Glycerin suppression of fluorescence self-quenching and improvement of heterogeneous fluoroimmunoassay sensitivity.
Fluorescent labels find wide application in immunoassays and immunosensors as well as in protein and DNA chips. However, the use of fluorescent labels in applications requiring high detection sensitivity is limited by fluorescence self-quenching observed when a relatively high number of fluorescent compounds is introduced in the recognition molecule. Here we describe a simple method that suppresses effectively fluorescence self-quenching observed when highly labeled antibodies are used as labels in immunoassays. This was achieved by treating the microtitration wells after the completion of the immunoassay with a glycerin solution followed by 15-min incubation of the emptied wells at 37 degrees C. The remedial action of this method on self-quenching was studied through a noncompetitive immunofluorometric assay for rabbit gamma-globulins employing a sheep anti-rabbit gamma-globulin antibody labeled with fluorescein at molar ratios ranging from 1.0 to 17.4. The glycerin/thermal treatment increased the fluorescence signal measured directly onto the solid surface by 9.2-117% for the antibodies with molar ratios of 1.0-17.4, compared with the values obtained prior to treatment. Furthermore, fluorescence self-quenching was completely removed for labeling ratios up to 14.0. The assay sensitivity was improved 2-4 times by the glycerin/thermal treatment when heavily fluoresceinated antibodies are used as labels (molar ratio >/=5.6). The proposed method resulted also in increased fluorescence signals when labels other than fluorescein were used and improved considerably the detection of protein spots on silicon dies.
Petrou
Panagiota S
PS
Immunoassay/Immunosensors Laboratory, Institute of Radioisotopes & Radiodiagnostic Products, NCSR "Demokritos", 15310 Athens, Greece.
Mastichiadis
Christos
C
Christofidis
Ion
I
Kakabakos
Sotirios E
SE
D000906:Antibodies / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015200:Fluoroimmunoassay / Q000592:standards*; D005990:Glycerol / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013696:Temperature
D000906:Antibodies; D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate; D005990:Glycerol