The characteristic stability of antibody binding to specific antigen lends itself as an immunological probe for localization of particular molecules in cells and tissues. Because fluorescent dyes such as fluorescein

and rhodamine can be coupled to antibodies without destroying their specificity, the conjugates can complex with antigen and be visualized via fluorescence microscopy. The microscope excites the chosen dyes by light of one or more wavelengths, which in turn emits light at a characteristic wavelength captured by a selective filter. Figure 1 shows the excitation and emission spectra for 8 different dyes used in FACS immunofluorescence experiments. The image is then projected with localized regions of fluorescence indicating different antigens labeled by antibodies of distinctive color.

Figure 1

The test can be carried out in three general ways (Figure 2): (1) Direct test, (2) Indirect test, and (3) Sandwich test.

The Direct Test

The antibody is itself conjugated with the fluorochrome and applied directly to a monolayer of cells or to frozen tissue on a slide. When examined with a fluorescence microscope, the antibody labelled with the fluorescent moiety identifies the localized antigen. Two different antigens can be identified simultaneously, in the same preparation by using antisera conjugated to dyes active at different wavelengths. Direct immunofluoresence imaging in Figure 3 indicates deposition of material containing polyclonal IgG and complement within capillary walls and mesangium, as well as tubular basement membranes.

Figure 3 : photographs by Maria Parizhskaya, M.D. and Sheldon Bastacky, M.D.

Click here for a link to a direct immunofluorescence staining protocol, including methods, materials, and equipment needed.

The Indirect Test

Unlike the direct test, the indirect test is a double-layer technique. The unlabelled antibody is applied directly to the tissue substrate and then treated with a fluorochrome-conjugated anti-immunoglobulin serum (figure 4). There are several advantages to this technique, and is recently much favoured over the direct test.

Figure 4 : Goat antisera, tagged with fluorscein, made against human IgG, was used to detect human autoantibody in thyroid tissue. The test in this case is positive for anti-thryroglobulin in the thyroid follicle colloid. (click here for link to source)

Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test. The method is also more flexible because a semi-quantitative assessment of the distribution of classes and subclasses of antibodies can be made by using antisera conjugates to individual immunoglobulin heavy chains. It is also more time-efficient since it is only one signal labelled reagent, the anti-immunoglobulin, is prepared during the lengthy conjugation process. The test is also not limited to localization of antibodies. Complement fixation can also be assessed by adding a mixture of the first antibody plus a source of complement, followed by a fluorescent anti-commplement reagent as the second layer. Application of a third layer increases the sensitivity of the test (unfortunately at the cost of specificity).

Click here to connect to an indirect immunofluorescence staining protocol, including methods, materials, and equipment needed.

The Sandwich Test

This test was designed to visualize a specific antibody produced within a cell. The tissue is first fixed with ethanol to prevent washing away the antibody during the test. Treatment with polysaccharide antigen constitutes the first layer. After washing, the fluorochrome-labelled antibody to the antigen is added to locate cells which have specifically bound the antigen (see Figure 2).

Cellular and molecular immunology. Abul K. Abbas, Andrew H. Lichtman, Jordan S. Pober. Philadelphia : W.B. Saunders, c1994 (60-61).

Essential immunology. Ivan M. Roitt. Oxford; Boston : Blackwell Scientific Publications; St. Louis, MO.: Distributors, USA, Mosby-Year Book, 1991 (96-100).

Immunobiology. Charles A. Janeway, Jr., Paul Travers. New York : Current Biology Ltd., 1997 (2:22-2:23).

Kansas University Medical College :

http://www.kumc.edu/instruction/medicine/pathology/ed/ch_4/c4_hashimoto_if.html

Stanford University :

http://cmgm.stanford.edu/~roederer/abcon/allspec.html

University of Florida, Flow Cytometry Core Lab :

http://www.biotech.ufl.edu/~fccl/indirect.html

http://www.biotech.ufl.edu/~fccl/direct.html

University of Pittsburgh Medical College :

http://www.pathology.pitt.edu/cases/case78/immuno.html

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