliable method to investigate such interactions. The underlying principle is to retrieve a bait protein from a protein extract using bait-specific antibodies and thereby indirectly capture the interacting partner proteins. However, bait-specific antibodies are not always available, and the genetic fusion of a peptide tag offers an alternative. An extensive range of peptide tags and the tag-specific antibodies are available nowadays. Fluorescent proteins are widely used protein tags for co-IP experiments. In this chapter, we describe a method to co-immunoprecipitate the fluorescently tagged candidate protein with its interacting partners from the crude plant cell extracts using green fluorescent protein (GFP)-trap magnetic beads.


Assuntos
Ligante de CD40 , Células Vegetais , Anticorpos , Extratos Celulares , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Peptídeos , Células Vegetais/metabolismo
2.
Methods Mol Biol ; 2578: 83-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152282

RESUMO

Microarray assay formats gained popularity in the 1990s, first implemented in DNA-based arrays but later adopted for use with proteins, namely antibodies, peptides, low molecular weight (LMW) molecules, such as lipids, and even tissues. In nucleic acid-based affinity assays and arrays, but not in protein or peptide arrays, the specificity and affinity of complementary strand interactions can be deduced from or adjusted through modifications to the nucleotide sequence. Arrays of LMW molecules are characterized by largely uniform but low binding affinities. Multiplexed protein-based affinity assays, such as microarrays, might present an additional challenge due to heterogeneity of antigen properties and of their binding affinities. The use of peptides instead of proteins reduces physical heterogeneity of these reagents through either the widened peptide selection options or rational sequence engineering. However, rational engineering of binding affinities remains an unmet need, and peptide-binding affinities to the respective antipeptide antibodies could vary by orders of magnitude. Hence, multiplexing of such assays by using a microarray format and data analysis and interpretation requires some knowledge of their binding affinities. Low-throughput binding assays to characterize such peptide-antipeptide antibodies interactions are widely available, but scaling-up of traditional protein- and peptide-binding assays might present practical challenges. Here, we describe fast label-free practical approach especially suitable for estimating peptide-binding affinities. The method in question relies on commercially available biolayer interferometry-based equipment with a protocol which can be easily scaled-up, subject to user needs and equipment availability.


Assuntos
Anticorpos , Ácidos Nucleicos , Anticorpos/metabolismo , DNA/metabolismo , Lipídeos , Ácidos Nucleicos/metabolismo , Peptídeos/química , Ligação Proteica , Proteínas/metabolismo
3.
Methods Mol Biol ; 2565: 57-75, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36205887

RESUMO

The localization and density of any plasma membrane or intracellular protein in chromaffin cells are prerequisites for those studies designed to elucidate their contribution to cellular function within the adrenal gland and can be achieved only by immunoelectron microscopy. The most popular immunoelectron microscopic techniques involved gold particles conjugated to secondary antibodies, leading to electron-dense markers and the so-called immunogold EM method. Two main immunogold electron microscopic techniques exist: the pre-embedding immunogold, whereby the immunolabeling steps take place before samples are embedded, and the post-embedding immunogold, where the immunolabeling steps take place on embedded and sectioned samples. Pre-embedding immunogold is a very sensitive technique useful for simultaneous observation of labeled tissue at the light and electron microscopic levels. Post-embedding immunogold enables the simultaneous localization of different molecules in the cell using secondary antibodies conjugated with gold particles of different size. In this chapter, we introduce pre-embedding and post-embedding immunogold procedures used for the identification of quantitative changes in a wide range of signaling molecules in different tissues and also discuss the limitations inherent to these approaches.


Assuntos
Células Cromafins , Ouro , Anticorpos , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia Imunoeletrônica
4.
Methods Mol Biol ; 2552: 61-79, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346585

RESUMO

The immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences and structural features of antibodies vary depending on species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies that lack the light chains. In antibodies that lack light chains, the C-terminal variable region is called the VHH domain. Antibodies recognize antigens through six complementarity-determining regions (CDRs). The third CDR of the heavy chain (CDR-H3) is at the center of the antigen-binding site and is diverse in terms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of CDR-H3s of antibodies that possess both light and heavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In this chapter, we describe current knowledge of sequence-structure-function correlations of single-domain VHH antibodies with emphasis on CDR-H3. Based on the 370 crystal structures in the Protein Data Bank, we also attempt structural classification of CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing number of the structures.


Assuntos
Anticorpos de Domínio Único , Animais , Anticorpos de Domínio Único/química , Modelos Moleculares , Regiões Determinantes de Complementaridade , Anticorpos/química , Bases de Dados de Proteínas , Antígenos , Conformação Proteica
5.
Methods Mol Biol ; 2552: 3-59, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346584

RESUMO

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org , the global reference in immunogenetics and immunoinformatics, was created in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS) to manage the huge diversity of the antigen receptors, immunoglobulins (IG) or antibodies, and T cell receptors (TR) of the adaptive immune responses. The founding of IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® standardized analysis of the IG, TR, and major histocompatibility (MH) genes and proteins bridges the gap between sequences and three-dimensional (3D) structures, for all jawed vertebrates from fish to humans. This is achieved through the IMGT Scientific chart rules, based on the IMGT-ONTOLOGY axioms, and primarily CLASSIFICATION (IMGT gene and allele nomenclature) and NUMEROTATION (IMGT unique numbering and IMGT Colliers de Perles). IMGT® comprises seven databases (IMGT/LIGM-DB for nucleotide sequences, IMGT/GENE-DB for genes and alleles, etc.), 17 tools (IMGT/V-QUEST, IMGT/JunctionAnalysis, IMGT/HighV-QUEST for NGS, etc.), and more than 20,000 Web resources. In this chapter, the focus is on the tools for amino acid sequences per domain (IMGT/DomainGapAlign and IMGT/Collier-de-Perles), and on the databases for receptors (IMGT/2Dstructure-DB and IMGT/3D-structure-DB) described per receptor, chain, and domain and, for 3D, with contact analysis, paratope, and epitope. The IMGT/mAb-DB is the query interface for monoclonal antibodies (mAb), fusion proteins for immune applications (FPIA), composite proteins for clinical applications (CPCA), and related proteins of interest (RPI) with links to IMGT® 2D and 3D databases and to the World Health Organization (WHO) International Nonproprietary Names (INN) program lists. The chapter includes the human IG allotypes and antibody engineered variants for effector properties used in the description of therapeutical mAb.


Assuntos
Imunogenética , Imunoglobulinas , Humanos , Animais , Imunogenética/métodos , Imunoglobulinas/genética , Imunoglobulinas/química , Anticorpos/genética , Biologia Computacional/métodos , Sequência de Aminoácidos , Receptores de Antígenos de Linfócitos T/genética
6.
Methods Mol Biol ; 2552: 83-100, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346586

RESUMO

Antibodies and T-cell receptors have been a subject of much interest due to their central role in the immune system and their potential applications in several biotechnological and medical applications from cancer therapy to vaccine development. A unique feature of these two lymphocyte receptors is their ability to bind a huge variety of different (pathogen) targets. This ability stems from six short loops in the binding domain that have hypervariable sequence due to genetic recombination mechanism. Particularly one of these loops, the third complementarity determining region (CDR3), has the highest sequence variability and a dominant role in binding the target. However, it has also been proven the most difficult to be modeled structurally, which is vitally important for downstream tasks such as binding prediction. This difficulty stems from its variability in sequence that both reduces the possibility of finding homologues and introduces unique structural features in the loop. We present here a general protocol for modeling such loops in antibodies and T-cell receptors. We also discuss the difficulties in loop modeling and the advantages and limitations of different modeling methods.


Assuntos
Regiões Determinantes de Complementaridade , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Anticorpos/química , Simulação por Computador , Receptores de Antígenos de Linfócitos T alfa-beta/genética
7.
Methods Mol Biol ; 2552: 109-124, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346588

RESUMO

Complex and coordinated dynamics are closely connected with protein functions, including the binding of antibodies to antigens. Knowledge of such dynamics could improve the design of antibodies. Molecular dynamics (MD) simulations provide a "computational microscope" that can resolve atomic motions and inform antibody design efforts.


Assuntos
Anticorpos , Simulação de Dinâmica Molecular , Anticorpos/química , Antígenos , Proteínas/química , Conformação Proteica
8.
Methods Mol Biol ; 2552: 125-139, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346589

RESUMO

This chapter describes the application of constrained geometric simulations for prediction of antibody structural dynamics. We utilize constrained geometric simulations method FRODAN, which is a low computational complexity alternative to molecular dynamics (MD) simulations that can rapidly explore flexible motions in protein structures. FRODAN is highly suited for conformational dynamics analysis of large proteins, complexes, intrinsically disordered proteins, and dynamics that occurs on longer biologically relevant time scales that are normally inaccessible to classical MD simulations. This approach predicts protein dynamics at an all-atom scale while retaining realistic covalent bonding, maintaining dihedral angles in energetically good conformations while avoiding steric clashes in addition to performing other geometric and stereochemical criteria checks. In this chapter, we apply FRODAN to showcase its applicability for probing functionally relevant dynamics of IgG2a, including large-amplitude domain-domain motions and motions of complementarity determining region (CDR) loops. As was suggested in previous experimental studies, our simulations show that antibodies can explore a large range of conformational space.


Assuntos
Proteínas Intrinsicamente Desordenadas , Simulação de Dinâmica Molecular , Conformação Proteica , Regiões Determinantes de Complementaridade , Anticorpos
9.
Methods Mol Biol ; 2552: 143-150, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346590

RESUMO

Immunogenicity is an important concern to therapeutic antibodies during antibody design and development. Based on the co-crystal structures of idiotypic antibodies and their antibodies, one can see that anti-idiotypic antibodies usually bind the complementarity-determining regions (CDR) of idiotypic antibodies. Sequence and structural features, such as cavity volume at the CDR region and hydrophobicity of CDR-H3 loop region, were identified for distinguishing immunogenic antibodies from non-immunogenic antibodies. These features were integrated together with a machine learning platform to predict immunogenicity for humanized and fully human therapeutic antibodies (PITHA). This method achieved an accuracy of 83% in a leave-one-out experiment for 29 therapeutic antibodies with available crystal structures. The web server of this method is accessible at http://mabmedicine.com/PITHA or http://sysbio.unl.edu/PITHA . This method, as a step of computer-aided antibody design, helps evaluate the safety of new therapeutic antibody, which can save time and money during the therapeutic antibody development.


Assuntos
Anticorpos , Regiões Determinantes de Complementaridade , Humanos , Formação de Anticorpos
10.
Methods Mol Biol ; 2552: 165-197, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346592

RESUMO

Engineering increased stability into antibodies can improve their developability. While a range of properties need to be optimized, thermal stability and aggregation are two key factors that affect the antibody yield, purity, and specificity throughout the development and manufacturing pipeline. Therefore, an ideal goal would be to apply protein engineering methods early-on, such as in parallel to affinity maturation, to screen out potential drug molecules with the desired conformational and colloidal stability. This chapter introduces our methods to computationally characterize an antibody Fab fragment, propose stabilizing variants, and then experimentally verify these predictions.


Assuntos
Anticorpos , Fragmentos Fab das Imunoglobulinas , Fragmentos Fab das Imunoglobulinas/genética , Engenharia de Proteínas , Conformação Molecular , Estabilidade Proteica , Afinidade de Anticorpos
11.
Methods Mol Biol ; 2552: 309-321, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346600

RESUMO

Affinity maturation is an important stage in biologic drug discovery as is the natural process of generating an immune response inside the human body. In this chapter, we describe in silico approaches to affinity maturation via a worked example. Both advantages and limitations of the computational methods used are critically examined. Furthermore, construction of affinity maturation libraries and how their outputs might be implemented in an experimental setting are also described. It should be noted that structure-based design of biologic drugs is an emerging field and the tools currently available require further development. Furthermore, there are no standardized structure-based strategies yet for antibody affinity maturation as this research relies heavily on scientific logic as well as creative intuition.


Assuntos
Anticorpos , Humanos , Afinidade de Anticorpos , Anticorpos/química
12.
Methods Mol Biol ; 2552: 323-331, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346601

RESUMO

Structure-based site-directed affinity maturation of antibodies can be expanded by multiple-point mutations to obtain various mutants. However, selecting the appropriate number of promising mutants for experimental evaluation from the vast number of combinations of multiple-point mutations is challenging. In this report, we describe how to narrow candidate mutants using the so-called weak interaction analysis such as CH-π and CH-O in addition to widely recognized interactions such as hydrogen bonds.


Assuntos
Anticorpos , Mutação Puntual , Anticorpos/genética , Ligação de Hidrogênio , Afinidade de Anticorpos
13.
Methods Mol Biol ; 2552: 219-235, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346594

RESUMO

A great effort to avoid known developability risks is now more often being made earlier during the lead candidate discovery and optimization phase of biotherapeutic drug development. Predictive computational strategies, used in the early stages of antibody discovery and development, to mitigate the risk of late-stage failure of antibody candidates, are highly valuable. Various structure-based methods exist for accurately predicting properties critical to developability, and, in this chapter, we discuss the history of their development and demonstrate how they can be used to filter large sets of candidates arising from target affinity screening and to optimize lead candidates for developability. Methods for modeling antibody structures from sequence and detecting post-translational modifications and chemical degradation liabilities are also discussed.


Assuntos
Anticorpos , Desenvolvimento de Medicamentos , Anticorpos/uso terapêutico
14.
Methods Mol Biol ; 2552: 255-266, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346596

RESUMO

The design of optimized protein antigens is a fundamental step in the development of new vaccine candidates and in the detection of therapeutic antibodies. A fundamental prerequisite is the identification of antigenic regions that are most prone to interact with antibodies, namely, B-cell epitopes. Here, we describe an efficient structure-based computational method for epitope prediction, called MLCE. In this approach, all that is required is the 3D structure of the antigen of interest. MLCE can be applied to glycosylated proteins, facilitating the identification of immunoreactive versus immune-shielding carbohydrates.


Assuntos
Biologia Computacional , Vacinas , Biologia Computacional/métodos , Anticorpos , Epitopos de Linfócito B , Antígenos , Mapeamento de Epitopos/métodos
15.
Methods Mol Biol ; 2552: 361-374, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346603

RESUMO

The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform guides the selection of mutants that improve/modulate the affinity of antibodies and other biologics. Predicted affinities are based on a consensus z-score from three scoring functions. Computational predictions are interleaved with experimental validation, significantly enhancing the robustness of the design and selection of mutants. A key step is an initial exhaustive virtual single-mutant scan that identifies hot spots and the mutations predicted to improve affinity. A small number of proposed single mutants are then produced and assayed. Only the validated single mutants (i.e., having improved affinity) are used to design double and higher-order mutants in subsequent rounds of design, avoiding the combinatorial explosion that arises from random mutagenesis. Typically, with a total of about 30-50 designed single, double, and triple mutants, affinity improvements of 10- to 100-fold are obtained.


Assuntos
Anticorpos , Afinidade de Anticorpos , Mutagênese , Mutação
16.
Methods Mol Biol ; 2552: 409-433, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346606

RESUMO

In the computational design of antibodies, the interaction analysis between target antigen and antibody is an essential process to obtain feedback for validation and optimization of the design. Kinetic and thermodynamic parameters as well as binding affinity (KD) allow for a more detailed evaluation and understanding of the molecular recognition. In this chapter, we summarize the conventional experimental methods which can calculate KD value (ELISA, FP), analyze a binding activity to actual cells (FCM), and evaluate the kinetic and thermodynamic parameters (ITC, SPR, BLI), including high-throughput analysis and a recently developed experimental technique.


Assuntos
Anticorpos , Antígenos , Ensaio de Imunoadsorção Enzimática , Cinética , Computadores
17.
Methods Mol Biol ; 2552: 375-397, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346604

RESUMO

Antibodies are essential experimental and diagnostic tools and as biotherapeutics have significantly advanced our ability to treat a range of diseases. With recent innovations in computational tools to guide protein engineering, we can now rationally design better antibodies with improved efficacy, stability, and pharmacokinetics. Here, we describe the use of the mCSM web-based in silico suite, which uses graph-based signatures to rapidly identify the structural and functional consequences of mutations, to guide rational antibody engineering to improve stability, affinity, and specificity.


Assuntos
Anticorpos , Software , Anticorpos/genética , Anticorpos/química , Engenharia de Proteínas , Mutação , Afinidade de Anticorpos/genética
18.
Methods Mol Biol ; 2552: 447-463, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346608

RESUMO

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.


Assuntos
Anticorpos , Biologia Computacional , Humanos , Biologia Computacional/métodos , Cadeias Leves de Imunoglobulina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos
19.
Methods Mol Biol ; 2552: 437-445, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346607

RESUMO

To ensure the functionalities of the antibodies in phage-displayed synthetic antibody libraries, we use computational method to evaluate the designs of the antibody libraries. The computational methodologies developed in our lab for designing antibody library provide rich information on the function of the designed antibody sequences-adequate antibody designs for a specific antigen type should have predicted paratopes for the antigen type. This computational assessment of the designed antibody sequences helps eliminate non-functional designs before proceeding to construct the library designs in the wet lab. As such, only reasonable antibody designs are constructed for antibody discoveries.


Assuntos
Anticorpos , Biblioteca de Peptídeos , Sítios de Ligação de Anticorpos , Antígenos
20.
Methods Mol Biol ; 2585: 211-225, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36331777

RESUMO

Since its discovery in 1937 in the West Nile district of Uganda, West Nile virus (WNV) has been one of the leading causes of mosquito-transmitted infectious diseases (Smithburn, Burke, Am J Trop Med 20:22, 1940). Subsequently, it spread to Europe, Asia, Australia, and finally North America in 1999 (Sejvar, Ochsner 5(3):6-10, 2003). Worldwide outbreaks have continued to increase since the 1990s (Chancey et al, Biomed Res Int 2015:376230, 2015). According to the Center for Disease Control and Prevention, more than 51,000 cases of WNV infection and nearly 2400 cases of WNV-related death were reported in the USA from 1999 to 2019. The estimated economic impact of WNV infections is close to 800 million dollars in the USA from 1999 to 2012 (Barrett, Am J Trop Med Hyg 90:389, 2014).


Assuntos
Culicidae , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Febre do Nilo Ocidental/prevenção & controle , Europa (Continente)/epidemiologia , Surtos de Doenças , Anticorpos
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