微生物学技术

Interferons are the antiviral early inflammatory proteins produced in the cells in response to the infectious agents. The characterization of the interferon genes, their expression, and their function was advanced with the development of novel techniques in molecular and cellular ...

The primary focus of this chapter is on providing an overview of how we use the tools of functional genomics to study virus infection, the interferon response, and the mechanisms by which viruses attenuate or evade this response to ensure successful replication. We provide examples of the types of ...

The clinical symptoms associated with rotavirus (RV) gastroenteritis are not sufficiently characteristic to distinguish between RV infection and other causes of gastroenteritis. Therefore, laboratory procedures, including electron microscopy (EM) (1), enzyme-linked ...

We provide procedures for the panning of fully humanized Fab antibodies using guided selection. Human heavy and light chain genes are amplified. A parental light chain is cloned into a phage display vector and combined with the heavy chain library. After several rounds of panning, positive clo ...

This chapter outlines a protocol for the selection by phage display of single-chain variable antibody fragments with dual properties-specificity for tumor cells and the ability to be internalized. The protocol is based on a direct incubation of living target cells with antibody phage di ...

Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against patho ...

Conventionally, antibody phage display has been used to isolate recombinant antibodies that are monovalent in their interaction with target antigens. These antibodies can be reengineered for expression in mammalian cell culture as full-length, monospecific immunoglobuli ...

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 ...