实验基础

DNA supercoiling in eukaryotic viruses was first described by Vinograd and co-workers more than 30 years ago (see ref. 1 for a review), but its origin was recognized to result from DNA complexation with host histones to form minichromosomes only 10 years later (2). Because they are in contact with endo ...

The assembly of DNA into the eukaryotic nucleus via an ascending hierarchy of intermediate chromatin structures has two major functional consequences: 1. The intermediate chromatin structures provide a filing system that greatly facilitates the search by RNA polymerase for regul ...

In the past five years, there have been numerous molecular and genetic investigations of the mechanisms by which chromatin condenses into interphase chromosomal fibers (1,2). In addition to traditional molecular techniques such as electron microscopy (3–5) and analytical ultrace ...

The ability of analytical ultracentrifugation to elucidate chromatin structure/function relationships originates directly from its capacity to accurately measure key structural properties of complex macromolecular assemblies in solution. Figure 1 schematically ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...