生物化学技术

Over the past 20 years, amino acid analysis methods based on precolumn derivatization procedures have become a popular alternative to the traditional postcolumn derivatization techniques based on ion-exchange separation of the underivatized amino acids. The major reasons for t ...

Precolumn derivatization of amino acids followed by their resolution by reverse-phase high-performance liquid chromatography (RP-HPLC) is now the preferred method for quantitative amino acid analysis. The derivatization step introduces covalently bound chromophores ...

During the 1980s and 1990s, electrochemical detection has gained acceptance as a method of choice for some important biomolecules. Initially, the technique was utilized with glassy carbon electrodes and in the mode of constant potential amperometry. The first widely used applicatio ...

The amino acids we have the most experience with are GABA, glutamate and arginine, though other amino acids can be measured. The method used is based on a derivatization reported by Jacobs (1) using o-phthalaldehyde in the presence of sulphite. The method was used by Pearson et al. (2), Smith and Sharp (3), and ...

There are several highly sensitive methods for identifying proteins: N-ter-minal sequencing, peptide mapping, and Western blotting, which are based on partial information about the structure of the protein. The amino acid composition of a protein is reflected in the primary structure ...

Gas chromatography (GC) has been widely utilized for amino acid analysis because of its inherent advantages of high resolving power, high sensitivity, simplicity, and low cost. However, the main problem is the necessity to derivatize the amino acids into less polar and more volatile compoun ...

Since its introduction in 1975 the methodology of Kohler and Milstein (1) for production of monoclonal antibody (MAb) from hybridoma cells has been widely used to provide antibodies with a defined specificity. One characteristic feature of this technology is that impure antigens can be us ...

The use of antigen fragments generated by specific chemical cleavage is a relatively simple “library” approach for epitope mapping in which overlapping fragments are screened with antibody on Western blots. It is widely applicable insofar as it is not restricted to recombinant antigens ...

Techniques that have been used for antigenic site mapping of linear epitopes in proteins include binding assays of protein components produced by synthetic chemistry (1,2) or by recombinant gene expression (3,4). More recently, epitope localization has been achieved through the use of s ...

Multiple peptide synthesis gives access to a set of reagents that permit thorough answers to such questions as: Where are all the linear epitopes in this protein? How long is the critical part of each epitope? Which epitopes are commonly recognized and which are rarely recognized? What are the affin ...

Positional scanning synthetic combinatorial libraries (PS-SCLs) are useful in identifying the amino acid sequences of antigenic determinants recognized by monoclonal antibodies (MAbs). These libraries are typically composed, in total, of tens of millions of nonsupport-bo ...

Monoclonal antibodies (MAbs) play a key role in defining structural, functional, and regulatory aspects of complex protein-protein interactions (1–4). The ability to identify the epitope recognized by an antibody and to understand how it relates to the primary or tertiary structure or t ...

Filamentous bacteriophage can tolerate the insertion of a foreign DNA into their gene encoding phage minor coat protein III. Such recombinant phage particles display on their surface foreign peptide sequences fused with the gene III product, which is a protein represented by five copies on ...

The high specificity of monoclonal antibodies (MAbs) enables them to discriminate subtle sequence and structural differences among homologous proteins. They have thus become important tools for serotyping of viruses and bacteria (1–3), and for typing of homologous proteins from m ...

The natural immune response against pathogens can be extremely varied, ranging from a strong, broad humoral and cellular response capable of stopping disease progression to a weak, inadequate, or even deleterious response inefficient at controlling replication of the infectious a ...

This technique was developed for mapping of epitopes of monoclonal antibodies (MAb) at the amino acid sequence level; i.e., the identification of amino acids of an antigenic protein involved in specific interactions with each antibody. The epitope mapping includes:

Two of the major aims of immunology are to understand the molecular basis of antibody-antigen interaction and the elicitation of the immune response. Epitope mapping techniques are useful in defining these processes.

The efficient detection of antigenic epitopes in a protein by screening a recombinant λ phage expression sublibrary of the antigen cDNA was first described by Mehra et al. (1). Subsequently, the method has found multiple applications. We have used it to identify the epitopes for a panel of monoclo ...

Phage-display peptide libraries (1–4) have become powerful tools for identification and characterization of peptide mimicries that bind to specific selector molecules, such as antibodies (5–7). The technology depends on random peptide sequences, displayed on the surface of fila ...

Antibodies induced by native antigens often crossreact with denatured antigen or with peptides that contain a segment of the antigen polypeptide chain. The epitopes recognized by these antibodies are linear or sequential, i.e., specified by the linear sequence of the amino acid residues ...