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CDR-FR Peptides

The non-immunogenic antigen binder has emerged as an important component in the research of cell recognition and targeting with the high degree of specificity inherited from prototypical antibody (Hoogenboom 2005; Holliger and Hudson 2005; Hu et al. 1996; Olafsen et al. 2004; Shahied et al. 20 ...

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Isolation of Antigen-Specific Nanobodies

Recombinant Nanobodies, derived from camelid heavy-chain antibodies, are a unique form of monoclonal antibodies composed of a single antigen-binding domain. Nanobodies are the smallest intact antigen-binding entities that can be derived from an in vivo affinity-matured antib ...

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Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig) Molecule

A dual-variable domain-immunoglobulin (DVD-Ig) is an engineered protein that combines the function and specificity of two monoclonal antibodies in one molecular entity. A DVD-Ig is designed as an IgG-like molecule, except that each light chain and heavy chain contains two variable doma ...

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Bispecific Diabodies and Single-Chain Diabodies

Bispecific antibodies can be applied for a broad range of diagnostic and therpaeutic applications, e.g., the retargeting of effector molecules and cells to tumor cells or other structures. This protocol describes the generation of bispecific diabodies and single-chain derivatives ...

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In Vivo Biotinylated scFv Fragments

Biotinylation of proteins is a rare process in almost all living organisms, whereas biotin-associated enzymes are key-enzymes for manifold essential metabolic pathways related to carboxyl transfer within cells. In vivo biotinylation of proteins via genetic engineering is high ...

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Fusion Proteins with Improved PK

Small recombinant antibody formats are rapidly cleared from circulation. The coupling of these molecules to human serum albumin (HSA) is an efficient strategy to prolong their in vivo half-life. Two methods are described in this chapter for the attachment of HSA to small antibody molecules: ...

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PEGylation of Antibody Fragments to Improve Pharmacodynamics and Pharmacokinetics

Covalent attachment of polyethylene glycol (PEG) with therapeutic proteins (PEGylation) e.g., antibodies, antibody fragments, and enzymes, has been emerged as an effective strategy to enhance the pharmacodynamics and pharmacological properties of biopharmaceuticals. F ...

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Disulfide-Stabilized Fv Fragments

The design and generation of disulfide-stabilized Fv fragments (dsFv’s) addresses stability and aggregation problems that are frequently associated with single-chain Fvs. VH and VL of dsFv’s are connected by an interdomain disulfide bond. To generate such molecules, one amino acid ea ...

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Phenotypic Knockdown with Intrabodies

Intracellular antibodies (intrabodies) are antibody molecules which are expressed intracellularly and which are located in defined subcellular compartments, for example, the endoplasmic reticulum (ER). The amount of any surface protein (target protein) on a mammalian cell can ...

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Expressing Intrabodies in Mammalian Cells

The intrabody approach is a gene-based technology developed to neutralize or modify the function of intracellular and extracellular target antigens. Since intrabodies can potentially target all different multiple isoforms of a protein, they can act as therapeutic molecules for t ...

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T Bodies

Genetic modification of T cells with antigen-specific chimeric receptors (CARs) has great potential to redirect their effector functions to a wide variety of therapeutic targets, including weakly expressed or poorly presented tumor antigens. The protocol describes the subclon ...

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Recombinant Immunotoxins for Treating Cancer

Targeted antibody therapy represents a promising approach for the treatment of cancer. However, antibodies that bind to cancer cells are often, by themselves, not cytotoxic. Recombinant immunotoxins (RITs) are chimeric proteins composed of the variable fragment (Fv) of a monoclonal ...

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Protein Sequence and Structure Analysis of Antibody Variable Domains

The protocols described here provide methods for computational analysis of antibody sequence and structure. With the availability of the World Wide Web, many online analysis tools have been made available, and URLs for these are cited throughout the text. The author has provided a Web site at ht ...

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Standardized Sequence and Structure Analysis of Antibody Using IMGT

Standardized sequence and structure analysis of antibody using IMGT�, the international ImMunoGeneTics information system� (http://www.imgt.org), allows to bridge, for the first time, the gap between antibody sequences and three-dimensional (3D) structures. This is achieved ...

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Analysis of Single Chain Antibody Sequences Using the VBASE2 Fab Analysis Tool

The VBASE2 database integrates sequences present in the various DNA sequence databases and selects germline-encoded variable gene segments from these databases. The sequences are grouped into three classes: the first class consisting of the variable gene segments that are proven g ...

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Binding Studies with Flow Cytometry

Flow cytometry allows the multiparametric physical and biochemical analysis of single cells within large cell suspensions which is most frequently for cell counting and for the measurement of antigen expression with fluorescence conjugated antibodies. Moreover, flow cytome ...

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Determination of Fc-Mediated Antibody-Effector Functions by Chromium Release Assay

Monoclonal antibodies (mAbs) can exert their biological functions by a large variety of mechanisms and have therefore become a major part in the treatment of diseases like viral infections and cancer. For the clinical efficacy of many mAbs Fc-mediated effector functions like antibody-d ...

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Quantification of Human IgG and Related Fc Fusion Proteins by a Human IgG/Fc Capture ELISA

The quantification of human immunoglobulin G (IgG) and recombinant derivatives is required for many applications in research and diagnostics. Particularly in research, robust and simple protocols are necessary to determine the concentration of recombinant human IgGs after pr ...

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Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo

Functional characterization of antibodies that inhibit soluble cytokines or chemokines requires robust, sensitive in vitro and in vivo bioassays. Testing an antibody in vitro requires consideration of antigen source, integrity, and concentration, as well as the magnitude of the b ...

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Neutralization Tests

Neutralization tests are performed in vitro, at the cellular level - including on cells in the form of organs - and at the sub-cellular level. They assess the efficacy of antibody fragments, or antibodies, to inhibit critical stages of activities. These activities are generally deleterious to H ...

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