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Oxidative Stress and Beta Cell Dysfunction

Autoimmune Type 1 A Diabetes (T1D) is characterized by dependence on exogenous insulin consequential to the autoimmune attack and destruction of insulin-producing islet beta cells. Pancreatic islet cell inflammation, or insulitis, precedes beta cell death and T1D onset. In the insul ...

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Pathogenesis of Multiple Sclerosis: What Can We Learn from the Cuprizone Model

Multiple sclerosis is an inflammatory demyelinating and neurodegenerative disorder of the central nervous system (CNS). The primary cause of the disease remains unknown, but an altered immune regulation with features of autoimmunity has generally been considered to play a critic ...

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Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models

Inflammatory diseases of the mucosal surfaces are rising worldwide and particularly in the Western world that is witnessing unprecedented increases in the number and incidence of both asthma and inflammatory bowel disease. The laboratory mouse allows the application of the full pan ...

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Tolerance Induction via B-Cell Delivered Gene Therapy

A master control of both the innate and adaptive immune system is the body’s ability to distinguish between self and foreign entities. This is accomplished by the elimination of autoreactive leukocytes through a series of checkpoints both in the thymus (central deletion) and in the circulat ...

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Mapping Susceptibility Gene in Systemic Lupus Erythematosus

Genome-wide association studies have identified many dozen genetic intervals that harbor single-nucleotide polymorphisms (SNPs) showing statistical association with systemic lupus erythematosus. Despite the wealth of data produced, there are limitations of these stu ...

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Methods and Protocols to Study T Cell Signaling Abnormalities in Human Systemic Lupus Erythematosus

Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). T cell receptor (TCR/CD3)-mediated stimulation of SLE T cells show increased protein tyrosine phosphoryla ...

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Immortalization of Human B Cells: Analysis of B Cell Repertoire and Production of Human Monoclonal Antibodies

One of the major challenges in human B cell immunology field has been the objective to establish stable monoclonal cells lines that express the B cell receptor (BCR) on their cell surface and secrete antibodies. Such a system is extremely attractive not only for studying various aspect of BCR signa ...

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Temperature-Dependent Antibody Kinetics as a Tool in Antibody Lead Selection

Antibody–antigen interactions can principally be classified into three different temperature-dependent kinetic rate profiles. The affinity K D can persist, decrease, or increase in the temperature gradient. Today, the impact of temperature-dependent antibody kinetics is r ...

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Kinetic Screening in the Antibody Development Process

Kinetic screening is of paramount importance when it is to select custom-made antibodies, tailored for their respective scientific, diagnostic, or pharmaceutical application. Here a kinetic screening protocol is described, using a Biacore A100 surface plasmon resonance biose ...

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Determination of Antibody Glycosylation by Mass Spectrometry

Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc �portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. Th ...

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PEGylation of Antibody Fragments for Half-Life Extension

Antibody fragments (Fab’s) represent important structure for creating new therapeutics. Compared to full antibodies Fab’ fragments possess certain advantages, including higher mobility and tissue penetration, ability to bind antigen monovalently and lack of fragment crys ...

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Cloning, Expression, and Purification of Monoclonal Antibodies in scFv-Fc Format

This protocol describes the generation of monoclonal antibodies in single-chain variable fragment (scFv)-Fc format. It includes the cloning of the scFv-Fc expression cassette into a mammalian expression vector followed by transient transfection of mammalian cells and purifi ...

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Bispecific Antibody Derivatives Based on Full-Length IgG Formats

Monoclonal antibodies have emerged as an effective therapeutic modality, and numerous antibodies have been approved for the treatment of several severe diseases or are currently in clinical development. To improve their therapeutic potential, monoclonal antibodies are cons ...

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Methods to Engineer and Identify IgG1 Variants with Improved FcRn Binding or Effector Function

Antibodies as therapeutic agents have gained broad acceptance as shown by the number of antibodies in clinical use and many more in clinical development. This utility is an outcome of the high specificity and affinity of the antigen-binding site comprised of the heavy and light chain variable d ...

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Generation of Fluorescent IgG Fusion Proteins in Mammalian Cells

The generation of recombinantly produced fluorescent antibody derivatives that are derived from full-length immunoglobulin G (IgG) has until now been problematic. One major reason for that lies in different and partially incompatible secretion- and folding-requirements of a ...

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Class-Specific Effector Functions of Therapeutic Antibodies

Physiology usually combines polyclonal antibodies of multiple classes in a single humoral response. Beyond their common ability to bind antigens, these various classes of human immunoglobulins carry specific functions which can each serve specific goals. In many cases, the functi ...

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Antigen Presentation for the Generation of Binding Molecules

In the last few decades, several new methods have been established to isolate full antibodies and fragments thereof, some even using alternative scaffolds from in vivo and in vitro sources. These methods encompass robust techniques including immunization and hybridoma technology or ...

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Recombinant Antibodies and In Vitro Selection Technologies

Over the past decade, the accumulation of detailed knowledge of antibody structure and function has enabled antibody phage display to emerge as a powerful in vitro alternative to hybridoma methods for creating antibodies. Many antibodies produced using phage display technology have ...

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Phage Display

Phage display has emerged as one of the leading technologies for the selection and generation of highly specific antibodies, offering a number of advantages over traditional ways of antibody generation such as mouse hybridoma techniques. While there are various possibilities to cond ...

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Selection of Human Fab Libraries by Phage Display

This protocol describes the selection of human antibody libraries in Fab format by phage display. It includes panning against immobilized antigens, biotinylated antigens in solution, and cell surface antigens.

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