抗体抗原实验

This chapter provides an introduction to the use of fluorescent probes in flow cytometry. Sample preparation for the use of surface labeling with antibodies as well as for the use of nucleic acid probes is discussed. The utility of cell sorting is also discussed.

The Clinical Laboratory Improvement Amendments (CLIA) set standards designed to improve the quality of all laboratory testing. In the first portion of this chapter, we discuss the CLIA requirements that apply to most Immunohistochemistry laboratories, and explain topics such as cer ...

Immunohistochemistry is widely used to identify, in situ, various components of cells and tissues in both normal and pathological conditions and is an exceptionally powerful method to demonstrate the localization of cellular components. Immunoglobulins (antibodies) are glyc ...

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies inclu ...

Antibodies can be purified by a variety of methods based on their unique physical and chemical properties such as size, solubility, charge, hydrophobicity and binding affinity. This chapter focuses on ammonium sulfate precipitation as a convenient first step in antibody purification ...

Affinity chromatography relies on the reversible interaction between a protein and a specific ligand immobilized in a chromatographic matrix. The sample is applied under conditions that favor specific binding to the ligand as the result of electrostatic and hydrophobic interact ...

Ion exchange chromatography techniques are the focus of this chapter and they showcase the power of this method for the purification of proteins and monoclonal antibodies. The technique is powerful and can separate biomolecules that have minor differences in their net charge, e.g., two pro ...

Live cells are often studied, in vitro, bathed in nutrient growth media. It is sometimes necessary to study individual compounds produced by these cells and antibodies work well for this purpose. These cells must first be concentrated and fixed before testing. There are a couple of ways to study cel ...

The development of heat-induced antigen (epitope) retrieval (HIER) technologies has led to dramatic improvements in our ability to detect antigens in formalin fixed, archival tissues. Paradoxically, wet heat treatment at temperatures greater than 95�C in appropriate buffer sol ...

Fixation is one of the most critical steps in immunostaining. The object of fixation is to achieve good morphological preservation, while at the same time preserving antigenicity. Tissue blocks, sections, cell cultures or smears are usually immersed in a fixative solution, while in other si ...

In order to detect intracellular antigens, cells must first be permeabilized especially after fixation with cross-linking agents such as formaldehyde and glutaraldehyde. Permeabilization provides access to intracellular or intraorganellar antigens. Two general types of ...

Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, ...

The ability to determine the expression dynamics of individual genes “in situ” by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular p ...

In an age of digital imaging, photographic film still provides a viable and effective means for recording fluorescence images by photomicrography. To maximize the quality of results that are obtained, a photographic emulsion with sufficient sensitivity for the low light level charac ...

Born out of the need to overcome an imaging problem in the 1950s, confocal microscopes today allow researchers to go beyond simple imaging and ask biochemical questions. This chapter provides background information on the development of modern confocal microscopes, their uses and appl ...

Laser capture microdissection (LCM) is a technical approach for obtaining microscopic samples as small as individual cells from tissues for molecular analysis. While the principles and details of the operation of LCM instruments, the technical requirements for obtaining identi ...

Immunoenzyme procedures take on many forms, including, simply, antibody coupled to enzyme. These direct techniques require the labeling of all the primary antibodies and can produce more background. A more economical method uses a secondary antibody or one that has the primary antibody as ...

Immunoenzyme methods can be enhanced by the use of the high affinity molecules, avidin and biotin. The binding of avidin to biotin is almost irreversible. By labeling a detection enzyme such as horseradish peroxidase with biotin, and a secondary antibody (reactive against the antigen detec ...

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is an useful technique for the quantitative analysis of cell surface antigen expression that was developed on the basis of enzyme immunohistochemistry (EIH) and ELISA. Since its development, which was made possible by the estab ...

The rapid detection of nucleotide mutations conferring drug resistance is especially important for organisms with long generation times. Mycobacterium tuberculosis, an organism thought to infect around one-third of the global population, is probably the most important of these ...