Labels indirect primary

The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (1). Later, the more sensitive and versatile indirect method (2) was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody. 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

In some cases, the preparation of a fluorescently labeled antibody is not even necessary. Particularly, if indirect methods are used to detect antibody binding to antigen, then preparing a fluorescently labeled primary antibody is not needed. Instead, the selection from a commercial source of a labeled secondary antibody having specificity for the species and class of primary antibody to be used is all that is required. However, if the primary antibody needs to be labeled and it is not manufactured commercially, then a custom labeling procedure will have to be done. 

Correspondingly, two principal immunohistochemistry methods are employed direct and indirect. The direct method is a straightforward one-step process that creates a direct reaction between the antigen and the labeled antibody. The indirect method requires the use of two antibodies a primary unlabeled antibody and a secondary labeled antibody. 

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species and accordingly can be visualized with differently labeled species-specific secondary antibodies (see Sect. 8.1). However, quite often the appropriate combination of primary antibodies from different host species is not available. A general problem relates to the fact that the available primary antibodies may originate only from one species either rabbit or mouse. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies (Ino 2004). 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

Fluorophore absorbs ultraviolet light (or violet, blue or green) and emits light of longer wavelength. Fluorophores are used in immunohistochemistry for labeling primary or secondary antibodies in direct and indirect immunostain-ing methods, respectively. They can be visualized in fluorescence microscopy using special filter sets. 

The specificity of antibodies can be exploited in order to probe the in situ organization of cells and tissues. Cellular antigens can be identified both in viable cells and in frozen or fixed tissue sections. Antibodies are used to identify the appropriate antigen in the section and then the position of this primary antibody may itself be detected either directly if it was initially labelled or indirectly using another secondary antibody or molecule to attach to the antibody (Figure 7.8). Samples need to be carefully washed after addition of the primary or labelled antibody in order to prevent any non-specific reactions. Labels that have been successfully linked to antibodies include the following ... 

Figure 7.8 Some examples of antibody detection techniques, (a) Direct labelling of the primary antibody, (b) indirect using labelled secondary antibody, (c) indirect using biotinylated secondary antibody and labelled streptavidin.

If the antigen of interest is abundant and additional sensitivity is not required, then a two-step direct method, rather than a three-step indirect method, can be anployed. With the two-step method, the primary antibody is a biotin-labeled mouse monoclonal antibody, which is followed directly by the ABC incubation step. Because of the abundance of human immunoglobulins in tissue sections, methods for staining immunoglobulin often employ biotin-labeled mouse antihuman immunoglobulin reagents. 

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