Historical background

The potential for employing labeled antibodies to localize antigens or antibodies in tissues at the microscopic level was first conceived by and realized through the experiments of Coons and colleagues in 1941 and 1942. These experiments described the first immunoconjugates wherein antibodies were chemically coupled to fluorescein and used to specifically bind target antigens in tissues. The resultant 'microprecipitates' were identified by their brilliant green color in a light microscope equipped with an ultraviolet light source and appropriate excitation and emission barrier filters for fluorescence microscopy. Novel fluorochromes such as rhoda-mine, Texas red and phycoerythrin have since been discovered that can be coupled to antibodies which, when excited by the appropriate wavelength of ultraviolet light, can be identified by their red color. The ability to couple fluorochromes with distinct emission spectra to antibodies specific for different epitopes has permitted dual-antigen localizations on the same sample. In addition to immunofluorescence microscopy, enzymes have also been coupled to antibodies and used to localize the antibody-antigen interaction. In immunoenzyme microscopy, the antibody-coupled enzyme converts a colorless chromogenic substrate into colored end-products at the site of the antibody-antigen reaction, which can then be visualized by conventional light microscopy.

With the advent of the transmission electron microscope and its greater resolving power, now in the range of 0.14 nm, valuable approaches complementary to those employed for the light micro scope were developed to localize specific antigen at the ultrastructural level. The first such immunoconjugates for electron microscopy utilized electron-dense molecules to replace the fluorochromes used in the ultraviolet microscope. Singer was the first, in 1959, to develop a method for coupling an electron-dense molecule, horse-spleen ferritin, to antibodies without a concomitant loss of immunobinding capacity. The experimental results of Rifkind and colleagues conclusively demonstrated in 1960 the utility of these electron-dense ferritin-antibody conjugates in the immunodetection of antigens in the electron microscope. These experiments formed the early foundation for the field of study now known as immunoelectron microscopy.

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