Fluorescein-conjugated anti-human primary antibodies are green-fluorescent immunoreagents used for detecting human antigens in various fluorescence-based applications. 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 fluorescein, a small-molecule xanthene dye with excitation and emission maxima at approximately 494 and 521 nm, respectively. Fluorescein exhibits a high molar extinction coefficient (~76,000 M−1cm−1) and quantum yield (~0.92), contributing to its widespread use in fluorescence-based assays.
Unlike FITC (fluorescein isothiocyanate), the most common isothiocyanate derivative used for conjugation, the term “fluorescein-conjugated” often refers to FITC-conjugated antibodies, as FITC is the reactive form that covalently bonds to primary amines on lysine residues. The conjugation process typically yields fluorophore-to-protein (F/P) ratios of 3–8, optimizing fluorescence intensity while preserving antigen-binding affinity. Available specificities include anti-human IgG (H+L), anti-human IgG (Fc), isotype-specific reagents (IgG1–IgG4, IgA, IgM, IgE), and anti-human CD markers (CD3, CD4, CD8, CD14, CD19, CD45, etc.), cytokines, and signaling proteins. Extensive cross-adsorption against serum proteins from mouse, rat, rabbit, and bovine eliminates nonspecific binding in multi-species samples.
Applications
Fluorescein conjugates are extensively employed in flow cytometry and FACS for immunophenotyping human leukocyte subsets, detecting surface and intracellular antigens using 488 nm laser excitation. In immunofluorescence microscopy and immunohistochemistry, these reagents enable antigen localization in human tissue sections and cultured cells with bright green emission. Additional applications include ELISA, fluorescent Western blotting, cell proliferation assays using CFSE labeling, and apoptosis detection.
However, fluorescein exhibits pH sensitivity, with optimal fluorescence at pH 7.0–9.0, significant signal reduction below pH 7.0, and susceptibility to photobleaching. These characteristics necessitate the use of antifade mounting media, reduced light exposure, and appropriate fixation protocols. 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, protected from light; do not freeze. These reagents are intended for research use only and are not for diagnostic procedures.