Development of a novel FRET immunosensor technique.
We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.
Lichlyter
Darcy J
DJ
Department of Biological Engineering, University of Missouri-Columbia, 250 Ag. Engineering Building, Columbia, MO 65211, USA.
Grant
Sheila A
SA
Soykan
Orhan
O
D000936:Antigen-Antibody Complex / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D016231:Fluorescence Polarization Immunoassay / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis / Q000737:chemistry / Q000276:immunology
D000936:Antigen-Antibody Complex; D000941:Antigens; C029152:G-substrate; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A