实验基础

Immunohistochemistry comprises methods used to recognize tissue components as antigens in situ by means of directly or indirectly labeled antibodies, usually (but not always) derived from another species. When applied to cell preparations, the same methods are called immunocyto ...

Cytokines are known to play a key regulatory role in immune responses. The onset or progression of immunopathology in various diseases is often associated with aberrant production of one or more cytokines. It is therefore of considerable interest to characterize cytokine “profiles” as ...

Celiac disease is characterized by mucosal changes in the small intestine, ranging from increased numbers of intraepithelial lymphocytes to complete villus effacement with various signs of malabsorption (1,2). Celiac disease is diagnosed by the demonstration of an altered intes ...

Celiac disease and the related condition dermatitis herpetiformis are caused by ingestion of wheat gluten (gliadin being the active moiety) and certain other cereal proteins.

Studies of intestinal mucosal immunity and inflammation are limited by the relative inaccessibility of most of the small intestine. Any new method of studying mucosal immunity and inflammation in patients should be minimally invasive, cost-effective, and provide information not r ...

Historically, the term celiac disease evolved within pediatric practice during the nineteenth century, defining children with severe wasting and putrid stools (1). In the earlier twentieth century, similar complaints in adults were categorized as “intestinal insufficiency” or ...

This chapter details DNA extraction through polymerase chain reaction (PCR) amplification, gel running, allele assignment, and data management so that the genotyping data produced is suitable for use in linkage analysis programs.

Many disorders such as celiac disease do not conform to a simple Mendelian model of inheritance and display a complex pattern of inheritance indicative of the interaction of a number of distinct susceptibility genes. Susceptibility to celiac disease is genetically determined by posse ...

Linkage analysis in families containing affected individuals can be used to identify the location of disease susceptibility genes. The aim of this chapter is to provide an overview of the molecular methods employed to clone these susceptibility genes on the basis of linkage data.

The wheat proteins, which are active in celiac disease and other glutenrelated conditions, are defined as prolamins, in that they are soluble as individual subunits in alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60–70% (v/v) aqueous ethanol. However, in wheat grain and flour, ...

In recent years there have been major developments in the field of mass spectrometry (MS) that permit the analysis and characterization of peptides and proteins at the femtomolar level (1–8). This chapter deals with the use of MS for the elucidation of peptide sequences of gliadin- and/or gluteni ...

This chapter describes a methodology for elucidating immunogenic epitopes stimulatory for CD4+ T-cell clones (Fig. 1). The methodology makes use of synthetic peptide libraries and must be regarded as an alternative to other approaches, such as peptide elution or the application of genet ...

Several immunological disorders display a striking association with particular HLA alleles. Although the basis for these HLA-disease associations is not completely understood, it is likely that peptides bound to the disease-associated molecules play a role in pathogenesis. The ...

Celiac disease is an immune-mediated disorder that primarily affects the small intestinal mucosa. It is one of the few human disorders of which it is possible, and ethically acceptable, to obtain samples from the disease-affected tissue. This chapter describes how small intestinal biopsy ...

Large-insert genomic DNA libraries are based on the Escherichia coli F factor, a low-copy plasmid that exits in a supercoiled circular form in the host cells. These libraries are used to provide a way to divide complex genomes into DNA segments, thereby reducing the complexity (1). The libraries are ...

Recent advances in the Human Genome Project have opened the door to new approaches in biologic research. The availability of the human draft sequence (1) now offers the tool for sequence-based genetic analyses on a genomewide level. However, owing to the fragmented and incomplete nature of the d ...

Shotgun cloning is a method to generate the templates needed for DNA sequencing. This process entails breaking a large target DNA randomly into smaller fragments; end sequencing these smaller fragments; and from the overlapping sequences of the randomly generated fragments, reassem ...

Robust and reproducible isolation of high-quality templates is a requirement for successful DNA sequencing. To date, approaches for template generation have been limited to purification of biologically propagated M13 or plasmid-based templates, or in vitro amplification of su ...

Transposon-mediated sequencing is an effective method for obtaining full-length high-quality DNA sequence. This method can be applied to the finishing stages of bacterial artificial chromosome (BAC) sequencing, allowing the user to expand a finishing repertoire of custom oligo ...

Pyrosequencing, a bioluminometric DNA sequencing technique based on sequencing by synthesis, is emerging as a widely applicable tool for detailed characterization of nucleic acids (1–3). This technique relies on the real-time detection of inorganic pyrophosphate (PPi) releas ...