FITC (fluorescein isothiocyanate) conjugated anti-human primary antibodies are green-fluorescent immunoreagents used for the detection of human antigens in standard fluorescence-based platforms. Produced by immunizing hosts such as goat, rabbit, mouse, or rat with purified human immunoglobulins or specific human proteins, these antibodies are affinity-purified and conjugated to FITC, a small-molecule fluorophore with excitation and emission maxima at approximately 495/519 nm. The isothiocyanate group reacts with primary amines on lysine residues, forming stable thiourea bonds. Typical fluorophore-to-protein (F/P) ratios range from 3 to 8, supporting fluorescence intensity while maintaining antigen-binding activity. FITC has a high molar extinction coefficient (≈ 70,000 M−1cm−1) and quantum yield (≈ 0.92), contributing to moderate fluorescence brightness. However, FITC is susceptible to photobleaching and exhibits pH-dependent fluorescence, with optimal emission generally observed between pH 7.0 and 9.0 and significant signal reduction below pH 7.0. Available specificities include anti-human IgG (H+L), anti-human IgG (Fc), isotype-specific reagents (IgG1–IgG4, IgA, IgM, IgE), anti-human CD markers such as CD3, CD4, CD8, CD14, and CD45, as well as antibodies targeting cytokines. Extensive cross-adsorption against serum proteins from mouse, rat, rabbit, and bovine sources helps minimize nonspecific binding.
Applications
FITC conjugates are extensively used in flow cytometry and fluorescence-activated cell sorting (FACS) for immunophenotyping human leukocyte subsets and detecting surface and intracellular antigens using standard 488 nm laser excitation. In immunofluorescence microscopy and immunohistochemistry, FITC enables visualization and localization of antigens in human tissue sections and cultured cells through its characteristic green fluorescence. Additional applications include ELISA, fluorescent Western blotting, and cell proliferation assays.
Because FITC is susceptible to photobleaching, antifade mounting media and minimized exposure to excitation light are recommended during fluorescence imaging. Typical working dilutions range from 1:50 to 1:500 for flow cytometry, 1:100 to 1:1,000 for immunofluorescence, and 1:1,000 to 1:5,000 for ELISA. Store at 4°C and protect from light; do not freeze. Intended for research use only.