Immunofluorescence

In 1944, Albert Coons showed that antibodies could be labeled with molecules that have the property of fluorescence. Fluorescent molecules absorb light of one wavelength

Magnetic bead

Magnetic bead

Albert Coons

Add specific antibody to cell extract

Add secondary antibody coupled to magnetic beads

Add specific antibody to cell extract

FIGURE 6-13

Add secondary antibody coupled to magnetic beads

Apply magnet and rinse to remove unbound material

Apply magnet and rinse to remove unbound material

Immunoprecipitates can be collected using magnetic beads coupled to a secondary antibody. (a) Treatment of a cell extract containing antigen A (red) with a mouse anti-A antibody (blue) results in the formation of antigen-antibody complexes. (b) Addition of magnetic beads to which a rabbit anti-mouse antibody is linked binds the antigen-antibody complexes (and any unre-acted mouse Ig). (c) Placing a magnet against the side of the tube

Attaching Heavy Beads With Chain

allows the rapid collection of the antigen-antibody complexes. After rinsing to remove any unbound material, the antigen-antibody complexes can be dissociated and the antigen studied. (d) An electron micrograph showing a cell with magnetic beads attached to its surface via antibodies. [Part (d), P. Groscurth, Institute of Anatomy, University of Zurich-Irchel.]

allows the rapid collection of the antigen-antibody complexes. After rinsing to remove any unbound material, the antigen-antibody complexes can be dissociated and the antigen studied. (d) An electron micrograph showing a cell with magnetic beads attached to its surface via antibodies. [Part (d), P. Groscurth, Institute of Anatomy, University of Zurich-Irchel.]

(excitation) and emit light of another wavelength (emission). If antibody molecules are tagged with a fluorescent dye, or fluorochrome, immune complexes containing these fluores-cently labeled antibodies (FA) can be detected by colored light emission when excited by light of the appropriate wavelength. Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized. The emitted light can be viewed with a fluorescence microscope, which is equipped with a UV light source. In this technique, known as im-munofluorescence, fluorescent compounds such as fluores-cein and rhodamine are in common use, but other highly fluorescent substances are also routinely used, such as phyco-erythrin, an intensely colored and highly fluorescent pigment obtained from algae. These molecules can be conjugated to the Fc region of an antibody molecule without affecting the specificity of the antibody. Each of the fluo-rochromes below absorbs light at one wavelength and emits light at a longer wavelength:

■ Fluorescein, an organic dye that is the most widely used label for immunofluorescence procedures, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence (517 nm).

■ Rhodamine, another organic dye, absorbs in the yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). Because it emits fluorescence at a longer wavelength than fluorescein, it can be used in two-color immunofluorescence assays. An antibody specific to one determinant is labeled with fluorescein, and an antibody recognizing a different antigen is labeled with rhodamine. The location of the fluorescein-tagged antibody will be visible by its yellow-green color, easy to distinguish from the red color emitted where the rhodamine-tagged antibody has bound. By conjugating fluorescein to one antibody and rhodamine to another antibody, one can, for example, visualize simultaneously two different cell-membrane antigens on the same cell.

■ Phycoerythrin is an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter of red fluorescence, stimulating its wide use as a label for immunofluorescence.

Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect (Figure 6-14). In direct staining, the specific antibody (the primary antibody) is directly conjugated with fluorescein; in indirect staining, the primary antibody is unlabeled and is detected with an additional fluorochrome-labeled reagent. A number of reagents have been developed for indirect staining.

Cells with membrane antigens (mAg)

Primary antibody c

Primary antibody

(a) Direct method with fluorochrome-labeled antibody to mAg

(a) Direct method with fluorochrome-labeled antibody to mAg

Primary antibody to mAg

Primary antibody to mAg

Indirect Method With Fluorochrome

Protein A

Secondary anti-isotype antibody

(b) Indirect method with fluorochrome-labeled anti-isotype antibody

(c) Indirect method with fluorochrome-labeled protein A

Secondary anti-isotype antibody

(b) Indirect method with fluorochrome-labeled anti-isotype antibody

Protein A

(c) Indirect method with fluorochrome-labeled protein A

FIGURE 6-14

Direct and indirect immunofluorescence staining of membrane antigen (mAg). Cells are affixed to a microscope slide. In the direct method (a), cells are stained with anti-mAg antibody that is labeled with a fluorochrome (Fl). In the indirect methods (b and c), cells are first incubated with unlabeled anti-mAg antibody and then stained with a fluorochrome-labeled secondary reagent that binds to the primary antibody. Cells are viewed under a fluorescence microscope to see if they have been stained. (d) In this micrograph, antibody molecules bearing ^ heavy chains are detected by indirect staining of cells with rhodamine-conjugated second antibody. [Part(d), H. A. Schreuder et al., 1997, Nature 386:196, courtesy H. Schreuder, Hoechst Marion Roussel.]

The most common is a fluorochrome-labeled secondary antibody raised in one species against antibodies of another species, such as fluorescein-labeled goat anti-mouse im-munoglobulin.

Indirect immunofluorescence staining has two advantages over direct staining. First, the primary antibody does not need to be conjugated with a fluorochrome. Because the supply of primary antibody is often a limiting factor, indirect methods avoid the loss of antibody that usually occurs during the conjugation reaction. Second, indirect methods increase the sensitivity of staining because multiple molecules of the fluorochrome reagent bind to each primary antibody molecule, increasing the amount of light emitted at the location of each primary antibody molecule.

Immunofluorescence has been applied to identify a number of subpopulations of lymphocytes, notably the CD4+ and CD8+ T-cell subpopulations. The technique is also suitable for identifying bacterial species, detecting Ag-Ab complexes in autoimmune disease, detecting complement components in tissues, and localizing hormones and other cellular products stained in situ. Indeed, a major application of the fluorescent-antibody technique is the localization of antigens in tissue sections or in subcellular compartments. Because it can be used to map the actual location of target antigens, fluorescence microscopy is a powerful tool for relating the molecular architecture of tissues and organs to their overall gross anatomy.

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  • franziska
    Do magnetic bead interfere with fluorescence?
    8 years ago

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