Methods in molecular medicine最新文献

After air sampling and elution, the air sample eluate contains an unknown amount of allergens together with other materials. The proteins of interest can be quantified using immunoassays, which are sensitive, economical, and can be used for high throughput. However, the amount of antigen or allergen in an air sample may be very low and consequently the assays must be very sensitive and specific. Immunoassays use antibodies both to capture and visualize the chosen antigen. High specificity and sensitivity can best be achieved by the use of purified, characterized, and specific antibodies. It is possible to choose between a wide variety of assay setups and reagents. The method described here has been developed for the measurement of airborne rodent allergens. It is a noncompetitive, two-site (sandwich) EIA that utilizes polyclonal antibodies. The detection system uses biotin and streptavidin for increased sensitivity and horseradish peroxidase as the substrate with 3,3',5,5'-tetramethylbenzidine (TMB) for rapid color development and high sensitivity.

Many naturally occurring proteins, peptides, carbohydrates, nucleic acids, and lipids, as well as synthetic peptides, are successful immunogens. To elicit an immune response, a compound must contain an antigenic determinant or epitope and must be of sufficient size to initiate lymphocyte activation necessary for an antibody response. In practice, small chemical compounds (haptens) are generally not good immunogens. However, when attached to macromolecules (carriers), they can become immunogenic. An immunogen must have epitopes that can be recognized by antigen-presenting cells and a T-cell receptor, and it must be degradable. Haptens and corresponding hapten-carrier conjugates have been essential to the development of sensitive quantitative and qualitative immunoassays. In the design of hapten conjugates, consideration must be given to the hapten, the carrier, the coupling strategy, and the hapten density because the amount of hapten attached to the carrier influences the strength of the immune response directed toward the newly created antigenic determinant. Hence the haptenic density of the conjugate is also important in the development of immunoassays. The optimal epitope density of a conjugate to elicit either a strong immune response or provide the best immunoassay is dependent on the structure of the epitope and the nature of the immunoassay. The aim of this chapter is to describe the diverse techniques used to couple haptens to carriers and provide guidance in the selection of the most appropriate procedure for a particular hapten.

The prevalence of allergic disease has dramatically increased over the past 30 years in Westernised countries. It is unlikely that the rapid increase in the prevalence of allergic disease is the result of genetic changes, which highlights the importance of environmental factors in the development of allergic disease. The 'hygiene hypothesis' was put forward in 1989 and focused attention on the notion that exposure to microbes and their products in early life can modify the risk for development of allergic disease. Infections were thought to polarize the immunological response towards a Th2-mediated immune responses causing allergic disease. However it is likely that the Th1/Th2 imbalance is too simplistic to explain the increased prevalence of allergic disease. Current research is focusing on understanding the role of T regulatory cells in inducing a state of tolerance and the resulting modified Th2 response observed in natural and induced tolerance.

The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.DNA polymerases are unique enzymes with respect to the five substrates (four dNTPs, one of which is radiolabeled, and primer:template DNA) that they typically utilize. Variations of the assay, e.g., by leaving out one dNTP but allowing measurable incorporation of the remaining substrates, or use of homopolymer primer:templates, may be used to simplify the assay or to obtain mechanistic information about inhibitors. Use of gel analysis of primer extension assays can also be applied to study alternate substrates of DNA polymerases. Methods to isolate pol IIIC from Gram-positive bacterial cells and to clone and express the polC gene are described in this chapter. In addition, the assay conditions commonly used to identify and study the mechanism of inhibitors of pol IIIC are emphasized.