The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported. These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity. Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared. These conjugates bind specifically to beads containing a covalently attached target molecule and render them highly fluorescent. Femtomole (10(-15) mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (epsilon M = 2.4 x 10(6) cm-1 M-1 for 2.4 x 10(5) daltons) and high fluorescence quantum yield (Q = 0.8) of the phycobiliprotein moiety. An important feature of these conjugates is that they emit in the orange-red spectral region, where background fluorescence is less than at shorter wavelengths. Phycoerythrin conjugates are well-suited for two-color flow cytofluorimetric analyses employing a single excitation line. The distributions of Leu antigens (also called OKT antigens) on the surface of T-lymphocytes were analyzed using fluoresceinated antibody as the green-fluorescent stain and biotinylated antibody counter-stained with phycoerythrin-avidin as the red one. This one-laser two-color analysis showed that cells express Leu-3a and Leu-3b or neither antigen. In contrast, the distributions of Leu-2a (a marker of suppressor and cytotoxic T-cells) and Leu-3a (a marker of helper and inducer T-cells) are mutually exclusive. These studies show that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay.

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