实验基础

The term “proteomics” describes the technologies collectively used to define the protein complement of the genome or “proteome” (1,2). The recent growth of this discipline is reflected in the many review articles available (3–7). In addition to describing all the proteins encoded by the geno ...

Completion of the sequence of the entire genome of strain H37Rv was a benchmark for Mycobacterium tuberculosis research (1). This achievement ushers in the era of genome-wide functional and comparative genomics for this organism. At present, the most powerful enabling technology of the p ...

The storage and maintenance of mycobacterial reference strains and clinical isolates are important parts of good laboratory practice in a mycobacterial laboratory. The storage of reference and control strains facilitates a reliable control on intra- and inter-test reproducib ...

Research into and identification of Mycobacterium tuberculosis can take on a number of facets, many of which involve the use of DNA at one stage or another. The quality and quantity of DNA required will depend on the end-use requirement. For example, good yields of pure, high-molecular-weight DNA un ...

The worldwide resurgence of tuberculosis (TB) has resulted in a rapid expansion in research efforts directed at a deeper understanding of the disease, the efficacy of vaccines, and improved drug targets. This has required the establishment of multiple new facilities to contain the pathog ...

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term per ...

Genetic analyses of pathogenic mycobacteria such as Mycobacterium tubeculosis and Mycobacterium bovis required improvement of existing methodologies for the generation of large representative libraries of mutants. Two basic methodologies have been used to generate mut ...

Gene replacement and transposon mutagenesis are two complementary tools that have been widely used to perform genetic studies in various living organisms. In mycobacteria, and especially in the Mycobacterium tuberculosis complex, the lack of these tools has severely hampered the g ...

Much progress has been made in mycobacterial research in general and in mycobacterial genetics in particular during the past 10 yr. The complete genome sequences of two isolates of Mycobacterium tuberculosis, the widely distributed laboratory strain M. tuberculosis H37Rv (1) and a clin ...

Gene replacement by homologous recombination (HR) is an invaluable tool in understanding the physiology and the significance of specific genes in the virulence of Mycobacterium tuberculosis. It will also allow for the development of rationally attenuated strains as candidate vac ...

The production of cytokines can be controlled by the regulation of transcription, by the regulation of translation, and by post-translational mechanisms. Therefore, to understand better the control of cytokine production, it is important to measure both concentration of the free cyt ...

The multiprobe ribonuclease protection assay (RPA) is a highly sensitive and specific method for simultaneous detection and quantification of several species of mRNA. Three most distinct advantages of the multiprobe RPA method are: high sensitivity and specificity, capacity to si ...

Cytokines belong to a family of immunoregulatory peptide growth factors that are produced mainly by immune cells after immune challenge. Various cells of nonimmune cell origin such as epithelial and muscle cells are also capable of producing cytokines, as well as many tumor cells (1, 2). We have r ...

The isolation of total and bioactive cytokines can be achieved by a combination of immunoaffinity and immobilized receptor chromatography. The former procedure isolates the total amount of a specific cytokine, and the latter procedure isolates the bioactive fraction of the immunoa ...

Circadian rhythmicity is a prominent feature of many important biologic functions. Failure to take such variation into account when performing assays may lead to increased assay variation or, most importantly, an erroneous result. Circadian rhythms of immunologic relevance incl ...

ELISA (enzyme-linked immunosorbent assay) is a powerful, versatile, precise, and reliable quantitative technique for the measurement of antigens or antibodies in biologic samples. The ELISA technique is a widely used tool in biologic and biomedical research; it has been modified and a ...

From their genesis as instruments designed to count and size particles, flow cytometers have, over the last 40 years, evolved into a range of sophisticated instruments. These instruments are now used widely in all branches of biologic science. Originally, the preserve of the research labora ...

The ordinary method for quantitative analysis of cytokines consists of measuring cytokines produced and accumulated in the supernatants of shortterm cultures, by means of enzyme immunoassay. However, this approach provides only cumulative amounts in a fixed time, thus limiting the ...

The enzyme-linked immunosorbent assay (ELISA) spot (ELISPOT) procedure is basically a modification of the plaque techniques, hence its initial synonym ELISA plaque assay. Plaque assays allow the enumeration of antibody-secreting cells by diluting them in an environment in which the ...

As an alternative to the ELISPOT assay described in previous chapters, the expression of known cytokines can be analyzed at the mRNA level. Although expression of mRNA for a particular cytokine does not imply corre-sponding expression of the protein, both techniques in combination provide ...