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丁香实验推荐阅读
Selection of Antibodies Against Biotinylated Antigens

Phage antibody (Ab) library selections on peptides or proteins are usually carried out using antigens (Ags) directly coated onto a plastic surface (e.g., Petri dishes, microtiter plate wells, and immunotubes). This straightforward method is easy to perform and has been shown to be successf ...

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Isolation of Anti-Hapten Specific Antibody Fragments from Combinatorial Libraries

The generation of high-affinity antibodies (Abs) against hapten targets (molecular weight below 1000 Dalton) presents particular problems not encountered with larger antigens (Ags). By their nature, haptens are invisible to the host immune system unless presented as an epitope con ...

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Rescue of a Broader Range of Antibody Specificities Using an Epitope-Masking Strategy

The use of combinatorial libraries displayed on the surface of filamentous bacteriophage offers an efficient route to obtain a diverse set of monoclonal antibodies (MAbs) with desired specificity (1,2). However, following selection of such libraries, certain epitope specificit ...

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Screening of Phage-Expressed Antibody Libraries by Capture Lift

Cloning and expression of functional antibody fragments (Fabs) in bacteria and on the surface of filamentous phage has revolutionized the processes by which antibodies (Abs) are discovered and engineered. Cloning, expression, and screening efficiencies of phage systems permit t ...

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Antibody-Guided Selection Using Capture-Sandwich ELISA

Antibody (Ab) phage display is a recently developed recombinant DNA technology for making human monoclonal antibodies (MAbs) from immune sources, such as bone marrow, lymph node, or peripheral blood lymphocytes from patients with various diseases, or from healthy individuals (1,2). M ...

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Isolation of Human Monoclonal Antibodies Using Guided Selection with Mouse Monoclonal Antibodies

Repertoires of antibody (Ab) V genes derived from nonimmunized human donors (1) or made synthetically (2 3) have been cloned for display on filamentous bacteriophage as either scFvs or Fabs fused to the minor phage coat protein (pIII) (4). Phage Ab repertoires can be subjected to multiple rounds of p ...

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Proximity-Guided (ProxiMol) Antibody Selection

Cell surfaces provide a rich source of potential antigen (Ag) targets for therapeutic and research reagent antibodies (Abs). However, in some circumstances, access to these targets may be difficult since it is technically challenging to purify individual Ags while retaining their nat ...

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Selecting Antibodies to Cell-Surface Antigens Using Magnetic Sorting Techniques

As described in other chapters, the selection of phage-displayed immunoglobulin (Ig) fragments with desired specificity can be accomplished through successive rounds of panning on purified antigen (Ag). However, for many applications, the target Ag may not be able to be purified becau ...

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Overview of Antibody Phage-Display Technology and Its Applications

The generation of new drugs has long involved the screening of hundreds of thousands of components with well defined in vitro tests, seeking compounds to mimic as closely as possible the desired in vivo activity of the new drug. New library methodologies offer many alternative routes that are at le ...

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Isolation of Human Tumor-Associated Cell Surface Antigen-Binding scFvs

Several approaches have been used to identify antibody (Ab)-defined human tumor-associated antigens (TAA) that can be used for passive and/or active specific immunotherapy of malignant diseases. For example, hybridoma technology has been successfully used to identify TAA recog ...

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Selection of Antibodies Based on Antibody Kinetic Binding Properties

Molecular evolution approaches to developing molecules with characteristics particularly suited for specific applications have become important tools in biomedicine and biotechnology. Not only is it possible to identify molecules with specificities that cannot easily ...

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Subtractive Isolation of Single-Chain Antibodies Using Tissue Fragments

Cell and/or tissue-specific phage antibodies (Abs) are usually generated using single-cell suspensions as a starting material for selection. However, isolation of Abs specific for certain cells and/or tissue components may be hampered because the natural architecture of the tiss ...

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Selection of Functional Antibodies on the Basis of Valency

Antibodies (Abs) displaying an agonist or antagonist activity are powerful tools for mimicking or blocking physiological functions in the cell. A number of applications of Abs in diagnosis and therapy require multivalent reagents, either because biological activity depends on the ...

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Targeting Random Mutations to Hotspots in Antibody Variable Domains for Affinity Improvement

The potential of Fvs as magic bullets for targeting therapeutic drugs or imaging agents is well-documented. However, the conversion of whole antibodies (Abs) into Fvs (scFvs or dsFvs) is often associated with a drastic reduction in antigen (Ag)-binding affinity. For efficient use of Fvs as tar ...

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Two-Step Strategy for Alteration of Immunoglobulin Specificity by In Vitro Mutagenesis

A two-step strategy for changing the specificity of antibodies (Abs) is presented, which we have used to change the specificity of an Ab from 11-deoxycortisol (11-DOC) to cortisol (CS). Two kinds of in vitro mutagenesis are utilized in this protocol: first, mutations are introduced at restricted ...

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Use of Escherichia coli Mutator Cells to Mature Antibodies

Despite the power of antibody (Ab) phage-display technology, a problem which can be commonly encountered is the recovery of Abs of low affinity for the antigen (Ag) of interest. Two general strategies can be applied to increase affinity: mutations can be scattered randomly throughout the gene ...

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Error-Prone Polymerase Chain Reaction for Modification of scFvs

The use of antibody (Ab) molecules and their fragments in research, diagnosis, and therapy has prompted the development of methods to improve their affinity and stability to increase their expression levels and to change or improve their specificity. This is easier to carry out on Ab fragments ( ...

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Chain Shuffling to Modify Properties of Recombinant Immunoglobulins

Combinatorial libraries and selection of variants from such libraries have proven to be a successful approach for identifying molecules with novel or improved properties. The importance of antibody (Ab) molecules in basic and applied research, as well as the extensive knowledge of how t ...

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High-Level Periplasmic Expression and Purification of scFvs

In the past few years, some of the limitations of monoclonal antibodies (MAbs) as therapeutic agents have been addressed by genetic engineering. Such an approach is particularly suitable because of the domain structure of the Ab molecule, where functional domains carrying antigen (Ag)-b ...

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Standard Protocols for the Construction of Fab Libraries

Fab libraries, in which light-chain (LC) and heavy-chain (HC) variableregion genes are cloned into a phagemid vector and subsequently displayed on the surface of the filamentous phage particle, have been widely used for the isolation of antibodies (Abs) with specificity for haptens, fore ...

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