Fluorescent-labeled antibodies are used as a histochemical or cytochemical tool for detection and localization of antigen and antibody reactions with use of a fluorescence microscope. In research, a wide range of disease-causing microorganisms has been identified, localized, and quantified by fluorescent-antibody staining. Some of the diseases in which the technique has been useful in research are toxoplasmosis, malaria, anthrax, bubonic plague, typhus, polio, and rabies. In clinical assays, the technique is used in screening tests for the detection of syphilis, neurosyphilis, lupus, rheumatoid arthritis, myasthenia gravis, rickettsia, typhus, chlamydia, psittacosis, Epstein-Barr virus, Herpes Simplex viruses, and streptococcal infections. The two dyes most widely used are fluorescein and rhodamine B because of their intensity of fluorescence. An isothiocyanate derivative of either dye is coupled to amino groups in the proteins of antibodies; these conjugated antibodies are usually used for staining, and result is observed on slide mounts. The rapid fading of the fluorescence of fluorescein is its greatest disadvantage. We propose to use several newly developed dyes already shown to have exceptional photostability as laser dyes, and their isothiocyanate derivatives. The new dyes are quaternary salts of oligophenylene derivatives, are water- soluble, have high fluorescence quantum yields, a huge Stokes shift, and absorption maxima matching the usual 366nm UV source. For preliminary screening of the new dyes, resistance to fading and compatibility with biological materials and buffers would be checked, along with fluorescein and rhodamine B, by making slide mounts of CV-1, monkey kidney cells, in phosphate buffer. Then reactive derivatives of the new dyes containing isothiocyanate, maleimide, or oxirane groups would be prepared. Anti-tubulin antibodies will be conjugated to the new dyes using standard procedures, and the fluorescent-labeled antibodies allowed to form antibody-antigen complex with CV-1, monkey kidney cells, in phosphate buffer: and the stained fibroblast cells observed for brilliance and fading.
