抗体应用

One of the major advantages of flow cytometry is the simultaneous evaluation of multiple markers, especially surface markers (1). The detection of intracellular proteins is less well developed, in large part because antibodies can bind nonspecifically to dying cells and dead cell compo ...

Ion-exchange chromatography is a rapid and inexpensive procedure employed to purify antibodies partially from different sources and species (1–4). It is a particularly useful tool for isolating antibodies that either do not bind or that bind only weakly to protein A (e.g., mouse IgG1) (3). This p ...

The combination of the specificity of the antigen-antibody interaction with the exquisite sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology (1). Within the last decade, flow cytometry (FCM) has become an i ...

The extracellular matrix of mammalian tissue is composed of a complex mix of constituitive proteins. This matrix must be broken down to recover single cells effectively for culture and/or staining (1). Tissue dissociation and its affiliated problems were described and defined over 80 yea ...

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so ...

Although gold particles are readily detectable by transmission electron microscopy, they can be difficult to visualize by bright-field light microscopy. If the particle size is large enough and the labeling dense enough, the gold particles will stain tissue red (1, 2). However, unless the go ...

Enzyme-linked immunosorbant assay (ELISA) has been a powerful technique in immunology since it was first developed in 1971 by Engvall and Perlman (1). The technique is based on the fact that proteins can be absorbed nearly irreversibly to plastic (2). The antigen (or antibody) is immobilized in t ...

Immunohistochemical staining techniques are widely used for the identification of a variety of diverse antigens in paraffin-embedded tissues (1). Nonetheless, a major limitation in the use of paraffin-embedded fixed tissue is that many potentially interesting antigens are den ...

The labeled avidin binding (LAB) method is the latest method to use the avidin-biotin technology. It provides even more sensitivity than either the standard peroxidase-antiperoxidase (PAP) or avidin-biotin complex (ABC) methods see Chapter 20 and Chapter 21) (1). The technological dif ...

This method, described in 1981 by Hsu and associates, makes fundamental use of the covalent and irreversible binding seen between avidin, an egg white protein, and biotin, a vitamin (1). By establishing a biotin link, through avidin, between the horseradish peroxidase enzyme and a secondary an ...

The peroxidase-antiperoxidase (PAP) method was pioneered by Sternberger in 1979 (1). The method uses an immunological sandwich amplification and the enzyme peroxidase to effect a signal. The unique feature of this procedure is the enzyme-antibody solution, the PAP immune complex. The h ...

The use of enzymes along with immunoglobulins to identify specific substances emerged with the work by Nakane and Pierce, who labeled an immunoglobulin with the peroxidase enzyme rather than with a fluorescent compound (1). The difficulty with this approach lies in attaching a relatively ...

During the last century, before the advent of microtomes capable of producing thin, even sections, whole-mounts and thick sections were used to study the peripheral nervous system. Tissues were either viewed directly, if sufficiently thin, or further separated into layers by acid macera ...

Cells in culture offer a unique opportunity to visualize intracellular organelles. Because of the ability of some cultured cells to attach and spread on a substratum, the cell’s cytoplasm can be spread over a large surface area, resulting in a thin, broad cytoplasmic layer. In this layer, optical m ...

In this chapter, two commonly used techniques that are utilized in many immunoglobulin purification schemes are described. The first procedure, ammonium sulfate fractionation, is generally employed as the initial step in the isolation of crude antibodies from serum or ascitic fluid ( ...

The surfaces of living cells contain a wide variety of molecular species that are characteristic of the type and physiological role of the cell. These surface molecules mediate cell-recognition events, receptorligand interactions and hormonal signaling events, surface homeost ...

In their original immunofluorescent labeling technique, Coons and colleagues employed a one-step, direct labeling of antigen using a fluorescein-conjugated antibody (1, 2). Although this was not the first published report of a chemically modified antibody, it was the first time the ant ...

Since its inception in the 1940s, the technique of immunofluorescence has provided a sensitive, high-resolution method for analyzing the binding of antibody to antigen. Fluorescent molecules, termed fluorophores or fluorochromes, can be conjugated directly to antibodies by cov ...

Generating an antitumor immune response can be thought of as eliciting an immune response to cells derived from self-tissue. As such, tumor immunity may result in autoimmunity. Melanoma patients undergoing immunotherapy often develop a form of autoimmune depigmentation referred to ...

The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and re ...