FITC (fluorescein isothiocyanate) and PE (R-phycoerythrin) conjugated anti-human primary antibodies are green- and orange-fluorescent immunoreagents, respectively, offered as separate conjugates for detecting human antigens in fluorescence-based applications. Produced by immunizing hosts such as goat, rabbit, mouse, or rat with purified human immunoglobulins or specific human proteins, the antibodies are affinity-purified and conjugated to either fluorophore. FITC (ex/em 495/519 nm) is a small-molecule dye compatible with 488 nm lasers, offering cost-effectiveness and a high quantum yield (~0.92). PE (ex/em 488 or 565/578 nm) is a 240 kDa phycobiliprotein with exceptional brightness (~25× brighter than FITC), a high extinction coefficient (1.96 × 106 M−1cm−1), and a quantum yield (~0.84), enabling detection of low-abundance human antigens.
Available specificities include anti-human IgG (H+L), anti-human IgG (Fc), isotype-specific reagents (IgG1–IgG4, IgA, IgM), and anti-human CD markers (CD3, CD4, CD8, CD19, CD20, CD45). Extensive cross-adsorption is used to minimize nonspecific binding. PE conjugates are more photostable but should be protected from light and not frozen.
Applications
FITC and PE conjugates are widely employed in flow cytometry and fluorescence-activated cell sorting (FACS) for immunophenotyping human leukocyte subsets using 488 nm laser excitation. PE is particularly suitable for low-abundance markers because of its superior fluorescence brightness. In immunofluorescence microscopy, FITC provides green emission, whereas PE produces bright orange fluorescence. Additional applications include ELISA, high-content screening, and immunohistochemistry.
FITC exhibits pH sensitivity and is susceptible to photobleaching; the use of antifade mounting media is therefore recommended for fluorescence microscopy. PE exhibits lower sensitivity to pH-related fluorescence changes and strong photostability, although protection from light remains important during handling and storage.