Fluorochrome streptavidin

Fig. 1 Diagram illustrating the molecular interactions of direct immunofluorescent labeling method. The fluorochrome-labeled antibody reacts with the cell or tissue antigen in a one-step staining procedure (a). Alternatively, the primary antibody, labeled with biotin, can be used with a secondary fluorochrome-streptavidin reagent to increase signal strength (b).

Fluorochrome-streptavidin conjugate (for laser confocal fluorescence microscopy). 

When one or more of the desired antibodies is not available as a direct conjugate and a conjugate cannot be synthesized, indirect methods have to be used. The simplest variation is to use a biotin-labeled antibody as a first layer, and then after washing the cells, incubate with fluorochrome-labeled streptavidin, and additional directly labeled antibodies labeled with complementary fluoro-chromes. The additional streptavidin step does not usually cause difficulty... 

By using a combination of antibody microarrays and streptavidin-QDs, several cytokines (TNF-a, IL-8, IL-6, macrophage inflammatory protein-1 p, EL-13, and EL-lp) were detected at 1 pM concentrations in cancer samples (62). In 2006, Ghazani et al. increased 30-fold the sensitivity in a few cancer biomarkers, such as CEA and Her-27Neu, in serum and plasma by using QDs instead of conventional fluorochromes (63). 

Fluorochrome labeling of streptavidin or antibody Conjugation procedures should yield optimal fluorochrome/protein (F/P) ratios. Most economically, the desired F/P ratio is regulated by the initial weight of dye in the reaction mixture and the reaction is allowed to go to completion. Alternatively, with relatively more dye, the reaction is interrupted after a specific incubation period. The efficiency of labeling depends on the protein, the protein concentration, the specific fluorochrome and the purity of the fluorochrome (some preparations only 30%). Over- or undercoupling leads to nonspecificity or low detectability, respectively. 

Streptavidin-fluorochrome conjugates provide higher sensitivity than two-step indirect techniques. The use of Alexa dyes instead of the traditional fluorochromes enhances brightness and detection sensitivity of immunofluorescence methods several-fold (12). 

In most cases, it is convenient to use a conjugate of streptavidin with the fluorochrome Cy3 which has a red flnorescence and the SYTOX Green dye for nuclear staining, which has a green fluorescence. Typically, most flnorescence microscopes have filters for these two fluorochromes, and confocal microscopes are equipped with lasers for the excitation of these dyes. 

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