实验基础

A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a ye ...

The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tes ...

We describe procedures for intracellular expression of scFv in eukaryotic cells. Starting from a scFv gene cloned in a phage-display vector, we describe the cloning step into a mammalian expression vector, the transient transfection of a HeLa cell line, and the monitoring of intrabody expr ...

The most frequently used approach to produce single-chain Fv fragments (scFv) and Fab in Escherichia coli is to express them in the periplasm of the bacteria. We present here an alternative procedure that uses cytoplasmic expression of soluble active scFv. This can be accomplished by using eit ...

Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platform ...

This chapter deals with relatively simple ways to use control charts to monitor the performance of ELISAs. A rinderpest competition ELISA, for the estimation of antibodies in serum samples, is used to demonstrate the methods. This assay is available in kit form. Constant evaluation of the use of t ...

Interferon (IFN)-αs constitute a family of proteins exhibiting high degree of homology in primary, secondary, and tertiary structure and display a high level of species specificity in their biological properties. However, small structural differences in these proteins may be respo ...

Inhibition of protein synthesis by interferon treatment is mediated by two major pathways: the 2′–5′-linked oligoadenylates synthetase-RNase L pathway and the double-stranded ribonucleic acid-dependent protein kinase-mediated pathway. 2–5 (A) synthetases are unique inte ...

Interferon action against viruses is mediated in part through a ribonucleic acid (RNA) decay pathway known as the 2–5A system. Unusual 5′-triphosphorylated, 2′,5′-linked oligoadenylates (2–5A) are produced in mammalian cells by interferon-inducible 2–5A synthetases (OAS) in resp ...

Despite a key role in the regulation of interferon (IFN) gene expression and in mediating many downstream actions of the IFNs, the regulation of IFN regulatory factor (IRF) gene expression in the normal and pathological central nervous system and, indeed, other tissues, is poorly defined. We so ...

There is recent evidence that in addition to the classic JAK/STAT pathways, mitogen-activated proteins (MAP) kinase pathways play important roles in Type I interferon signaling and the generation of interferon responses. In particular, the p38 MAP kinase cascade exerts positive regu ...

Small cationic peptides with antibiotic properties have been isolated from a diverse array of evolutionarily divergent organisms, including insects, amphibians, mammals, and plants. They contribute to the innate immunity of the host by fending off opportunistic (i.e., environme ...

It is now over 20 years since dendritic cells (DC) were first identified in and isolated from the spleens of mice (1,2) and they continue to be a much-studied population. Only a small proportion of spleen cells are DC, but the large size of the organ means that useful numbers of DC can still be purified. In recent yea ...

Lymph nodes are the primary sites of T-cell stimulation by dendritic cells (DC). After contact with antigens, DCs migrate to draining lymph nodes from the skin and other tissues (1-3). Investigation of the morphology and function of lymph node DCs may provide important information about the role ...

The method described in this chapter for the isolation of mouse thymic dendritic cells (DC) is an optimization of our previously published methods (1,2) and involves the following major steps:

Dendritic cells (DC) are rare cells in peripheral tissues, and their isolation from tissues is fraught with problems. Thus, the proportion of DC within a tissue that is extracted is unknown, isolation procedures may select for subpopulations, and the isolation procedure itself may affect th ...

Langerhans cells are the epidermal variant of the dendritic cell system (1-5). They were-unknowingly, though-the first dendritic cell to be described: In 1868 Paul Langerhans published his observations of a dendritically shaped cell in the human epidermis (6). Until the early 1990s (i.e., the ...

Dendritic cells (DC) are a trace cell population found in most tissues that display potent antigen acquisition and presentation capabilities and are unique among antigen-presenting-cell types in their ability to activate unprimed T cells (1). DC are particularly prominent at the muco ...

This chapter describes techniques for the isolation of dendritic cell-enriched single-cell suspensions from the lamina propria (LP) of both small and large intestine and from Peyer’s patches (PP). This technique can also be used to obtain intestinal macrophage-enriched populatio ...

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) derived from precursors within the bone marrow (BM). They are distributed ubiquitously throughout the body, and are few in number (1). They are classified as lymphoid-related or myeloid DC depending on their dev ...