Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.
Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.
复制标题
DOI：
10.1083/jcb.93.3.981
复制
发表时间：
1982-06
期刊：
影响因子：
--
通讯作者：
Stryer L
中科院分区：
文献类型：
--
作者：
Oi VT;Glazer AN;Stryer L
报道了一类新型分子和细胞荧光分析试剂的合成。这些化合物由与具有生物特异性的分子缀合的高荧光藻胆蛋白组成。制备藻蓝蛋白-免疫球蛋白、藻蓝蛋白-蛋白A和藻蓝蛋白-抗生物素蛋白缀合物。这些缀合物特异性地结合到含有共价连接的靶分子的珠上，并使其具有高度荧光。由于藻胆蛋白部分的高消光系数（对于2.4 × 10（5）道尔顿，λ M = 2.4 × 10（6）cm-1 M-1）和高荧光量子产率（Q = 0.8），可以检测到毫微微摩尔（10（-15）摩尔）量的藻红蛋白缀合物。这些缀合物的一个重要特征是它们在橙红色光谱区域中发射，其中背景荧光小于较短波长。藻红蛋白偶联物非常适合采用单一激发线的双色流式细胞荧光分析。用荧光素标记的抗体作绿色荧光染色，生物素标记的抗体与藻蓝蛋白-亲和素复染作红色荧光染色，分析T淋巴细胞表面Leu抗原（又称OKT抗原）的分布。这种单激光双色分析显示细胞表达Leu-3a和Leu-3b或两者都不表达。相比之下，Leu-2a（抑制性和细胞毒性T细胞的标志物）和Leu-3a（辅助性和诱导性T细胞的标志物）的分布是相互排斥的。这些研究表明藻胆蛋白偶联物可应用于荧光激活细胞分选和分析、荧光显微镜和荧光免疫分析。
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.