生物化学技术

It is often the case that one has a cloned gene for a protein and a monoclonal antibody (MAb) that recognizes an epitope within that protein. Determination of the amino acid sequences constituting an epitope recognized by the MAb can be problematic. Producing ever and ever smaller fragments of the pr ...

The knowledge of the distribution of ligand binding sites on extracellular domains of cell-surface molecules is important both in basic research and in the context of drug development. Mapping of binding sites of pathogens on the cell-surface molecules that they use for entry into the cell may ...

Epitope mapping of proteins defines the structural elements of a molecule needed for recognition by individual antibodies. X-ray crystallography of anti- body-antigen complexes (1) is perhaps the most precise means of defining an epitope. However, several quicker but less detailed m ...

In vitro protein synthesis often leads to the production of polypeptide chains of intermediate lengths in addition to the full-size polypeptide. This accumulation of nascent chains of discrete sizes has been observed for a variety of proteins and has been attributed mainly to ribosome pau ...

X-ray crystallography provides a powerful tool for the study of antigen- antibody interactions. Information provided by crystallographic studies of antigen-antibody complexes includes the topological description of intermolecular contacts and the nature of interacti ...

The epitopes recognized by the antigen receptors on T-lymphocytes (T-cells) consist of a molecular complex made up of a self-protein and a short linear peptide (1). The self protein is a product of the major histocompatibility gene complex (MHC), and is classified as either a Class I or a Class II molecul ...

This brief chapter is less of a protocol and more of a reminder that valuable information on the location of epitopes can be obtained by studying antibody binding to naturally occurring variants of the antigen. These variants are usually antigens from different species or different isoforms ...

Since the first description by Kohler and Milstein (1), many variations on this method for the production of monoclonal antibodies (MAbs) have appeared (e.g., 2–4, and it may seem superfluous to add another. The variation we describe here, however, includes a number of refinements that enable rap ...

NMR spectroscopy has been used to study antibody complexes with antigenic peptides. A crucial parameter in NMR studies of large biological complexes is the rate of exchange of a ligand between its free and bound states. In cases where the peptide off-rate is fast (faster than 10 s−1) relative to the dec ...

The development of genetic engineering has enabled the production of antibodies in Escherichia coli (1,2). An essential requirement for a good antibody expression system is that an antibody fragment is folded and in a functional state so that the selection or purification procedure can ma ...

The mapping of B-cell epitopes on antigen molecules is best done with monoclonal antibodies (MAbs). Most methods require physical chemical procedures for purifying, binding, or labeling the antigen or the MAb. These procedures require some technical proficiency, may present method ...

The BIAcore (biomolecular interaction analysis) analytical system consists of a detection unit, an autosampler, and a liquid delivery system, controlled by a computer. Karlsson et al. (1) have given a detailed description of the analytical system, the detection principle, and the theor ...

Antibodies are directed against three-dimensional features of proteins, and the recognition of an epitope by an antibody is always a fit of structures in three-dimensional space. In the case of antibodies to native proteins, most or perhaps all epitopes are discontinuous (1). Because of the l ...

Chemical modification of the side-chains of residues in protein antigens was one of the first methods developed to investigate epitopes. Together with proteolytic fragmentation, it played a major role in the pioneering efforts of Atassi and others to assign antigenic determinants on t ...

Asymmetric aryl alkyl sulfides (R-S-R′) are metabolized by flavin-containing monooxygenase (FMO) and cytochrome P450 enzymes to enantiomerically enriched sulfoxide products (R-SO-R′) that are readily analyzed with a host of commercially available chiral stationary phases. ...

An approach to raising antibodies is described that can be applied to the majority of cytochrome P450 (P450) enzymes. Its application is limited only by the availability of suitable protein sequence information. The method is based on immunizing animals with synthetic peptides that mimic ...

Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) ana ...

When a new chemical entity is tested for its safety for humans and their environment, in vivo experiments on living animals are usually conducted. However, in the early preclinical stage of drug development, in vitro techniques, and more specifically hepatocyte-based in vitro models, are cu ...

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of t ...

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the “gold standard” for investigating xenobiotic biotransformat ...