Fluorescent dyes such as fluorescein or rhodamine can be attached to known specific antibodies, which are then used to detect the presence of an antigen in serum or microorganisms in a sample. There are two basic versions of these quick and sensitive tests.
In this test, the microorganism to be tested is fixed to a slide. Known antibodies labeled with fluorescein are added, the mixture is incubated, and the slide is washed. Antibody that binds to the microorganism will not wash away. The slide is then examined under a fluorescence microscope with a special light source to permit light of the desired wavelength to excite the dye. The dye then emits light of a different wavelength, which is viewed by using special filters in the microscope. If the microorganisms have bound the antibody, they will glow a yellow-green color (figure 17.8a). If rhodamine is used instead of fluorescein, the microorganisms will glow a red color. By using various fluorescent dyes, it is possible to locate different antigens in the same cell or preparation. ■ fluorescence microscope, p. 43
This test is used to find a specific antibody in human serum following an infection. A known organism is fixed to the slide, a sample of the test serum is added, and the mixture on the slide is incubated. If specific antibodies are present in the serum, they will bind to the antigens of the organism; but the complex cannot be seen until it is labeled or tagged in some way, as with a fluorescent dye. In order for the antigen-antibody complexes formed to be seen, any unfixed material is washed off and anti-human-gamma-globulin (anti-HGG) antibodies that have been labeled with fluorescein are added to the slide. Anti-HGG is prepared by immunizing animals with the gamma globulin portion of human serum, the portion that contains the antibodies. Such anti-human antibodies are useful in many
Fluorescent dye molecule
Fluorescent dye molecule
For direct testing, antigen is fixed to the slide. Antibody, to which fluorescence has been attached, is added. When viewed with a fluorescence microscope, antibodies appear yellow-green.
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