Glycerin Suppression of Fluorescence Self-Quenching and Improvement of Heterogeneous Fluoroimmunoassay Sensitivity

Abstract

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.

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

“…Finally, there is a limit to the number of fluorescence molecules that can be attached to an antibody. The presence of multiple fluorophores in close proximity can decrease fluorescence via quenching mechanisms; increased labeling may produce a reagent that is dimmer then one with less labeling [6; 7; 10; 11; 12; 13; 14]. …”

Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples

“…In addition, as mentioned in a previous publication, fluorescence self-quenching, which may affect the sensitivity of fluorescence immunoassay, is sometimes observed when a relatively large number of fluorescent compounds are introduced in the recognition molecule, such as antibodies. In this part, according the method developed by Petrou et al, , the fluorescence self-quenching was evaluated by comparing the fluorescence intensity of this assay treated or not with 30% glycerin. As shown in Figure D, fluorescence self-quenching was suppressed effectively after treatment with 30% glycerin when antibody dilution was from 500 to 4000 but the fluorescence intensity was nearly the same under a low antibody concentration.…”

Environment-sensitive benzoxazole based fluorescein derivatives: Synthesis and application to the design of ON–OFF fluorescent chemosensors for microenvironment

“…However the development of fluorescence labels based on the heterogeneous assay presented several problems, mainly low signal to noise ratio when proteins are labeled with multiple fluorescence molecules [22]. This phenomenon has been attributed to the small Stoke's shift of the fluorescence molecule that permits energy transfer from one fluorescent molecule to another causing self-quenching [23].…”